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Cetuximab-Activated Natural Killer and Dendritic Cells Collaborate to Trigger Tumor Antigen–Specific T-cell Immunity in Head and Neck Cancer Patients
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Tensile behavior of humid aged advanced composites for helicopter external fuel tank development
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© 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/). Tensile behavior of humid aged advanced
composites for helicopter external fuel tank
development Mihaela Condruz1*, Alexandru Paraschiv2, Cristian Pușcașu2, and Ionuț Sebastian Vintilă1
1COMOTI - Romanian Research & Development Institute for Gas Turbines, Composite Materials
Laboratory for Aeronautical Field and Space, 220 D Iuliu Maniu Av., Bucharest, Romania
2COMOTI - Romanian Research & Development Institute for Gas Turbines, Gas Turbine Special
Components. Physics and Mechanic Testing Laboratory, 220 D Iuliu Maniu Av., Bucharest, Romania Abstract. Influence of humid aging on tensile properties of two polymeric
composites was studied. The purpose of the study was to evaluate the
suitability of the materials for a naval helicopter external fuel tank. Due to
the application, the humid environment was kerosene and saline solution to
evaluate the sea water effect on the composite tensile strength. The
composite samples were immersed in kerosene for 168 hours, respective
1752 hours and in saline solution for 168 hours. Tensile tests were
performed after the immersion. The composite sample tensile tests showed
that kerosene and saline solution had no influence on the elastic modulus
of the materials, but it was observed a slight improvement of the tensile
strength of the two polymeric composites. * Corresponding author : raluca.condruz@comoti.ro MATEC Web of Conferences 145, 02004 (2018)
NCTAM 2017 MATEC Web of Conferences 145, 02004 (2018)
NCTAM 2017 https://doi.org/10.1051/matecconf/201814502004 1 Introduction Plastic fuel tanks should be fuel and environmental resistant (corrosion due to salt,
moisture, ultraviolet radiation, thermal cycling in atmospheric conditions during normal
service use and storage between -54°C and +71°C) [8-11]. Durability of FRP can be
affected by the contact with water or other fluids [12]. Most polymeric materials (in the
form of a composite matrix or a polymeric fibre) are capable of absorbing relatively small
but potentially significant amounts of moisture from the surrounding environment. The
physical mechanism for moisture gain, assuming there are no cracks or other wicking paths,
is generally assumed to be the mass diffusion following Fick's Law (the moisture analogue
to thermal diffusion) [13]. The humid aging is one of the main causes of long-term failure
of organic composites, which can affect the material in several modes: matrix
microcracking, plasticization, hydrolysis, delamination, blistering, void growth, swelling,
localized damage at the fibre-matrix interface [12-18]. Epoxy resin is known to have
affinity for water [12], moisture has significant effects on physical and chemical properties
of the resin as well as on the final composite structures performance, especially in long-
term utilization [19]. The moisture absorption effect on polymeric composites mechanical
properties had been studied for decades [20,21], but until now it’s still a challenge to
predict how a fibre reinforced composite will behave under a certain humid environment. In early technical studies regarding environmental influence on composites, it was
observed that in case of carbon/epoxy composites, the filament properties show very little
environmental sensitivity, while epoxy resin is affected by the absorbed moisture and
temperature effects which can lead to a change in failure mode [20], also the mechanical
properties of composites are reduced after humid exposure, but all moisture effects were
completely reversed upon re-drying the specimens [21]. Most of the authors studied water
humid aging influence on polymeric composites [22-28] and as a general conclusion, the
water causes mechanical properties degradation of thermosetting composites in case of
extended period of immersion which can be noticed by matrix osmotic, cracking, swelling,
mass loss, fibre exposure, chemical alterations etc. Many information about the effect of
other fluids than water on composite properties are not available in the literature. p
p
p
Souza et.al. 1 Introduction Nowadays, fibre reinforced polymers (FRP) are a common material used in the aerospace
field (for commercial/military aircrafts, helicopters, satellites etc.) due to their remarkable
properties, such as strength, stiffness, low density and low coefficient of thermal expansion
[1, 2]. An important factor that stays behind the greater use of composites is the cost
reduction experienced by carbon fibres [1]. Also, in case of aerospace applications, the
reduction of weight, costs and fuel along with the increase of resistance and durability of
the components are key aspects. Usually, in helicopter applications, composite materials are
used to manufacture rotor blades, which ensure a significant weight reduction over
aluminium and provide a better control over the vibration characteristics of the blades [3]. Excepting rotor blades, polymeric composites were integrated in tank assemblies for launch
vehicles and helicopters. Researches were conducted to manufacture and validate external
composite fuel tanks for helicopters to replace the metallic tanks [4]. External fuel tanks
have been introduced to extend the range of a helicopter or to provide refuelling capacity at
a remote location (it can provide at least an additional hour of flight depending on its
capacity [5]). The first attempt to replace metallic external tanks was in the middle of 1970
and they were replaced by a hybrid model, a Composite Overwrapped Pressure Vessel - MATEC Web of Conferences 145, 02004 (2018)
NCTAM 2017 https://doi.org/10.1051/matecconf/201814502004 COPV [4] (structure with a thin metal liner that acts as a leakage barrier, overwrapped by
composite winding layers [6]). Afterwards, the COPV tanks were replaced by all composite
external tanks. For researchers, an opportunity is represented by designing and
manufacturing of an external composite fuel tank for F-35 Lightning II fighters [4,7]. An
important aspect for designing and manufacturing external and auxiliary fuel tanks is to
study structure's requirements and specification, which are stated in MIL-T-7378A [8]. COPV [4] (structure with a thin metal liner that acts as a leakage barrier, overwrapped by
composite winding layers [6]). Afterwards, the COPV tanks were replaced by all composite
external tanks. For researchers, an opportunity is represented by designing and
manufacturing of an external composite fuel tank for F-35 Lightning II fighters [4,7]. An
important aspect for designing and manufacturing external and auxiliary fuel tanks is to
study structure's requirements and specification, which are stated in MIL-T-7378A [8]. 2 Experimental procedure Two types of polymeric composite laminates were manufactured for this experiment. The
laminates were manufactured by using metallic moulds and an autoclave (curing process
under temperature, vacuum, pressure). The composite consisted in two prepreg precursors,
a HexPly M49/42%/200T2X2/CHS-3K (referred as HexPly) and an EP 127-C20-45 T2
(referred as EP). The HexPly consisted in a twill 2x2 carbon fabric 3K HS preimpregnated
with an epoxy resin system (42% resin content), and the EP was a twill 2x2 carbon fabric
3K HTA preimpregnated with a blend of epoxy resin and cyanate ester (45±3% resin
content). After curing, samples were cut from laminates using a water jet cutting machine. The samples dimensions (250x25x3 mm with 1 mm thick tabs, type 3) were established
based on SR EN ISO:527-4/2000 [33], and ASTM D3039/D 3039M/2000 [34]. To ensure
the water removal from the cutting process and the curing of the tabs, all the samples were
post-cured in a vacuum assisted oven (curing cycle: heating for 35 minutes until 120°C
were reached, heating rate of 2°C/min., 90 minutes at 120°C and oven assisted cooling). All the samples were weighed before the immersion in saline solution and in kerosene at
room temperature. To analyse the sea water effect on the composite tensile strength a saline
solution was made by dissolving 35 g sea salt in a litre of distilled water to obtain a saline
solution with 35 ‰ salinity (typical seawater salinity). The specimens were immersed in
saline solution in an open glass recipient and in kerosene in a sealed container. The samples
were kept at room temperature (20±2°C) in kerosene for 168 hours (7 days), respective
1752 hours (73 days) and in saline solution for 168 hours. In Fig. 1 can be observed the
immersed samples in kerosene and saline solution. After immersion, the samples were wiped out with a cloth, weighed out to calculate the
moisture content (or percent weight gain) of the material to establish if the material has kept
its dimensional measurements (or has swollen). a
b
Fig. 1. Samples immersed in kerosene (a) and saline solution (b) a Fig. 1. Samples immersed in kerosene (a) and saline solution (b) Tensile tests were carried out by using a Universal mechanical testing machine
Instron 8802 with a displacement rate of 2 mm/min, according to SR EN ISO: 527-4 [33]. 1 Introduction [29] evaluated the creep behaviour of an E-glass/polyester composite in
water and lubricant oil and it was observed that after 6 and 14 months of exposure, Young’s
modulus of the composite was reduced by 20% regardless the aging fluid when the test was
performed at room temperature and in case of tests performed at 60°C, the effects of
immersion in lubricant oil were more severe than those after immersion in water. Areef [30]
performed an experimental study on different solution absorption behaviour of a sandwich
glass fibres/epoxy composite. The results showed a maximum value of mass gain of the
samples immersed in organic solutions (kerosene, benzene) compared with samples
immersed in other solutions, the samples immersed in distilled water had a minimum mass
gain. Curliss [31] studied the effect of jet fuel JP-4 on thermoplastic and thermosetting
composites. The thermoplastic matrix composites absorbed more fuel compared with the
thermoset matrix composites, but the absorption appeared to be reversible without notable
degradation of the matrix polymer. There was no evidence that any component of the
matrix polymers was leaching into the jet fuel or that any fraction of the polymers was
dissolving into the jet fuel. Taghavi [32] studied the diffusion of moisture into glass/epoxy,
carbon/epoxy and carbon/polyimide composites immersed in water and kerosene. The glass
epoxy composites showed a loss of about 21% of their transverse tensile strength after 100 2 MATEC Web of Conferences 145, 02004 (2018)
NCTAM 2017 https://doi.org/10.1051/matecconf/201814502004 hours of immersion in 80°C kerosene, but after this point no further changes were observed
up to 3500 hours. The transverse modulus was not significantly changed even after 3500
hours of immersion. For carbon polyimide composites, even after 3500 hours of immersion
in 80°C kerosene neither the transverse modulus nor the strength changed. The present research was focused on studying the kerosene and saline solution influence
on the tensile properties of polymeric composite materials suitable for the manufacturing of
an external fuel tank for a naval helicopter (IAR 330 PUMA). 2 Experimental procedure Example representation of typical failure modes in tensile tests of composites [34 2 Experimental procedure For evaluating the influence of the immersion in kerosene and salted water as a function of
time it were used five specimens for each type of sample and for comparison were used 3 3 MATEC Web of Conferences 145, 02004 (2018)
NCTAM 2017 https://doi.org/10.1051/matecconf/201814502004 reference samples from each lot of samples (not immersed). The fracture’s modes were
coded using typical modes from ASTM D3039/D 3039M/2000 [34], in Table 1 are
presented the tensile test failure codes. In Table 2 can be observed the fracture’s modes of
composite specimens after tensile tests. reference samples from each lot of samples (not immersed). The fracture’s modes were
coded using typical modes from ASTM D3039/D 3039M/2000 [34], in Table 1 are
presented the tensile test failure codes. In Table 2 can be observed the fracture’s modes of
composite specimens after tensile tests. Table 1. Tensile test failure codes/typical modes [34] Table 1. Tensile test failure codes/typical modes [34]
First character
Code
Second character
Code
Third character
Code
Angled
A
Inside grip/tab
I
Bottom
B
edge Delamination
D
At grip/tab
A
Top
T
Grip/tab
G
<1W from grip/tab
W
Left
L
Lateral
L
Gage
G
Right
R
Multi – mode
M
Multiple areas
M
Middle
M
long. Splitting
S
Various
V
Various
V
eXplosive
X
Unknown
U
Unknown
U
Other
O
-
-
-
Fig. 2. Example representation of typical failure modes in tensile tests of composites [34] Table 1. Tensile test failure codes/typical modes [34]
First character
Code
Second character
Code
Third character
Code
Angled
A
Inside grip/tab
I
Bottom
B
edge Delamination
D
At grip/tab
A
Top
T
Grip/tab
G
<1W from grip/tab
W
Left
L
Lateral
L
Gage
G
Right
R
Multi – mode
M
Multiple areas
M
Middle
M
long. Splitting
S
Various
V
Various
V
eXplosive
X
Unknown
U
Unknown
U
Other
O
-
-
-
Fig. 2. Example representation of typical failure modes in tensile tests of composites [34] Table 1. Tensile test failure codes/typical modes [34]
First character
Code
Second character
Code
Third character
Code
Angled
A
Inside grip/tab
I
Bottom
B
edge Delamination
D
At grip/tab
A
Top
T
Grip/tab
G
<1W from grip/tab
W
Left
L
Lateral
L
Gage
G
Right
R
Multi – mode
M
Multiple areas
M
Middle
M
long. Splitting
S
Various
V
Various
V
eXplosive
X
Unknown
U
Unknown
U
Other
O
-
-
- Fig. 2. MATEC Web of Conferences 145, 02004 (2018)
NCTAM 2017 MATEC Web of Conferences 145, 02004 (2018)
NCTAM 2017 https://doi.org/10.1051/matecconf/201814502004 In comparison with their reference samples, the HexPly specimens showed an increase
of the tensile strength with 3% after 1752 hours of kerosene immersion at room
temperature. EP specimens showed an increase of the tensile strength with 5% after 1752
hours of kerosene immersion at room temperature. A critical factor that can affect the tensile strength of the polymeric composite materials
immersed in kerosene is the temperature of kerosene, fact proved by Taghavi [32] in his
thesis, in which the specimens immersed for 100 hours in kerosene at 80°C showed a 21%
reduction of transverse tensile strength, but after this point no further changes were
observed up to 3500 hours. Thereby, in case of room temperature immersion for this period
of time (max. 1752 h), the kerosene had an opposite effect, it leads to a slight increase of
tensile strength. It can be concluded that kerosene absorption could slightly improve the tensile strength
on short term due to modifications of the internal structure of the polymer and on long term
it could weaken the polymer due to breaking of hydrogen bonds because a decrease number
of hydrogen bonds is a factor which influences the mechanical properties of the polymeric
matrix. Regarding the influence of the immersion on saline solution the tensile properties of
composites weren’t affected by the saline solution immersion on short term (168 h), results
confirmed also by the literature. The studies regarding saline solution/seawater immersion of composite materials are
limited, Roy Xu et. al. [28] observed that compression-after-impact strengths of the wet
specimens were reduced by around 10% compared to the baseline dry specimens. Visco et. al. [22] studied seawater absorption ability and mechanical performance of
two glass–fibre reinforced composites with different thermoset matrices (isophthalic
polyester, vinyl ester) and concluded that vinyl ester resin composite had a lower water
diffusion coefficient but higher chemical stability, stiffness and strength after immersion in
seawater than the isophthalic one. Even if the tensile strength of the composite materials was increased it was observed
that they maintain their Young’s modulus value, 68 GPa for HexPly samples, respective 62
GPa for EP samples. The elastic modulus could be maintained over a period of time in
moist environment and afterwards it could be altered, the polymer becoming more brittle
and easy to break. 3 Results and discussions Both materials (HexPly and EP) showed a moist weight gain of 0,10% after the first 168
hours of immersion in kerosene and 0,16% after the 1752 hours of immersion in kerosene. Regarding the immersion in saline solution, the moist weight gain of both composites was
0,13% after 168 hours of immersion. The composite materials gained more weight after the
immersion in saline solution compared with kerosene immersion, those were different
results compared with the findings of Areef [30] who observed a higher weight gained by
the composite immersed in organic solution (kerosene) then in distilled water (this could be
influenced by the fact that the composite material used by Areef [30] consisted in glass
fibre reinforced polymer and the samples were immersed only in distilled water not in
distilled water with salt). The moisture absorption of the HexPly and EP materials could increase after more
hours of immersion, but the overall capacity of absorption of the composite wasn’t the goal
of this study. The composite samples maintained their dimensional measurements after the
immersion in kerosene and saline solution immersion, they didn’t modify the physical
aspect of the samples (no peels or cracks were observed) and the polymeric materials 4 MATEC Web of Conferences 145, 02004 (2018)
NCTAM 2017 https://doi.org/10.1051/matecconf/201814502004 weren’t dissolved after immersion in saline solution or kerosene. Similar physical
observations regarding the effect of jet fuel on polymeric composite materials were made
by Curtis et.al. [31]. g
g
j
p y
p
by Curtis et.al. [31]. Table 2. Fracture’s modes of composite specimens after tensile tests
Immersion
conditions
HexPly
EP
Reference -
unimmersed
168 h saline
168
h
kerosene
1752
h
kerosene
The tensile test results showed a slight increase of the tensile strength of laminates after
each immersion phase in kerosene. by Curtis et.al. [31]. Table 2. Fracture’s modes of composite specimens after tensile tests
Immersion
conditions
HexPly
EP
Reference -
unimmersed
168 h saline
168
h
kerosene
1752
h
kerosene
The tensile test results showed a slight increase of the tensile strength of laminates after
each immersion phase in kerosene. The tensile test results showed a slight increase of the tensile strength of laminates after
each immersion phase in kerosene. 5 5 MATEC Web of Conferences 145, 02004 (2018)
NCTAM 2017 Regarding the analysis of the tensile failure modes, it was observed that in case of
HexPly samples, the majority presented a lateral inside grip top (LIT) typical failure mode
followed by lateral gage top (LGT) failure mode, while EP samples presented an angle gage
middle (AGM) failure mode followed by angle gage bottom (AGB) failure mode. In Table 3 are specified the results of tensile test failure mode analysis of the composite
specimens. It can be observed that in case of HexPly, the tested specimens present less
scattered failure modes compared with the EP specimens. The predominant failure modes
are typical for the matrix shear failure and brittle failure of polymeric matrix composites
reinforced with fibres along with fibre pull-out. In Fig. 3 a, b it can be summarized the influence of kerosene immersion on the tensile
strength of the two composite materials and in Fig. 3 c can be observed the influence of
saline solution on their tensile strength after 168 hours of immersion. 6 6 https://doi.org/10.1051/matecconf/201814502004 MATEC Web of Conferences 145, 02004 (2018)
NCTAM 2017 Table 3. Results of the tensile test failure mode analysis
a
b
c
Fig. 3. Influence of kerosene and saline solution immersion on composite tensile strength:
a) in case of HexPly; b) in case of EP; c) results in case of saline immersion of the
Material
Typical failure mode
Number of specimens
HexPly
LIT
10 specimens
HexPly
LGB
2 specimens
HexPly
LGT
3 specimens
HexPly
LAB
2 specimens
HexPly
LAT
1 specimen
HexPly
LIB
1 specimen
HexPly
MIT
1 specimen
EP
AGM
6 specimens
EP
AGB
3 specimens
EP
LIB
1 specimen
EP
LAT
2 specimens
EP
AMM
1 specimen
EP
LGT
1 specimen
EP
AGT
1 specimen
EP
LGB
2 specimens
EP
LIT
2 specimens
EP
LAB
1 specimen a
b
c
Fig. 3. MATEC Web of Conferences 145, 02004 (2018)
NCTAM 2017 Influence of kerosene and saline solution immersion on composite tensile strength:
a) in case of HexPly; b) in case of EP; c) results in case of saline immersion of the
composites
Material
Typical failure mode
Number of specimens
HexPly
LIT
10 specimens
HexPly
LGB
2 specimens
HexPly
LGT
3 specimens
HexPly
LAB
2 specimens
HexPly
LAT
1 specimen
HexPly
LIB
1 specimen
HexPly
MIT
1 specimen
EP
AGM
6 specimens
EP
AGB
3 specimens
EP
LIB
1 specimen
EP
LAT
2 specimens
EP
AMM
1 specimen
EP
LGT
1 specimen
EP
AGT
1 specimen
EP
LGB
2 specimens
EP
LIT
2 specimens
EP
LAB
1 specimen b c Fig. 3. Influence of kerosene and saline solution immersion on composite tensile strength:
a) in case of HexPly; b) in case of EP; c) results in case of saline immersion of the
composites 7 7 https://doi.org/10.1051/matecconf/201814502004 MATEC Web of Conferences 145, 02004 (2018)
NCTAM 2017 4 Conclusions The present study was focused on the tensile behaviour of two composite materials before
and after aging in humid environment (saline solution and kerosene). The humid
environments selected for the tests were kerosene due to the fact that jet fuels consist in
kerosene and different types of additives and saline solution due to the fact that the tank is
designed for a navel helicopter. Two types of composite laminates were tested and it was
observed that kerosene slightly improves the tensile properties on short term exposure. g
y
p
p
p
p
HexPly M49/42%/ 200T2X2/CHS-3K showed an increase in tensile strength of 3%
after 1752 hours of kerosene immersion and EP 127-C20-45 T2 showed an increase in
tensile strength of 5% after 1752 hours of kerosene immersion. The saline solution
exposure on short term (168 h) didn’t influence the tensile properties of the HexPly
M49/42%/ 200T2X2/CHS-3K, but in case of EP 127-C20-45 T2 it was observed that the
saline absorption increased the tensile properties with 5%. However, it was observed that
the composite laminates maintain their Young modulus 68 GPa for HexPly samples,
respective 62 GPa for EP samples. Peeling, cracking or matrix dissolution of polymeric
composite immersed in saline solution or kerosene weren’t observed. Due to the fact that it can’t be predicted how a fibre reinforced composite will behave
under a certain humid environment further studies will be made regarding the fuel
compatibility with polymeric composites. Acknowledgment This study was carried out within Grant no. 293/2014, supported by the Romanian Minister
of Research and Innovation. References [1] A.P. Mouritz, Introduction to aerospace materials, Woodhead Publishing, 303-337
(2012) [1] A.P. Mouritz, Introduction to aerospace materials, Woodhead Publishing, 303-337
(2012)
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1, Springer (2017)
[3] P.K. Mallick, Fiber-reinforced composites: Materials, manufacturing and design, CRC
Press, 19 - 48 (2007)
[4] M.R. Legault, http://www.compositesworld.com/articles/carrier-capable-all-composite-
external-fuel-tank(2) (2011)
[5] UH-60 A/L/M CRASHWORTHY EXTERNAL FUEL SYSTEM (CEFS) Brochure,
http://www.robertsonfuelsystems.com/defense/crashworthy-external-fuel-system-cefs/
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[7] E. Elkhershi, The Telegraph, http://www.telegraph.co.uk/news/2016/03/18/inside-the-f-
35-lightning-ii---the-invisible-fighter-jet/ (2015)
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Removable (1958)
[9] MIL-DLT-5578D. Detail Specification Tanks, Fuel, Aircraft, Self-sealing (2008)
[10] B. Wei, H. Cao, S. Song, Corros Sci, 53, 426-431 (2011)
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Springer, 1-38 (2012) (
)
[2] R.J.H. Wanhill, Aerospace Materials and Material Technologies : Aerospace Materials,
1, Springer (2017) [3] P.K. Mallick, Fiber-reinforced composites: Materials, manufacturing and design, CRC
Press, 19 - 48 (2007) [4] M.R. Legault, http://www.compositesworld.com/articles/carrier-capable-all-composite-
external-fuel-tank(2) (2011) [5] UH-60 A/L/M CRASHWORTHY EXTERNAL FUEL SYSTEM (CEFS) Brochure,
http://www.robertsonfuelsystems.com/defense/crashworthy-external-fuel-system-cefs/
[6] M. Ren, X. Chang, H.Y. Xu, T. Li, Compos Struct, 160, 1339-1347 (2017)
[7] E. Elkhershi, The Telegraph, http://www.telegraph.co.uk/news/2016/03/18/inside-the-f-
35-lightning-ii---the-invisible-fighter-jet/ (2015) [5] UH-60 A/L/M CRASHWORTHY EXTERNAL FUEL SYSTEM (CEFS) Brochure,
http://www.robertsonfuelsystems.com/defense/crashworthy-external-fuel-system-cefs/
[6] M R
X Ch
H Y X
T Li C
St
t 160 1339 1347 (2017) E. Elkhershi, The Telegraph, http://www.telegraph.co.uk/news/2016/03/18/inside-the-f-
-lightning-ii---the-invisible-fighter-jet/ (2015) g
g
g
j
(
)
[8] MIL-T-7378A. Military specification Tanks, Fuel, Aircraft, external, Auxiliary,
Removable (1958) [9] MIL-DLT-5578D. Detail Specification Tanks, Fuel, Aircraft, Self-sealing (2008)
[10] B. Wei, H. Cao, S. Song, Corros Sci, 53, 426-431 (2011) [10] B. Wei, H. Cao, S. Song, Corros Sci, 53, 426-431 (2011)
[11] S. Lampman, Characterization and Failure Analysis of Plastics, ASM International,
146-152 (2003) [11] S. Lampman, Characterization and Failure Analysis of Plastics, ASM International,
146-152 (2003) [12] J.A. Hinkley, J.W. Connell, Long-Term Durability of Polymeric Matrix Composites,
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(2002), pp. 487-492 [19] E. Perez-Pacheco, J.I. Cauich – Cupul, A. Valadez-Gonzalez, P.J. Herrera-Franco, J. Mater. Sci, 48, (2013), pp. 1873-1882, [20] C.E. Browning, C.E. Husman, J.M. Whitney, Moisture effects in epoxy matrix
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J.D.D Melo, Compos Part B-Eng., 110, (2017), pp. 298-306 [27] J. Naveen, T.P. Sathish Kumar, S. Satheesh Kumar, 3, (2014), pp. 1091-1095 [28] L. Roy Xu, A. Krishnan, H. Ning, U. Vaidya, Compos Part B-Eng, 43, (2012), pp. 2480-2486 [29] L.R. de Souza, A.T. marques, J.R.M. D’Almeida, Compos Struct, 168, (2017), pp. 285-291 [30] S.R. Areef, Eng. & Technology, 26, (2008), pp. 1235-1239 [30] S. . ee ,
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Polymer Matrix Composite Materials 9 9
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Grey and white matter volume changes after preterm birth: A meta-analytic approach
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medRxiv (Cold Spring Harbor Laboratory)
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Department of Psychiatry, School of Medicine, Technical University of Munich, Ismaninger Str. 22,
81675 Munich, Germany Citation: Schmitz-Koep, B.; Haller,
B.; Coupé, P.; Menegaux, A.; Gaser, C.;
Zimmer, C.; Wolke, D.; Bartmann, P.;
Sorg, C.; Hedderich, D.M. Grey and
White Matter Volume Changes after
Preterm Birth: A Meta-Analytic
Approach. J. Pers. Med. 2021, 11, 868. https://doi.org/10.3390/jpm11090868 *
Correspondence: benita.schmitz-koep@tum.de Abstract: Cross-sectional studies have reported lower brain grey matter volumes (GMV) and white
matter volumes (WMV) in preterm (PT) born individuals. While large MRI studies in the normative
population have led to a better understanding of brain growth trajectories across the lifespan, such
results remain elusive for PT born individuals since large, aggregated datasets of PT born individuals
do not exist. To close this gap, we investigated GMV and WMV in PT born individuals as reported in
the literature and contrasted it against individual volumetric data and trajectories from the general
population. Systematic database search of PubMed and Web of Science in March 2021, and extraction
of outcome measures were conducted by two independent reviewers. Individual data on full-term
(FT) controls was extracted from freely available databases. Mean GMV, WMV, total intracranial
volume (TIV), and mean age at scan were the main outcome measures. Of 532 identified records, nine
studies were included with 538 PT born subjects between 1.1 and 28.5 years of age. Reference data
was generated from 880 FT controls between 1 and 30 years of age. GMV was consistently lower in PT
born individuals from infancy to early adulthood with no evidence for catch-up growth. While GMV
changes followed a similar trajectory as FT controls, WMV was particularly low in adolescence after
PT birth. Results demonstrate altered brain volumes after PT birth across the first half of lifespan. Future studies should address this issue in large aggregated datasets of PT born individuals. Received: 18 July 2021
Accepted: 27 August 2021
Published: 30 August 2021 Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations. Keywords: brain development; grey matter volume; white matter volume; structural magnetic
resonance imaging; preterm birth Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article
distributed
under
the
terms
and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/). Journal of
Personalized
Medicine Journal of
Personalized
Medicine Journal of
Personalized
Medicine Article
Grey and White Matter Volume Changes after Preterm Birth:
A Meta-Analytic Approach Benita Schmitz-Koep 1,2,*
, Bernhard Haller 3
, Pierrick Coupé 4, Aurore Menegaux 1,2, Christian Gaser 5,6,
Claus Zimmer 1,2, Dieter Wolke 7,8
, Peter Bartmann 9, Christian Sorg 1,2,10 and Dennis M. Hedderich 1,2 1
Department of Neuroradiology, School of Medicine, Technical University of Munich, Ismaninger Str. 22,
81675 Munich, Germany; aurore.menegaux@tum.de (A.M.); claus.zimmer@tum.de (C.Z.);
christian.sorg@tum.de (C.S.); dennis.hedderich@tum.de (D.M.H.) 1
Department of Neuroradiology, School of Medicine, Technical University of Munich, Ismaninger Str. 22,
81675 Munich, Germany; aurore.menegaux@tum.de (A.M.); claus.zimmer@tum.de (C.Z.);
christian.sorg@tum.de (C.S.); dennis.hedderich@tum.de (D.M.H.) 2
TUM-NIC Neuroimaging Center, School of Medicine, Technical University of Munich, Ismaninger Str. 22,
81675 Munich, Germany 3
Institute of Medical Informatics, Statistics and Epidemiology, School of Medicine,
Technical University of Munich, Ismaninger Str. 22, 81675 Munich, Germany; bernhard.haller@tum.de y
,
g
,
,
y;
4
Laboratoire Bordelais de Recherche en Informatique (LaBRI) UMR 5800, CNRS, Bordeaux INP, y
g
y
4
Laboratoire Bordelais de Recherche en Informatique (LaBRI) UMR 5800, CNRS, Bordeaux INP,
University of Bordeaux, F-33400 Talence, France; pierrick.coupe@u-bordeaux.fr Laboratoire Bordelais de Recherche en Informatique (LaBRI) UMR 5800, CNRS, Bordeaux INP,
University of Bordeaux, F-33400 Talence, France; pierrick.coupe@u-bordeaux.fr 5
Department of Psychiatry, University Hospital Jena, Am Klinikum 1, 07747 Jena, Germany;
christian.gaser@uni-jena.de 6
Department of Neurology, University Hospital Jena, Am Klinikum 1, 07747 Jena, Germany
7
Department of Psychology, University of Warwick, University Road, Coventry CV4 7AL, UK;
D.Wolke@warwick.ac.uk 7
Department of Psychology, University of Warwick, University Road, Coventry CV4 7AL, UK;
D.Wolke@warwick.ac.uk 8
Warwick Medical School, University of Warwick, University Road, Coventry CV4 7AL, UK
9 9
Department of Neonatology, University Hospital Bonn, Venusberg-Campus 1, 53127 Bonn, Germany;
Peter.Bartmann@ukbonn.de 2.2. Study Selection Criteria The following inclusion criteria were used to assess eligibility: (1) participants were
born PT (<37 weeks of gestation); (2) MRI was used to determine mean total intracranial
volume (TIV), total GMV, and total WMV; (3) mean age at MRI scan was reported and
>1 year; and (4) the study was published in a peer-reviewed journal. If multiple studies
reported on volumetric data of the same cohort at the same age, the study with the larger
sample was included. Records were screened by two independent reviewers. p
y
p
Studies with mean age lower than one year were excluded because of particular rapid
brain growth in this period, in which small changes in postnatal age correspond to large
volumetric changes [13]. 1. Introduction Preterm (PT) birth, defined as birth <37 weeks of gestation, has a high worldwide
prevalence of almost 11% [1]. Prematurity is related to alterations in brain development in
general, and in grey matter volume (GMV) and white matter volume (WMV) in particular, https://www.mdpi.com/journal/jpm J. Pers. Med. 2021, 11, 868. https://doi.org/10.3390/jpm11090868 J. Pers. Med. 2021, 11, 868 2 of 11 which has been documented at various age groups from infancy and childhood until early
adulthood [2–7]. Large population studies and open datasets have led to a better understanding of
brain development and aging in the general population across the lifespan [8–10]: GMV
rapidly increases during infancy and early childhood until it peaks at school age. A fast
decrease until around 40 years of age follows with a subsequent plateau phase. Lastly,
there is an accelerated decrease of GMV around 80 years of age. Development of WMV
follows an inverted U-shape. After fast growth in early ages, WMV peaks at around 30 to
40 years of age followed by a volume decrease. Characterization of age-dependent grey
and white matter development in healthy subjects has enabled modelling pathological
alterations in patients. For example, deviations from normal brain volume trajectories
have been described in pathologic states such as Alzheimer’s disease and may be used as a
diagnostic tool [11]. However, brain structure alterations after PT birth have mostly been studied in cross-
sectional designs with narrow age ranges. Data from longitudinal studies with several
magnetic resonance imaging (MRI) examinations are scarce and shared large datasets do
not exist for PT populations. Hence, aggregated evidence about brain growth trajectories
of the lifespan after PT birth remains elusive. To close this gap, we investigated GMV and WMV changes observed in cross-sectional
studies of PT subjects over the first half of lifespan from infancy to early adulthood and
contrasted it against brain growth trajectories of a large normative cohort of FT individuals. 2.1. Search Strategy The electronic databases PubMed and Web of Science were systematically searched
for articles published before 30 March 2021. Key words: (birth OR born) AND (preterm
OR prematur *) AND (magnetic resonance imaging OR MRI) AND (brain OR intracranial)
AND (volume). 2.2. Study Selection Criteria 2. Materials and Methods This meta-analysis was conducted following the Preferred Reporting Items for Sys-
tematic Reviews and Meta-Analyses (PRISMA) guidelines, see Table S1 [12]. It was not
registered. A review protocol was not prepared. 3.1. Study Selection and Characteristics The search strategy identified 532 records (Figure 1, Table 1). After screening of
abstract and title, 167 articles were identified as potentially relevant. Based on the inclusion
criteria, nine studies were eligible for the meta-analysis [5,7,17–23]. Most of the studies
excluded did not report on all variables of interest, i.e., TIV, GMV and WMV. For example,
a recent study tracking regional brain growth up to age 13 in children born term and very
preterm (VP) appeared to meet the inclusion criteria. However, while it did report mean
age at scan, mean TIV and mean WMV, it did not report mean global GMV [24]. Therefore,
it was excluded. Another study investigating regional brain volume abnormalities and
long-term cognitive outcome in PT infants reported adjusted marginal mean of specific
brain regions, however, it did not report mean TIV, WMV or GMV [25]. Therefore, it was
excluded. Seven studies were excluded because they reported on data from cohorts that
were already covered by other studies. In summary, 538 PT subjects collected in studies
with mean age between 1.1 and 28.5 years, and data from 880 FT controls between 0.7 and
30 years of age were analyzed. Please see Figure 1 for a flowchart depicting study selection. Study characteristics of the nine studies included are shown in Table 1. 2.3. Data Collection Process and Data Extraction Data was extracted from the studies by two independent reviewers using a standard-
ized form including author and year of publication, sample size, age at MRI scan (in years),
mean and SD of TIV, GMV and WMV (in cm3), gestational age (GA, in weeks), birth weight
(BW, in grams), percentages of male participants, year of birth, country of origin, and
methodology of brain volume estimation. If PT samples were separated into groups (e.g.,
GA subgroups, positive or normal cranial ultrasound (cUS) findings and with or without
intrauterine growth restriction (IUGR), volumes were reported and analyzed separately. If
age was reported in months, it was divided by twelve to obtain age in years. If volume was
reported in mm3, it was divided by 1000 to obtain volume in cm3. If volume was reported J. Pers. Med. 2021, 11, 868 3 of 11 in dm3, it was multiplied with 1000 to obtain volume in cm3. One study [7] reported mean
GMV/TIV-ratio and mean WMV/TIV ratio. Mean GMV and WMV values were calculated
from these ratios. If BW was reported in kilograms, it was multiplied with 1000 to obtain
weight in grams. 2.4. Data on Full-Term Controls Data on FT controls was extracted from freely available databases: Cincinnati MR
Imaging of NeuroDevelopment (C-MIND, https://research.cchmc.org/c-mind/ (accessed
on February 2015)), National Database for Autism Research—NIH MRI Study of Normal
Brain Development, Pediatric MRI Data Repository (NDAR-NIHPD, https://www.bic.mni. mcgill.ca/nihpd_info/info2/data_access.html (accessed on February 2015)), International
Consortium for Brain Mapping (ICBM, http://www.loni.usc.edu/ICBM/ (accessed on
February 2015)), and Information eXtraction from Images (IXI, http://brain-development. org/ixi-dataset/ (accessed on February 2015)). TIV, GMV and WMV were calculated
from these four datasets as previously described by Coupé et al. [10] Overall, 880 FT con-
trols between 0.7 and 30 years of age were included: 236 participants from C-MIND
(mean age = 8.4 years, age range = 0.7–18.9 years, male = 45.3%), 375 from NDAR-
NIHPD (mean age = 11.9 years, age range = 1.1–29.0 years, male = 53.6%), 169 from ICBM
(mean age = 23.9 years, age range = 18–30 years, male = 52.7%), and 100 from IXI (mean
age = 25.7 years, age range = 20.0–29.8 years, male = 44%). 2.5. Statistical Analysis Statistical analysis was performed with R [14]. A nonlinear regression model (Model 6,
the cubic hybrid model, from Coupé et al. [10]: Vol = β4 (1 −e−Age/β5) + β0 + β1Age +
β2Age2 + β3Age3 + ε) was used to model GMV, WMV, TIV, GMW/TIV, and WMV/TIV
for FT controls in dependence of age using the R library minpack.lm [15]. Grey areas sur-
rounding the curves show 95%-confidence intervals (CI), dotted lines show 95%-prediction
intervals (PI), which were calculated using the R library propagate [16]. 3.2. Development of Grey Matter Volume after Preterm Birth While mean absolute GMV decre
between adolescence and early adulthood in both PT subjects and FT controls, this
crease appeared steeper in the PT studies. Figure 3a illustrates GMV/TIV, i.e., relative GMV, from the PT studies contra
against individual data of FT controls. Relative GMV decreased from infancy thro
early adulthood. Relative GMV calculated from data on infants with and without IU
reported by Padilla et al. [20] was above the curve describing FT development. All o
values were below the 95%-CI. Data from three PT studies of school-aged children
adolescents with brain injury [19] and adults [17] were below the 95%-PI. In summary GMV was lower after PT birth from infancy through early adulth between adolescence and early adulthood in both PT subjects and FT controls, this decrease
appeared steeper in the PT studies. REVIEW
4 of 12 ean absolute GMV extracted from the PT studies c
f
l
b
l
ll
f h below mean GMV in FT development. O
infants appropriate for gestational age school age. While mean absolute GM
hood in both PT subjects and FT con
dies. y adulthood. Relative GMV calcul
orted by Padilla et al. [20] was abov Figure 1. Flowchart of study selection. Figure 1. Flowchart of study selection. mmary, GMV was lower after PT birth
owing a similar trajectory as FT control Figure 1. Flowchart of study selection. Figure 1. Flowchart of study selection. mmary, GMV was lower after PT birth
owing a similar trajectory as FT control Figure 2. Mean absolute grey and white matter volumes after preterm birth compared to full-term controls. The four
different datasets on FT controls are illustrated in different colors. C-MIND is presented in red, NDAR-NIHPD in in cyan,
ICBM in green, and IXI in blue. Mean absolute volumes from the PT studies are included as diamonds. The size of each
diamond is proportional to the sample size of the study. (a) Mean absolute GMV after PT birth compared to FT controls. The graph shows mean absolute GMV extracted from the PT studies contrasted against the GMV trajectory of FT controls
with 95%-CI (grey areas) and 95%-PI (dotted lines). (b) Mean absolute WMV after PT birth compared to FT controls. The
graph shows mean absolute WMV extracted from the PT studies contrasted against the WMV trajectory of FT controls
with 95%-CI (grey areas) and 95%-PI (dotted lines). Figure 2. 3.2. Development of Grey Matter Volume after Preterm Birth C-MIND is presented in red, NDAR-NIHPD in in cyan,
ICBM in green, and IXI in blue. Mean absolute volumes from the PT studies are included as diamonds. The size of each
diamond is proportional to the sample size of the study. (a) Mean absolute GMV after PT birth compared to FT controls. The graph shows mean absolute GMV extracted from the PT studies contrasted against the GMV trajectory of FT controls
with 95%-CI (grey areas) and 95%-PI (dotted lines). (b) Mean absolute WMV after PT birth compared to FT controls. The
graph shows mean absolute WMV extracted from the PT studies contrasted against the WMV trajectory of FT controls
with 95%-CI (grey areas) and 95%-PI (dotted lines). Figure 2. Mean absolute grey and white matter volumes after preterm birth compared to full-term controls. The four
different datasets on FT controls are illustrated in different colors. C-MIND is presented in red, NDAR-NIHPD in in cyan,
ICBM in green, and IXI in blue. Mean absolute volumes from the PT studies are included as diamonds. The size of each
diamond is proportional to the sample size of the study. (a) Mean absolute GMV after PT birth compared to FT controls. The graph shows mean absolute GMV extracted from the PT studies contrasted against the GMV trajectory of FT controls
with 95%-CI (grey areas) and 95%-PI (dotted lines). (b) Mean absolute WMV after PT birth compared to FT controls. The
graph shows mean absolute WMV extracted from the PT studies contrasted against the WMV trajectory of FT controls with
95%-CI (grey areas) and 95%-PI (dotted lines). between adolescence and early adulthood in both PT subjects and FT controls, this de
appeared steeper in the PT studies. Med. 2021, 11, x FOR PEER REVIEW
ers. Med. 2021, 11, x FOR PEER REVIEW
6
3.2. Development of Grey Matter Volume after Preterm Birth
Figure 2a illustrates mean absolute GMV extracted from the PT studies contra
against individual GMV data of FT controls. Mean absolute GMV in all of the PT stu
was below mean GMV in FT development. One study [20] reported mean absolute G
of PT infants appropriate for gestational age (AGA) at about 1 year of age just within
95%-CI. All other studies reported mean absolute GMV below the 95%-CI. The hig
mean absolute GMV was reported at school age. 3.2. Development of Grey Matter Volume after Preterm Birth Figure 2a illustrates mean absolute GMV extracted from the PT studies contrasted
against individual GMV data of FT controls. Mean absolute GMV in all of the PT studies
was below mean GMV in FT development. One study [20] reported mean absolute GMV
of PT infants appropriate for gestational age (AGA) at about 1 year of age just within the
95%-CI. All other studies reported mean absolute GMV below the 95%-CI. The highest
mean absolute GMV was reported at school age. While mean absolute GMV decreased J. Pers. Med. 2021, 11, 868 4 of 11 Pers. Med. 2021, 11, 868
4 of
between adolescence and early adulthood in both PT subjects and FT controls, this decrea
appeared steeper in the PT studies. Pers. Med. 2021, 11, x FOR PEER REVIEW
4 of
Figure 1. Flowchart of study selection. Figure 1. Flowchart of study selection. J. Pers. Med. 2021, 11, x FOR PEER REVIEW
6 of 12
3.2. Development of Grey Matter Volume after Preterm Birth
Figure 2a illustrates mean absolute GMV extracted from the PT studies contrasted
against individual GMV data of FT controls. Mean absolute GMV in all of the PT studies
was below mean GMV in FT development. One study [20] reported mean absolute GMV
of PT infants appropriate for gestational age (AGA) at about 1 year of age just within the
95%-CI. All other studies reported mean absolute GMV below the 95%-CI. The highest
mean absolute GMV was reported at school age. While mean absolute GMV decreased
between adolescence and early adulthood in both PT subjects and FT controls, this de-
crease appeared steeper in the PT studies. Figure 3a illustrates GMV/TIV, i.e., relative GMV, from the PT studies contrasted
against individual data of FT controls. Relative GMV decreased from infancy through
early adulthood. Relative GMV calculated from data on infants with and without IUGR
reported by Padilla et al. [20] was above the curve describing FT development. All other
values were below the 95%-CI. Data from three PT studies of school-aged children [5],
adolescents with brain injury [19] and adults [17] were below the 95%-PI. In summary, GMV was lower after PT birth from infancy through early adulthood
while following a similar trajectory as FT controls. Figure 2. Mean absolute grey and white matter volumes after preterm birth compared to full-term controls. The four
different datasets on FT controls are illustrated in different colors. 3.2. Development of Grey Matter Volume after Preterm Birth Mean absolute grey and white matter volumes after preterm birth compared to full-term controls. The four
different datasets on FT controls are illustrated in different colors. C-MIND is presented in red, NDAR-NIHPD in in cyan,
ICBM in green, and IXI in blue. Mean absolute volumes from the PT studies are included as diamonds. The size of each
diamond is proportional to the sample size of the study. (a) Mean absolute GMV after PT birth compared to FT controls. The graph shows mean absolute GMV extracted from the PT studies contrasted against the GMV trajectory of FT controls
with 95%-CI (grey areas) and 95%-PI (dotted lines). (b) Mean absolute WMV after PT birth compared to FT controls. The
graph shows mean absolute WMV extracted from the PT studies contrasted against the WMV trajectory of FT controls with
95%-CI (grey areas) and 95%-PI (dotted lines). J. Pers. Med. 2021, 11, 868 5 of 11 Table 1. Characteristics of preterm studies included. Abbreviations: AGA, appropriate for gestational age; BW, birth weight; cUS, cranial ultrasound; GA, gestational age; GMV, grey matter volume; IUGR, intrauterine growth restriction; SD, standard deviation; TIV,
total intracranial volume; WMV, white matter volume. * corrected age. und; GA, gestational age; GMV, grey matter volume; IUGR, intrauterine growth restriction; SD, standard deviation; TIV, Abbreviations: AGA, appropriate for gestational age; BW, birth weight; cUS, cranial ultrasound; GA, gestational age; GM
total intracranial volume; WMV, white matter volume. * corrected age. 3.2. Development of Grey Matter Volume after Preterm Birth Relative GMV calculated from data on infants with and without IUGR reported
by Padilla et al. [20] was above the curve describing FT development. All other values
were below the 95%-CI. Data from three PT studies of school-aged children [5], adolescents
with brain injury [19] and adults [17] were below the 95%-PI. j
y
In summary, GMV was lower after PT birth from infancy through early adulthood
while following a similar trajectory as FT controls. REVIEW
7 of 12 Figure 3. Relative grey and white matter volumes, and total intracranial volume after preterm birth compared to full-term
controls. The four different datasets on FT controls are illustrated in different colors. C-MIND is presented in red, NDAR-
NIHPD in in cyan, ICBM in green, and IXI in blue. Data points from the PT studies are included as diamonds. The size of
each diamond is proportional to the sample size of the study. (a) Relative GMV after PT birth compared to FT controls. The graph shows GMV/TIV extracted from the PT studies contrasted against the GMV/TIV trajectory of FT controls with
95%-CI (grey areas) and 95%-PI (dotted lines). (b) Relative WMV after PT birth compared to FT controls. The graph shows
WMV/TIV extracted from the PT studies contrasted against the WMV/TIV trajectory of FT controls with 95%-CI (grey areas)
and 95%-PI (dotted lines). (c) TIV after PT birth compared to FT controls. The graph shows mean TIV extracted from the
PT studies contrasted against the TIV trajectory of FT controls with 95%-CI (grey areas) and 95%-PI (dotted lines). 3.3. Development of White Matter Volume after Preterm Birth
Figure 2b illustrates mean absolute WMV extracted from the PT studies contrasted
Figure 3. Relative grey and white matter volumes, and total intracranial volume after preterm birth compared to full-term
controls. The four different datasets on FT controls are illustrated in different colors. C-MIND is presented in red, NDAR-
NIHPD in in cyan, ICBM in green, and IXI in blue. Data points from the PT studies are included as diamonds. The size of
each diamond is proportional to the sample size of the study. (a) Relative GMV after PT birth compared to FT controls. The graph shows GMV/TIV extracted from the PT studies contrasted against the GMV/TIV trajectory of FT controls with
95%-CI (grey areas) and 95%-PI (dotted lines). (b) Relative WMV after PT birth compared to FT controls. 3.2. Development of Grey Matter Volume after Preterm Birth Author (Year)
Group
Sample
(n)
Age at
Scan
(Years)
TIV
Mean
(cm3)
TIV
SD
(cm3)
GMV
Mean
(cm3)
GMV
SD
(cm3)
WMV
Mean
(cm3)
WMV
SD
(cm3)
GA
(Weeks)
BW
(g)
Male
(%)
Year of Birth
Country of Origin
Methodology of Brain
Volume Estimation
Pascoe (2019) [17]
150
28.5
1504
140
669
61
513
61
28.8
1077
41.3
1986
New Zealand
CAT12 toolbox (SPM12)
Lemola (2017) [18]
57
10.0
1397
148
777
70
464
67
29.7
1447
65.3
1998–2006
Switzerland
New segment toolbox (SPM8)
Meng (2016) [7]
85
26.5
1385
609.4
554
30.67
1356
55.3
1985–1986
Germany
VBM8 toolbox (SPM8)
Northam (2011) [19]
positive
cUS
27
16
1601
222
757
57
418
46
27.1
1081
44.4
1989–1994
United Kingdom
VBM5 toolbox (SPM5)
Northam (2011) [19]
normal cUS
22
16.3
1480
193
765
65
438
49
28.1
1098
31.8
1989–1994
United Kingdom
VBM5 toolbox (SPM5)
Padilla (2011) [20]
IUGR
18
1.1 *
969.6
101.8
683.7
64.5
243.5
36.9
32.1
1060
38.9
2006–2007
Spain
VBM5 toolbox (SPM5)
Padilla (2011) [20]
AGA
15
1.1 *
1001.1
95.4
714.0
57.0
240.7
38.0
31
1580
73.3
2006–2007
Spain
VBM5 toolbox (SPM5)
Soria-Pastor (2009) [5]
20
9.3
1641.2
172.6
821.7
84.9
419.2
53.8
32.5
1754
55
1996–1998
Spain
SPM5
Narberhaus (2007) [21]
GA ≤27
9
14.1
1354.8
174.1
733.4
54.7
312.1
57.9
26.4
899
77.8
1983–1994
Spain
SPM2
Narberhaus (2007) [21]
GA 28–30
19
14.6
1488.5
164.9
771.3
133.2
377.2
57.9
29
1140
42.1
1983–1994
Spain
SPM2
Narberhaus (2007) [21]
GA 31–33
25
13.8
1445.3
146.4
778.1
72.1
372.0
52.1
31.7
1534
44
1983–1994
Spain
SPM2
Narberhaus (2007) [21]
GA 34–36
11
13.55
1473.3
148.4
780.3
69.9
389.8
45.1
34.6
2446
63.6
1983–1994
Spain
SPM2
Gimenez (2006b) [23]
50
14.5
1488.8
148.8
787.8
80.8
377.2
47.4
29.9
1327
48
1982–1994
Spain
SPM2
Gimenez (2006a) [22]
30
14.3
1460
140
780
70
360
50
29.1
1108
n.a. n.a. Spain
SPM2
Abbreviations: AGA, appropriate for gestational age; BW, birth weight; cUS, cranial ultrasound; GA, gestational age; GMV, grey matter volume; IUGR, intrauterine growth restriction; SD, standard deviation; TIV,
total intracranial volume; WMV white matter volume * corrected age Table 1. Characteristics of preterm studies included. J. Pers. Med. 2021, 11, 868 6 of 11 Figure 3a illustrates GMV/TIV, i.e., relative GMV, from the PT studies contrasted
against individual data of FT controls. Relative GMV decreased from infancy through early
adulthood. 3.2. Development of Grey Matter Volume after Preterm Birth (c) TIV after PT birth compared to FT controls. The graph shows mean TIV extracted from
the PT studies contrasted against the TIV trajectory of FT controls with 95%-CI (grey areas) and 95%-PI (dotted lines). ve grey and white matter volumes, and total intracranial volume after preterm birth compared to full-term
diff
d
FT
l
ill
d i
diff
l
C MIND i
d i
d NDAR
e grey and white matter volumes, and total intracranial volume after preterm birth compared to full-term Figure 3. Relative grey and white matter volumes, and total intracranial volume after preterm birth compared to full-term
controls. The four different datasets on FT controls are illustrated in different colors. C-MIND is presented in red, NDAR-
NIHPD in in cyan, ICBM in green, and IXI in blue. Data points from the PT studies are included as diamonds. The size of
each diamond is proportional to the sample size of the study. (a) Relative GMV after PT birth compared to FT controls. The graph shows GMV/TIV extracted from the PT studies contrasted against the GMV/TIV trajectory of FT controls with
95%-CI (grey areas) and 95%-PI (dotted lines). (b) Relative WMV after PT birth compared to FT controls. The graph shows
WMV/TIV extracted from the PT studies contrasted against the WMV/TIV trajectory of FT controls with 95%-CI (grey areas)
and 95%-PI (dotted lines). (c) TIV after PT birth compared to FT controls. The graph shows mean TIV extracted from the
PT studies contrasted against the TIV trajectory of FT controls with 95%-CI (grey areas) and 95%-PI (dotted lines). 3 3 Development of White Matter Volume after Preterm Birth
Figure 3. Relative grey and white matter volumes, and total intracranial volume after preterm birth compared to full-term
controls. The four different datasets on FT controls are illustrated in different colors. C-MIND is presented in red, NDAR-
NIHPD in in cyan, ICBM in green, and IXI in blue. Data points from the PT studies are included as diamonds. The size of
each diamond is proportional to the sample size of the study. (a) Relative GMV after PT birth compared to FT controls. The graph shows GMV/TIV extracted from the PT studies contrasted against the GMV/TIV trajectory of FT controls with
95%-CI (grey areas) and 95%-PI (dotted lines). (b) Relative WMV after PT birth compared to FT controls. 3.2. Development of Grey Matter Volume after Preterm Birth The graph shows
WMV/TIV extracted from the PT studies contrasted against the WMV/TIV trajectory of FT controls with 95%-CI (grey
areas) and 95%-PI (dotted lines). (c) TIV after PT birth compared to FT controls. The graph shows mean TIV extracted from
the PT studies contrasted against the TIV trajectory of FT controls with 95%-CI (grey areas) and 95%-PI (dotted lines). 3.3. Development of White Matter Volume after Preterm Birth Figure 3. Relative grey and white matter volumes, and total intracranial volume after preterm birth compared to full-term
controls. The four different datasets on FT controls are illustrated in different colors. C-MIND is presented in red, NDAR-
NIHPD in in cyan, ICBM in green, and IXI in blue. Data points from the PT studies are included as diamonds. The size of
each diamond is proportional to the sample size of the study. (a) Relative GMV after PT birth compared to FT controls. The graph shows GMV/TIV extracted from the PT studies contrasted against the GMV/TIV trajectory of FT controls with
95%-CI (grey areas) and 95%-PI (dotted lines). (b) Relative WMV after PT birth compared to FT controls. The graph shows
WMV/TIV extracted from the PT studies contrasted against the WMV/TIV trajectory of FT controls with 95%-CI (grey areas)
and 95%-PI (dotted lines). (c) TIV after PT birth compared to FT controls. The graph shows mean TIV extracted from the
PT studies contrasted against the TIV trajectory of FT controls with 95%-CI (grey areas) and 95%-PI (dotted lines). 3 3 Development of White Matter Volume after Preterm Birth
Figure 3. Relative grey and white matter volumes, and total intracranial volume after preterm birth compared to full-term
controls. The four different datasets on FT controls are illustrated in different colors. C-MIND is presented in red, NDAR-
NIHPD in in cyan, ICBM in green, and IXI in blue. Data points from the PT studies are included as diamonds. The size of
each diamond is proportional to the sample size of the study. (a) Relative GMV after PT birth compared to FT controls. The graph shows GMV/TIV extracted from the PT studies contrasted against the GMV/TIV trajectory of FT controls with
95%-CI (grey areas) and 95%-PI (dotted lines). (b) Relative WMV after PT birth compared to FT controls. The graph shows
WMV/TIV extracted from the PT studies contrasted against the WMV/TIV trajectory of FT controls with 95%-CI (grey
areas) and 95%-PI (dotted lines). 3.2. Development of Grey Matter Volume after Preterm Birth The graph shows
WMV/TIV extracted from the PT studies contrasted against the WMV/TIV trajectory of FT controls with 95%-CI (grey
areas) and 95%-PI (dotted lines). (c) TIV after PT birth compared to FT controls. The graph shows mean TIV extracted from
the PT studies contrasted against the TIV trajectory of FT controls with 95%-CI (grey areas) and 95%-PI (dotted lines). 3.3. Development of White Matter Volume after Preterm Birth
Figure 2b illustrates mean absolute WMV extracted fr
3.3. Development of White Matter Volume after Preterm Birth 4. Discussion This meta-analysis investigated changes of GMV and WMV after PT birth compared
to FT controls across the first half of lifespan. We found lower GMV after PT birth from
infancy until adulthood, indicating lastingly altered brain development. GMV changes
of PT born individuals followed a similar trajectory as FT controls with no evidence for
catch-up growth. WMV was particularly low in adolescence after PT birth. Furthermore,
the present study highlights the need for large longitudinal datasets to compare PT and FT
brain development across the lifespan. 4.1. Changes of Grey and White Matter Volume after Preterm Birth across the First Half of Lifespan We found lower GMV after PT birth compared to FT controls from infancy through
early adulthood. Similar to their FT peers, GMV in PT subjects peaked at school age
and decreased between adolescence and early adulthood. However, relative GMV in
infancy was higher compared to FT peers, possibly reflecting delayed maturation. Yet
this speculation is based on only one study. At school age, in adolescence and in early
adulthood GMV was lower. There was no evidence for catch-up growth. Furthermore,
WMV after PT birth was particularly low in adolescence. p
y
Comparing our results to the scarce longitudinal data available is difficult because of
methodological differences. Ment et al. [26] observed reduction in cerebral GMV and an
increase in WMV between 8 and 12 years of age. However, compared to FT controls, there
was lower GMV reduction and less WMV gain over time [26]. In contrast, our results show
that GMV in PT individuals mostly follow the FT reference curve, albeit on an overall lower
level and that WMV is particularly low in early adolescence. Mostly in line with our results,
Parker et al. [27] reported significantly smaller GMV and WMV in VP subjects compared
to FT controls in adolescence (15 years) and in early adulthood (19 years). Furthermore,
they reported similar growth patterns between these two timepoints [27]. Karolis et al. [28]
observed GMV alterations in VP subjects between adolescence (15 years), early adulthood
(20 years) and adulthood (30 years), indicating accelerated brain maturation. Similarly,
our findings of lower relative GMV at school age, in adolescence and early adulthood
might indicate early aging. Consistent with our findings, Karolis et al. [28] found no
evidence for developmental catch-up. In line with our results, de Kieviet et al. 3.4. Total Intracranial Volume after Preterm Birth Figure 3c illustrates mean TIV extracted from the PT studies contrasted against indi-
vidual TIV data of FT controls. While TIV reported by some studies in infancy, childhood
and early adulthood was below the curve describing FT development, other PT studies
reported higher values. 3.3. Development of White Matter Volume after Preterm Birth
Figure 2b illustrates mean absolute WMV extracted f
3.3. Development of White Matter Volume after Preterm Birth Figure 2b illustrates mean absolute WMV extracted from the PT studies contrasted
against individual WMV data of FT controls. Except for one study in early adulthood [7],
mean absolute WMV in all of the PT studies was below the curve describing mean FT
development. Mean absolute WMV of infants with and without IUGR reported by Padilla
et al. [20] was within the 95%-CI. All other studies reported mean absolute WMV below
Figure 2b illustrates mean absolute WMV extracted from the PT studies contrasted
against individual WMV data of FT controls. Except for one study in early adulthood [7],
mean absolute WMV in all of the PT studies was below the curve describing mean
FT development. Mean absolute WMV of infants with and without IUGR reported by J. Pers. Med. 2021, 11, 868 7 of 11 Padilla et al. [20] was within the 95%-CI. All other studies reported mean absolute WMV
below the 95%-CI. Mean absolute WMV reported in adolescence was particularly low,
moving away from the curve describing mean FT development. Padilla et al. [20] was within the 95%-CI. All other studies reported mean absolute WMV
below the 95%-CI. Mean absolute WMV reported in adolescence was particularly low,
moving away from the curve describing mean FT development. Figure 3b illustrates WMV/TIV, i.e., relative WMV, from the PT studies contrasted
against individual data of FT controls. Relative WMV calculated from data on infants with
and without IUGR reported by Padilla et al. [20] and from data in early adulthood [7] was
above the curve describing FT development. Data from one PT study at school age [18] was
below the curve describing FT development but just within the 95%-CI. All other values
were below the curve. The other PT study at school age [5] and all PT studies in adolescents
were below the 95%-PI. In summary, we observed particularly low WMV in adolescence, while WMV in
infancy and early adulthood were closer to the trajectory describing FT development. 3.4. Total Intracranial Volume after Preterm Birth 4. Discussion [29] found
reductions in GMV and WMV compared to FT controls in their investigation of brain
volume throughout childhood and adolescence in subjects born VP and with very low
birthweight in a meta-analysis. J. Pers. Med. 2021, 11, 868 8 of 11 8 of 11 Proposed reasons for aberrant brain development after PT birth include several pro-
cesses: Inflammatory, hypoxic-ischemic and/or stress-related events are potential causes
for injury to preoligodendrocytes, axons, thalamus, subplate neurons or migrating gamma-
aminobutyric acid-ergic neurons [6,30]. Remarkably, our results suggest a significant
impact of prematurity on WMV in adolescence. On a cellular level, pre-oligodendrocytes,
precursors of mature oligodendrocytes, are critical for white matter myelination. Pre-
oligodendrocytes are specifically vulnerable to insults, such as ischemia and inflammation,
resulting in cell injury or death and subsequent replenishment but dysmaturation [6,30–32]. Hence, our results showing preferential alteration of WMV might emphasize the signifi-
cance of pre-oligodendrocyte vulnerability and dysmaturation in the context of prematurity. GMV and WMV changes have been associated with functional outcome such as
cognitive development in children, adolescents, and young adults [5,7,25,33]. Therefore,
characterization of GMV and WMV changes across the first half of lifespan could help
identify PT individuals at increased risk for impaired cognitive development. In conclusion, PT birth has lasting effects on the development of GMV and WMV
compared to FT controls with particularly low WMV in adolescence. We found no evidence
for GMV catch-up growth. 4.2. What Is Needed in the Future? PT cohorts have been followed from birth and well investigated, however, to date
longitudinal imaging data on PT subjects are scarce and only cover specific age ranges. Shared large datasets do not exist. Methodological differences in image processing as well
as statistical analysis make it difficult to compare results. Thanks to large, open datasets on
healthy subjects, FT developmental trajectories have been well characterized [10], facili-
tating insights in diseases affecting brain structure, for example Alzheimer’s Disease [11]. Similarly, PT developmental trajectories could be used to identify PT adults with acceler-
ated aging at risk for cognitive decline and/or neurodegenerative diseases. To achieve
these goals, access to original imaging data of large PT populations across all age groups as
well as shared individual patient data with information on parameters like birthweight,
duration of neonatal intensive care unit exposure, and days of mechanical ventilation, and
information about possible brain damage, such as intraventricular hemorrhage, is critical. 4.3. Limitations There are limitations of this meta-analysis. First, only few studies fit the inclusion
criteria (see also Sections 2.2 and 2.3). One reason for exclusion was that raw volumes
were not reported. For example, one study [2] investigated GMV and WMV in adolescents
born VP, however, not raw volumes but volumes after controlling for whole brain volume
were reported. Furthermore, not TIV but whole brain volume was reported [2]. Another
study [26] investigated longitudinal brain volume changes in PT and FT subjects during
late childhood and adolescence, however, volumes were not reported as raw values but
as model-based least square means. Furthermore, not TIV but whole brain volume was
reported [26]. A third study [34] investigated brain volumes and developmental outcome
in childhood following fetal growth restriction leading to VP birth, however, mean age at
scan was not reported. Another reason for exclusion was the use of other analysis methods,
for example, investigating regional group differences instead of global volumes [25]. Second, only data on limited age groups was available. Hence, PT volume changes of
GMV and WMV in comparison to FT trajectories remain less clear in some age groups, for
example between about 2 and 7 years. However, it is inherently difficult to obtain a brain
MRI in this age group since this is generally not possible without general anesthesia. Third, as mentioned above, different methods of processing images make it very
difficult to compare data. All PT studies included in this meta-analysis used toolboxes
within SPM (https://www.fil.ion.ucl.ac.uk/spm/) for segmentation, while FT images were
processed with volBrain [35]. Volumes obtained with volBrain showed high correlation
with volumes obtained by manual segmentations [35]. Comparing subcortical segmenta- J. Pers. Med. 2021, 11, 868 9 of 11 9 of 11 tion of volBrain with state-of-the-art methods like Freesurfer or the FSL-based tool FIRST,
and with manual segmentation showed competitive results in terms of accuracy and repro-
ducibility. However, the possibility of systematic error cannot be ruled out. Only access
to raw imaging data could improve this major limitation and, as discussed, is needed. However, contrasting individual MRI scans and derived measures of GMV and WMV to
reference data from open datasets is increasingly used in clinical routine. Lastly, GMV and WMV are global measures representing brain development. 4.3. Limitations How-
ever, determination of developmental changes of other measures such as gyrification
and individual volumes of subcortical structures are necessary to characterize PT brain
development, but data is not available. 5. Conclusions Conflicts of Interest: The authors declare no conflict of interest. 5. Conclusions In conclusion, GMV is lower after PT birth from infancy through early adulthood,
indicating lastingly altered brain development. GMV changes were similar to the trajectory
of FT controls with no evidence for catch-up growth. WMV was particularly low in
adolescence after PT birth. Large longitudinal datasets are crucial to compare PT with FT
brain development across the lifespan. Supplementary Materials: The following are available online at https://www.mdpi.com/article/10
.3390/jpm11090868/s1, Table S1: PRISMA Checklist. Author Contributions: Conceptualization, D.M.H.; methodology, B.H., P.C., D.M.H.; software, B.H.;
formal analysis, B.H., P.C.; investigation, D.M.H., B.S.-K.; resources, P.C., C.Z.; data curation, B.H.,
P.C., D.M.H., B.S.-K.; writing—original draft preparation, B.S.-K.; writing—review & editing, D.M.H.,
B.H., P.C., A.M., C.G., C.Z., D.W., P.B., C.S.; visualization, B.H., B.S.-K.; supervision, C.S., D.M.H.;
project administration, D.M.H.; funding acquisition, D.W., P.B., C.S., D.M.H., B.S.-K. All authors have
read and agreed to the published version of the manuscript. Author Contributions: Conceptualization, D.M.H.; methodology, B.H., P.C., D.M.H.; software, B.H.;
formal analysis, B.H., P.C.; investigation, D.M.H., B.S.-K.; resources, P.C., C.Z.; data curation, B.H.,
P.C., D.M.H., B.S.-K.; writing—original draft preparation, B.S.-K.; writing—review & editing, D.M.H.,
B.H., P.C., A.M., C.G., C.Z., D.W., P.B., C.S.; visualization, B.H., B.S.-K.; supervision, C.S., D.M.H.;
project administration, D.M.H.; funding acquisition, D.W., P.B., C.S., D.M.H., B.S.-K. All authors have
read and agreed to the published version of the manuscript. Funding: This work was supported by the Deutsche Forschungsgemeinschaft (SO 1336/1-1 to
C.S.), German Federal Ministry of Education and Science (BMBF 01ER0801 to P.B. and D.W., BMBF
01ER0803 to C.S.), the RECAP preterm project, an EU Horizon 2020 study (supported by grant 733280;
D.W. and P.B.), and the Kommission für Klinische Forschung, Technische Universität München (KKF
8765162 to C.S., KKF 8700000474 to D.M.H., and KKF 8700000620 to B.S.-K.). Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Please see references for the studies included in Section 3.1, and for
data on full-term controls in Section 2.4. Data Availability Statement: Please see references for the studies included in Section 3.1, and for
data on full-term controls in Section 2.4. Acknowledgments: We would like to thank all investigators involved in collecting following datasets:
C-MIND (https://research.cchmc.org/c-mind/ (accessed on February 2015)), NDAR-NIHPD (https:
//www.bic.mni.mcgill.ca/nihpd_info/info2/data_access.html (accessed on February 2015)), ICBM
(http://www.loni.usc.edu/ICBM/ (accessed on February 2015)), and IXI (http://brain-development. org/ixi-dataset/ (accessed on February 2015)). Conflicts of Interest: The authors declare no conflict of interest. 1.
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fluency impairment in those born prematurely. Neuroreport 2006, 17, 463–466. [CrossRef] 23. Giménez, M.; Junqué, C.; Narberhaus, A.; Bargalló, N.; Botet, F.; Mercader, J.M. White matter volume and concentration reductions
in adolescents with history of very preterm birth: A voxel-based morphometry study. Neuroimage 2006, 32, 1485–1498. [CrossRef] 24. Thompson, D.K.; Matthews, L.G.; Alexander, B.; Lee, K.J.; Kelly, C.E.; Adamson, C.L.; Hunt, R.W.; Cheong, J.L.Y.; Spencer-Smith,
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Volpe, J.J. Dysmaturation of premature brain: Importance, cellular mechanisms, and potential interventions. Pediatr. Neurol. 2019,
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Manjón, J.V.; Coupé, P. volBrain: An online MRI brain volumetry system. Front. Neuroinform. 2016, 10, 30. [CrossRef] [PubMed] References Parker, J.; Mitchell, A.; Kalpakidou, A.; Walshe, M.; Jung, H.-Y.; Nosarti, C.; Santosh, P.; Rifkin, L.; Wyatt, J.; Murray, R.M.; et al. Cerebellar growth and behavioural & neuropsychological outcome in preterm adolescents. Brain 2008, 131, 1344–1351. [CrossRef]
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S.C.R.; Thompson, P.M.; et al. Volumetric grey matter alterations in adolescents and adults born very preterm suggest accelerated
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S.C.R.; Thompson, P.M.; et al. Volumetric grey matter alterations in adolescents and adults born very preterm suggest accelerated
brain maturation. Neuroimage 2017, 163, 379–389. [CrossRef] 29. de Kieviet, J.F.; Zoetebier, L.; van Elburg, R.M.; Vermeulen, R.J.; Oosterlaan, J. Brain development of very preterm and very
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late oligodendrocyte progenitors to hypoxia-ischemia. J. Neurosci. 2002, 22, 455–463. [CrossRef] g
y
p
g
yp
J
[
]
32. Segovia, K.N.; McClure, M.; Moravec, M.; Luo, N.L.; Wan, Y.; Gong, X.; Riddle, A.; Craig, A.; Struve, J.; Sherman, L.S.; et al. Arrested oligodendrocyte lineage maturation in chronic perinatal white matter injury. Ann. Neurol. 2008, 63, 520–530. [CrossRef] 33. Nosarti, C.; Giouroukou, E.; Healy, E.; Rifkin, L.; Walshe, M.; Reichenberg, A.; Chitnis, X.; Williams, S.C.R.; Murray, R.M. Grey
and white matter distribution in very preterm adolescents mediates neurodevelopmental outcome. Brain 2008, 131, 205–217. [CrossRef] 34. Morsing, E.; Malova, M.; Kahn, A.; Lätt, J.; Björkman-Burtscher, I.M.; Maršál, K.; Ley, D. Brain volumes and developmental
outcome in childhood following fetal growth restriction leading to very preterm birth. Front. Physiol. 2018, 9, 1583. [CrossRef]
[PubMed] 35. Manjón, J.V.; Coupé, P. 32.
Segovia, K.N.; McClure, M.; Moravec, M.; Luo, N.L.; Wan, Y.; Gong, X.; Riddle, A.; Craig, A.; Struve, J.; Sherman, L.S.; et al.
Arrested oligodendrocyte lineage maturation in chronic perinatal white matter injury. Ann. Neurol. 2008, 63, 520–530. [CrossRef] g
y
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g
yp
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Dimensions of Online Conflict: Towards Modeling Agonism
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Abstract Not all conflicts are equal, and the confusion
over the political and social value of conflict partly
stems from its diverse nature. Conflict exists on a
sliding scale ranging from antagonistic conflict be-
tween enemies (which is often silencing, undemo-
cratic, and hateful because it is focused on delegit-
imizing opponents’ rights and status) to agonistic
conflict between adversaries (which has productive
potential for the emergence of democratic dialogue,
dissent, and trust in the public and political sphere,
since the struggle is over interpretation and not le-
gitimacy to speak) (Mouffe, 2002; Wenman, 2013). As many across the political spectrum have noted,
democracy needs agonism to widen voices and pre-
vent public spaces from becoming totalitarian or
meaningless consensus hubs. But how do we know
which conflicts are agonistic and which hateful? How do we know what kinds of counterspeech ef-
fectively facilitate dialogue and which destroy it? Which follow the spirit of platforms’ terms of use
and which do not? Agonism plays a vital role in democratic di-
alogue by fostering diverse perspectives and
robust discussions. Within the realm of online
conflict there is another type: hateful antago-
nism, which undermines constructive dialogue. Detecting conflict online is central to platform
moderation and monetization. It is also vital
for democratic dialogue, but only when it takes
the form of agonism. To model these two types
of conflict, we collected Twitter conversations
related to trending controversial topics. We
introduce a comprehensive annotation schema
for labelling different dimensions of conflict in
the conversations, such as the source of con-
flict, the target, and the rhetorical strategies
deployed. Using this schema, we annotated ap-
proximately 4,000 conversations with multiple
labels. We then trained both logistic regression
and transformer-based models on the dataset,
incorporating context from the conversation,
including the number of participants and the
structure of the interactions. Results show that
contextual labels are helpful in identifying con-
flict and make the models robust to variations
in topic. Our research contributes a conceptu-
alization of different dimensions of conflict, a
richly annotated dataset, and promising results
that can contribute to content moderation. To begin answering these questions, we present
an in-depth exploration of conflict online, using the
platform once known as Twitter (now “X”) as a
case study. Our work builds upon related Natural
Language Processing (NLP) research fields such
as abusive language, persuasion, and constructive
comments. Abstract We use Twitter because, unlike the
controlled environment of a priori conflictual dis-
cussions like those in Reddit’s ‘Change My View’
(Monti et al., 2022; Srinivasan et al., 2019), it offers
a more organic setting. Dimensions of Online Conflict: Towards Modeling Agonism 1Digital Democracies Institute, Simon Fraser University, Canada, matthew_canute@sfu.ca
2Department of Computer Science, University of Sheffield, UK, m.jin@sheffield.ac.uk Findings of the Association for Computational Linguistics: EMNLP 2023, pages 12194–12209
December 6-10, 2023 ©2023 Association for Computational Linguistics 2.1
Conflict and agonism Within this project, we understand conflict as the
generative ground that spans hate to agonism. Ac-
cording to political theorist Chantal Mouffe (2013),
democratic speech or dialogue always bears traces
of the conflicts from which it emerges. Democracy,
therefore, necessarily entails conflict and negotia-
tion; to expand who counts as a citizen, to negotiate
differing claims to freedom or rights, and to vali-
date collective decisions. This is agonistic conflict. Conflict can also be antagonistic: unproductive
and undemocratic, when it silences individuals and
groups, by shutting them out or harassing them
with hateful speech. Democratic institutions are
responsible for creating the space to allow con-
flicts to take an agonistic form, in which opponents
are not enemies but adversaries among whom con-
flictual consensus may emerge (see also Mouffe,
2002; Rancière, 1999, 2010; Wenman, 2013). This
generative aspect of conflict has been neglected in
discussions around content moderation on social
media platforms, which frame the problem as free-
dom of speech versus censorship (Douek, 2022;
Gillespie et al., 2020; Gillespie, 2018). Next, we trained logistic regression and trans-
former models (BERT and GPT-3) on this dataset,
to predict dimensions of conflict. The models are
trained on human annotations of the conversations,
enhanced with a) previously proposed labels for
online conversation: constructiveness and toxicity;
and b) contextual aspects such as cardinality (partic-
ipant counts) and topology (interaction structure). We show that all models are sensitive to the specific
words in the conversation, making them less gener-
alizable across topics and domains. Incorporating
conversational context, however, makes the models
more robust, showing that cardinality and topol-
ogy are important dimensions in the prediction of
online conflict. The potential use cases of this work include the
measurement of productive (agonistic) versus un-
productive (antagonistic) conflicts, providing in-
sights into where learning and constructive dis-
course can be fostered. This work ties in with
attempts at reflective content moderation, where
the goal is not simply to delete harmful content
or make it less visible (Zeng and Kaye, 2022), but
also to identify and promote content that can be con-
structive and productive towards democratic goals
(Mouffe, 2013; Gillespie et al., 2020; Morrow et al.,
2022). This kind of analysis has implications for
detecting early signs of scapegoating and unproduc-
tive disputes—cases where patterns of discourse
do not necessarily break the terms of service, but
can nonetheless bring harm over time. 1
Introduction Conflict is everywhere online. From political
protests to spirited debates over the latest TikTok
trend, these conflicts are simultaneously celebrated
as promoting democracy and condemned for fos-
tering polarization and undermining public insti-
tutions. Conflicts—ideas, arguments, or attitudes
that oppose each other—are also central to platform
moderation and monetization: from the amplifica-
tion of certain controversies to provoke user en-
gagement, to content takedowns to comply with a
platform’s terms of use or national laws (Gillespie,
2022, 2018; Morrow et al., 2022; Douek, 2022;
Zeng and Kaye, 2022). To determine the nature of a conflict, context is
central (Zosa et al., 2021; Hu et al., 2022; Ghosh
et al., 2018). Currently, though, automatic content
moderation mainly focuses on a single utterance
rather than on the ongoing conversation. Whether
a message fosters agonism depends on various di-
mensions, including which groups are participating,
how many participants, their level of interaction,
and their relative power differences, all subject to 12194 Findings of the Association for Computational Linguistics: EMNLP 2023, pages 12194–12209
December 6-10, 2023 ©2023 Association for Computational Linguistics ual conversations on Twitter; c) a detailed schema
to annotate conversations for various dimensions
of conflict and the resulting annotations, with good
inter-annotator agreement; and d) a set of experi-
ments that show the usefulness of contextual infor-
mation in predicting online conflict. change over time. We introduce a methodology for collecting and
curating a dataset of English Twitter conversations
embodying various aspects of dimensions of con-
flict. We collected conversations about trending
events, as these often serve as catalysts, prompting
individuals to interrogate their stance on current is-
sues and deliberate their self-conception in relation
to their views. We then annotated the conversations
following our own coding schema, created with
antagonistic and agonistic conflict in mind. These
annotations took context into consideration rather
than focusing on an isolated tweet. 2.1
Conflict and agonism Our findings
will be instrumental in shaping online discourse,
aiming to harness conflict as a driver of democratic
conversation (agonism) rather than as a destructive
silencing element (antagonism). 2.4
Constructive comments Research into high-quality online content has
shown that constructiveness is a useful dimension. Research into high-quality online content has
shown that constructiveness is a useful dimension. Constructive comments build on and contribute to
the conversation, providing points of view and jus-
tification for a particular opinion. They are not
necessarily conflictual in nature, since they may
simply build on the ongoing conversation. In a
study of online news comments, Kolhatkar et al. (2023) propose that constructive comments seek
to create a civil dialogue, with remarks that are
relevant to the article and not merely emotional
provocations. Comments identified as constructive
can be presented to future posters as prompts or
examples of desirable behaviour or as nudges to de-
polarize conversations (Stray, 2022). Our work on
identifying conflict can contribute to the growing
body of research on how to present content in such
a way that it contributes to productive, civil, and
also agonistic discussion. We emphasize, however, that we do not necessar-
ily correlate the absence of toxicity or abuse with
the presence of productive conflict. Abusive lan-
guage research tends to characterize healthy and/or
civil conversations as those that are absent of tox-
icity (e.g., Smith et al., 2021; Hede et al., 2021). While that may be the case, healthy conversations
are not necessarily agonistic. Agonism requires a
certain level of disagreement as a source of political
discussion and engagement. 3.1
Data collection We are interested in online conversations on con-
tentious topics, so we used the Twitter Academic
API v2 elevated access to gather replies containing
certain keywords, starting with controversial topics. Then we consulted subject experts and representa-
tives of equity-seeking groups as a way to increase
the topic diversity within the dataset. This led to a
set of keywords as search terms (in Appendix A). After selecting tweet replies in English contain-
ing the relevant keywords from each topic, we then
extracted their surrounding conversation trees us-
ing two traversal methods: depth and breadth. The
former involved recursively collecting a reply’s ref-
erenced tweet until it reached the root message, or
the 7-message limit (since length 7 is the last most
frequent distribution before the start of the long tail
of conversation thread). Breadth traversal involved
capturing adjacent messages of a conversation by
recursively creating new queries based on each re-
ply’s tagged author and the conversation ID of the
reply. We are interested in online conversations on con-
tentious topics, so we used the Twitter Academic
API v2 elevated access to gather replies containing
certain keywords, starting with controversial topics. Then we consulted subject experts and representa-
tives of equity-seeking groups as a way to increase
the topic diversity within the dataset. This led to a
set of keywords as search terms (in Appendix A). Modeling work includes attempts to find argu-
ments in text, an area known as argumentation min-
ing (Mochales and Moens, 2011; Lawrence and
Reed, 2019; Harris and Di Marco, 2017). The
goals include: a) identifying controversial topics
in debates, news, Wikipedia articles, or online dis-
cussions (Boltuži´c and Šnajder, 2014; Kittur et al.,
2007; Choi et al., 2010; Swanson et al., 2015; Stab
and Gurevych, 2017); b) forecasting conversational
derailment (Zhang et al., 2018); c) identifying con-
versational strategies that will change the interlocu-
tor’s mind as in r/ChangeMyView (Monti et al.,
2022; Srinivasan et al., 2019); d) detecting conflict
outside the conversation, as in r/AmITheAsshole
posts (Welch et al., 2022). After selecting tweet replies in English contain-
ing the relevant keywords from each topic, we then
extracted their surrounding conversation trees us-
ing two traversal methods: depth and breadth. 2.3
Persuasion, argumentation, derailed
conversations Persuasion styles, rhetorical strategies, and argu-
mentation styles all play a role in how we perceive
and interpret conflict. Research in this area has pro-
duced manual annotations of rhetorical strategies
such as framing, hedging, modality, repetition, and
rhetorical questions (Peldszus, 2014; Green, 2014;
Hirst et al., 2014). These approaches tend to fo-
cus on understanding which rhetorical approaches
will be most effective in changing someone’s mind
(Habernal and Gurevych, 2016; Hidey and McKe-
own, 2018). 2.2
Abusive language Abusive language online is a broad term that covers
various forms of harmful or offensive communica-
tion on the internet, such as hate speech, cyber-
bullying, trolling, or flaming (Fortuna et al., 2020;
Pachinger et al., 2023). Detecting and preventing
abusive language online is an important challenge
for natural language processing (NLP) and social
computing, and an extensive literature on the topic
exists. In addition to the challenges of detecting a
social phenomenon that the perpetrators often try
to disguise, current industry solutions suffer from a
lack of interpretability, undermining their credibil-
ity (MacAvaney et al., 2019). Equally dangerous is
the over-policing of certain communities and topics
online (Saleem et al., 2016).i Our main contributions are: a) a conceptualiza-
tion of conflict online on a scale between antago-
nism and agonism; b) a process to retrieve conflict- Further, supervised classifiers require high-
quality annotated data that may harm the annotators 12195 sarily have a successful outcome in terms of per-
suading interlocutors. and that may contain their biases (Sap et al., 2022;
Vidgen and Derczynski, 2021). We know context
is also crucial in obtaining high-quality annotations
(Ljubeši´c et al., 2022), and that some disagreement
among annotators is to be expected (Leonardelli
et al., 2021). All this prior work informs our annota-
tions and explorations of machine learning models. 3.2
Coding schema To label the dataset, we developed an original cod-
ing protocol based on an interdisciplinary review
of literature on conflict, including media studies,
political science, conflict resolution studies, and
critical race theory (e.g., Oetzel and Ting-Toomey,
2006; Lamberti and Richards, 2019; D’Errico et al.,
2015; Itten, 2019; Yardi and boyd, 2010; Han et al.,
2023). Figure 1: Coding schema for conflict session during which annotators were introduced to
the project and its goals and were taught how to use
the labelling platform (LabelStudio; see Appendix
E for screenshots). During the first week, the an-
notators and two members of the research team
worked together and labelled a subset of about 400
tweets. The annotation was conducted in person,
and each session had planned moments for dis-
cussing disagreements and clarifying the gray areas
of the annotation protocol. At the end of the train-
ing session, we assigned a subset of the dataset to
be codde. The research co-leaders held weekly in-
dividual check-in meetings with annotators to trou-
bleshoot issues and gather their feedback. These
meetings also served to assess the emotional im-
pact of annotation and to externalize the thoughts
and emotions annotators encountered during their
work. The entire research team also met every two
weeks, to compare different annotation styles and
discuss edge cases as a way to test the protocol’s
reliability. Each message was annotated by two
annotators. The initial protocol was first tested on a subset
of conversations (see Section 3.3). We revisited the
coding protocol twice throughout the course of the
project based on coders’ feedback and discussions
over disagreements. The final coding protocol fol-
lowed a decision tree structure, where answering in
the positive to one question led to a set of follow-
up questions, as shown in Figure 1. Appendix E
contains an extensive discussion of each of the con-
cepts in the figure, with examples. 3.1
Data collection The
former involved recursively collecting a reply’s ref-
erenced tweet until it reached the root message, or
the 7-message limit (since length 7 is the last most
frequent distribution before the start of the long tail
of conversation thread). Breadth traversal involved
capturing adjacent messages of a conversation by
recursively creating new queries based on each re-
ply’s tagged author and the conversation ID of the
reply. While this previous research on persuasiveness
informs ours, its goal is to identify successful and
unsuccessful argumentation styles. We are, first,
looking for conflict, to then try and pinpoint exam-
ples of agonistic discussions, which do not neces- The annotated dataset contains an equal mix of
depth and breadth traversals. While the former en-
ables more efficient data collection, the latter is
useful for capturing the chaotic nature of conversa- 12196 Figure 1: Coding schema for conflict tions on most platforms, such as the one depicted
in Figure 2. Only conversations of length 3-7 messages in
English were stored. This iterative process contin-
ued over a period of three years (January 2020 -
December 2022), yielding a total of 220,626 con-
versations.1 Based on a random sample of 1,000 conversa-
tions, roughly 30% of these conversations likely
involved first-time interactions2, suggesting that
these topics were contentious enough to spark de-
bates among strangers in the comments of large
accounts, creating virtual public forums. 1Raw data, annotations, and code used to extract
the conversations are available in our repository, which
also
includes
all
the
code
for
the
experiments
in
Section 4: https://github.com/Digital-Democracies-
Institute/Dimensions-of-Online-Conflict 1Raw data, annotations, and code used to extract
the conversations are available in our repository, which
also
includes
all
the
code
for
the
experiments
in
Section 4: https://github.com/Digital-Democracies-
Institute/Dimensions-of-Online-Conflict
2First-time interactions were approximated by examining
each account’s prior 200 messages and checking if any of the
accounts had interacted with each other previously. 2First-time interactions were approximated by examining
each account’s prior 200 messages and checking if any of the
accounts had interacted with each other previously. 3.3
Annotation and agreement We recruited a team of four annotators. In selecting
candidates, we aimed to maximize demographic di-
versity and cultural background. The team included
two graduate and two undergraduate students from
three departments at our university: Communica-
tion, Political Science, and International Studies. The self-identified gender split was three women
and one man, and the age ranges were: three 20-25
and one 26-30. At the beginning of the project,
three members of the research team led a training Throughout the duration of the annotation cam-
paign (May-December 2022), annotators labelled
4,022 conversations involving 9,472 individual au-
thors with 22 labels. It was a two-tiered labelling
system, where the annotators would first read the
entire conversation and indicate whether there was
conflict in the last message of the conversation
given the context of the previous messages. They 12197 would then annotate further aspects if the answer
was ‘yes’, following Figure 1. age’). Appendix C provides further information
on the rate of conflict per topic. In summary, most
topics contained some form of conflict, due to the
way they were collected (trending topics). This
makes the dataset possibly unbalanced, but also a
rich source of how conflict develops online. Since each binary label was annotated by two
annotators, we can compute inter-annotator agree-
ment using Cohen’s kappa, κ (Cohen, 1960). The
initial question, ‘did the last message in the conver-
sation contain conflict?’ had κ = 0.65, a moderate
to substantial level of agreement (Landis and Koch,
1977). The kappa values for the follow-up ques-
tions in Table 1 show that some of the other labels
foster less consensus. Annotators agreed, in gen-
eral, whether the conflict is internal or external to
the tweet (with internal more difficult to adjudicate). A conflict is internal when all involved parties are
also engaged in the conversation, i.e., it is conflict
among the participants. An external conflict is dis-
agreement about somebody else not in the conversa-
tion, e.g., a public figure (see Appendix E for more
detail). Annotators also often agreed that conversa-
tional context was needed. The level of agreement
for rhetorical strategies (sarcasm, explicit directives
and calls to action, association and metaphor analo-
gies) was quite low, although consistent with the
well-documented difficulty in annotating sarcasm
(González-Ibáñez et al., 2011; Oprea and Magdy,
2020). 3.3
Annotation and agreement In this paper, we mainly use the binary label
for conflict in the tweet, leaving other features for
further study. We kept only conversations where
the two annotators agreed on whether there was
conflict. This yielded a total of 3,577 data points. One could argue that, by keeping only cases with
clear agreement, we are in effect making the task
‘easier’. Given the small size of the dataset, we
follow this approach in order to eliminate noise. 3Names from the LEDIR repository (Sanders et al., 2020). 4
Conflict predictive models If there is
a path from C to A, that means that A received a no-
tification from C. For the above example, Deniz’s
message to Boróka sends a notification to both
Amal and Boróka, but not to Carlu. Intuitively, it
should help to know the directed graph’s structure
of a conversation. It seems important to know, for
example, that an utterance is part of a larger con-
versation sending out a notification to five different
people versus a back-and-forth conversation be-
tween just two people. For the experiments, we en-
coded this feature as a binary ‘has_bidirectionality’
feature (i.e., is there back-and-forth interaction),
but the dataset we will release has a full representa-
tion of this dynamic. For instance, the conversation
in Figure 2 is represented as: [(B, A), (D, B), (B,
D), (C, A), (E, D), (E, C)], which is also converted
to a matrix form in the annotated dataset. who received a notification from whom. If there is
a path from C to A, that means that A received a no-
tification from C. For the above example, Deniz’s
message to Boróka sends a notification to both
Amal and Boróka, but not to Carlu. Intuitively, it
should help to know the directed graph’s structure
of a conversation. It seems important to know, for
example, that an utterance is part of a larger con-
versation sending out a notification to five different
people versus a back-and-forth conversation be-
tween just two people. For the experiments, we en-
coded this feature as a binary ‘has_bidirectionality’
feature (i.e., is there back-and-forth interaction),
but the dataset we will release has a full representa-
tion of this dynamic. For instance, the conversation
in Figure 2 is represented as: [(B, A), (D, B), (B,
D), (C, A), (E, D), (E, C)], which is also converted
to a matrix form in the annotated dataset. We also postulate (Hypothesis 1c) that the bag-
of-words approach can be enhanced with additional
labels. These labels are derived from other models
that classify messages according to their construc-
tiveness (Kolhatkar et al., 2023) and toxicity (Hanu
and Unitary Team, 2020).4 We added logit scores
from each of these existing models into the same
feature matrix. 4
Conflict predictive models Table 2 shows a confusion matrix
for the bag-of-words (BOW) model with construc-
tiveness and toxicity on the 716 conversations in
our test set (we used a standard 80-20 split for train-
ing and testing). We can see that this model is quite
good at identifying both non-conflict and conflict,
but it overpredicts conflict, as it is the majority in
the imbalanced dataset. Non-Conflict Conflict Total
Non-Conflict
27
87
114
Conflict
0
602
602
Total
27
689
716
Table 2: Confusion matrix for BOW LR model with
constructiveness and toxicity (Hypothesis 1c) 4https://github.com/unitaryai/detoxify
5https://huggingface.co/bert-base-uncased 4
Conflict predictive models For now, we focus only on finding conflict, leaving
the issue of whether the conflict is agonistic or not
for future work (although we make some sugges-
tions in Section 5). Our starting hypothesis is that
we can find signals of conflict in the data. The tar-
get variable of interest in this paper is the presence
of conflict. Predictive features include: words in
the text (bag of words model); constructiveness and
toxicity labels; and context from the conversation,
namely cardinality (number of participants) and
topology (structure of participant interactions). By conversational topology we refer to the multi-
threaded nature of online conversations, which
have been described as polylogues (Marcoccia,
2004). Let us examine it with an example, rep-
resented in Figure 2. Amal sends out a message
about a new mural in their city. Boróka angrily
replies that this mural is a waste of tax-payer money. Deniz replies to Boróka with a meme making fun
of Amal, and Boróka sends the laugh emoji back
to Deniz in response. Carlu tells Amal that a sec-
tion of the mural is controversial and divisive and
shouldn’t have been publicly-funded. Eryl replies
to Deniz clarifying that the section of the mural
they are referring to was not actually funded by the
city. Eryl then says the same thing to Carlu.3 Table 1: Cohen’s kappa κ values for different features Table 1: Cohen’s kappa κ values for different features
Feature
κ
Conflict (overall)
0.65
Target - Individual
0.90
External
0.58
Context - Conversational
0.58
Internal
0.48
Rhetorical strategy - Sarcasm
0.36
Rhetorical strategy - Explicit
0.35
Rhetorical strategy - Association 0.19
Context - Media
0.18
Context - Cultural
0.02 Amal
Boróka
Carlu
Deniz
Eryl
Figure 2: An example conversation represented as a
graph Amal Eryl Appendix B provides a trend line for level of
activity per topic over time. We saw that some top-
ics, like ‘Social Distancing’ were discussed over
long periods of time, whereas other topics peaked
and declined quickly, perhaps having to do with
specific events (‘Will Smith Slap’, ‘Rogers Out- Figure 2: An example conversation represented as a
graph Each directed edge embeds information about
3Names from the LEDIR repository (Sanders et al., 2020). Each directed edge embeds information about 3Names from the LEDIR repository (Sanders et al., 2020). 12198 who received a notification from whom. 4.1
Bag of Words model Table 2: Confusion matrix for BOW LR model with
constructiveness and toxicity (Hypothesis 1c) Table 2: Confusion matrix for BOW LR model with
constructiveness and toxicity (Hypothesis 1c) The first basic hypothesis (Hypothesis 1a) is that
the presence of some words (unigrams and bi-
grams) is predictive of conflict. We used a logis-
tic regression classifier with L2 regularization to
predict whether the entire conversation represented
conflict or non-conflict. It used a TF-IDF vectorizer
to extract unigram and bigram features from the
last message in the conversation, the message that
we first extracted using keywords. We also used not
just the last message, but the entire conversational
context, with the same model (Hypothesis 1b). When we ran this model, we saw that some terms
had coefficients with very high absolute values in
predicting the presence of conflict. Although some
of these terms are intuitively indicative of general
conflict, such as the unigram ‘people’, some of the
terms are most likely hyper-specific for particular
conflicts that will have only happened once, such
as the unigram ‘smith’, referring to the Will Smith
slapping incident. To reduce the risk of overfitting
this model to particular topics on a new dataset or
domain, and to have the model learn topic-agnostic
linguistic patterns of conflict, we removed topic-
related unigrams and bigrams by selecting the top
10 c-TF-IDF words from each topic and then re-
moving those that seemed highly topic-specific
(see Appendix D for lists of words removed by
topic). We use this topic-agnostic dataset (Dataset
2) for the fine-tuning experiments in the next sec-
tion. Dataset 1 contains all the words, without
filtering. 4.3
Comparison of model results Even more interesting is the effect of additional
labels. When we incorporate constructiveness and
toxicity, there is an improvement over the baseline
of the last message and a slight decline from just
including all the messages. However, the model
is more robust to the removal of topic words. The
models with cardinality and topology have similar
topic-independent robustness. Cardinality leads to
the lowest drop in performance (3.47 points) for
Dataset 2, and topology also seems to show some
topic independence. The GPT-3 model performs
worse compared to all BERT combinations, and
also suffers from a drop in Dataset 2. We should
note that the GPT-3 model that we use was trained
on data collected roughly up to the end of 2019
(Brown et al., 2020), so it lacks knowledge about
most of the topics in our data, which was collected
later. It serves, thus, as a good test for topic inde-
pendence. Table 3 shows F1 score results for the two main
approaches we took, following the hypotheses de-
scribed above. We first show results for a BOW
logistic regression model under the three different
conditions (last message, all messages, all mes-
sages with constructiveness and toxicity). We then
test the performance of BERT with the same condi-
tions, plus we incorporate cardinality and topology. We also tested a simple GPT-3 fine-tuned model
using OpenAI’s Curie fine-tuning API. Our test
data comes from the human annotations (Dataset
1), and the same data but with topic-related words
removed (Dataset 2). The latter is more likely to
be generalizable to new data about different top-
ics, which is why we are interested in performance
changes relative to Dataset 1. Results in the table
are F1 score averages (average of three runs with
random seeds 42, 43, 44). Model
Dataset 1 Dataset 2
LR Last Msg (H1a)
38.30
33.82
LR All Msg (H1b)
26.87
23.73
LR All Msg + constr, tox (H1c)
38.30
24.81
BERT Last Msg (H2a)
94.58
85.26
BERT All Msg (H2b)
96.06
85.42
BERT All Msg + constr, tox (H2c)
95.43
87.26
BERT All Msg + cardinality (H2d)
92.83
89.36
BERT All Msg + topology (H2e)
94.40
88.92
GPT-3 Fine-tune All Msg
91.95
85.85
Table 3: F1 scores across datasets. 4.2
Transformer model Our second main hypothesis (Hypothesis 2a) is
that we can detect conflict using a transformer
model, the BERT (Devlin et al., 2019) implementa-
tion from HuggingFace.5 Further, we propose that,
beyond the specific words in the message, the con-
text of the conversation contributes to its likelihood
of becoming conflictual. We examine different
types of contextual information: the previous mes-
sages (Hypothesis 2b); the previous messages with
constructiveness and toxicity labels, as we saw in
Section 4.1 (Hypothesis 2c); the cardinality of the
conversation (Hypothesis 2d); and the topology, or
structure of the conversation (Hypothesis 2e). We use a technique inspired by Jin and Aletras
(2021) to incorporate contextual information into
the BERT model. This approach has proven effec-
tive for complaint severity classification by inject-
ing linguistic features. The word representations
from the embedding layer are combined with the
contextual information using an attention gate to
control the influence of different features. The com-
bined representations are passed through the BERT
encoder, followed by an output layer. We set the 12199 max length to 256 and keep the parameters in the
attention gate the same as Jin and Aletras (2021). with no topic words. This improvement with all
the messages likely results from BERT’s ability to
account for contextual sequences, as compared to
the LR model. 4.3
Comparison of model results constr = construc-
tiveness labels; tox = toxicity labels We conclude that information about the conver-
sational context is useful in pinpointing conflict, ad-
ditionally contributing the type of contextual infor-
mation that the model needs to be robust to changes
in topics and individual words. 6
Conclusions and future work qualitative analysis of all the conversations in that
green zone, to investigate whether they have traces
of agonism. The long-term goal of our program of research is to
identify agonistic conflict and distinguish it from
less productive and democratic forms of conflict. The work presented here contributes a definition
of agonism and its operationalization in a coding
schema, an important step in approaching content
moderation as a task of fostering agonistic dialogue,
a productive form of conflict that is essential to
democratic dissent. Furthering our analysis, we sampled the top 100
bidirectional conversations from the highest PU
and PA scores. A different set of annotators was
then tasked with categorizing each conversation as
either agonistic, antagonistic, or neither. This re-
sulted in an inter-annotator agreement quantified by
κ = 0.44, indicating a moderate level of agreement. The distribution of resulting labels in agreement is
shown in Table 4. The work presented here contributes a definition
of agonism and its operationalization in a coding
schema, an important step in approaching content
moderation as a task of fostering agonistic dialogue,
a productive form of conflict that is essential to
democratic dissent. We introduce a richly annotated dataset of online
conversations containing conflict. Using this data,
we test methods that can identify conflict from con-
versations, crucially incorporating contextual in-
formation. We experiment with dimensions of the
context that we believe can be proxies for agonistic
conflict, including the presence of constructiveness
and toxicity, the number of participants, and the
topology of the conversation, which includes the
level and direction of interaction. We show that the
contextual information is key to identifying con-
flict, especially because it helps the model remain
topic-agnostic. This contextual approach can be
helpful not just in identifying conflict and agonism,
but also in detecting abusive language, as it pro-
vides a wider view of the conversation, rather than
whether an utterance is abusive or not in isolation. Table 4: Distribution of conversational labels Table 4: Distribution of conversational labels Label
Percentage
Agonistic
34%
Antagonistic
32%
Neither
35% 76.5% of the conversations coded as Agonism
were sampled from the top 100 PA set, and 80%
of the conversations coded as Antagonism were
sampled from the top 100 PU set. These findings
suggest that while our proxy scores could do mod-
erately well at discerning between agonistic and
antagonistic conversations, there is still room for
improvement. 6https://github.com/Digital-Democracies-
Institute/Dimensions-of-Online-Conflict 5
Approximating agonism We can attempt to approximate agonism as well as
other categories of these conversations given the
three dimensions shown in Figure 3 by defining PA
as the Possibly Agonism Score, PU as the Possibly
Unproductive Score, and PS as the Possibly Small-
Talk Score as follows: Table 3: F1 scores across datasets. constr = construc-
tiveness labels; tox = toxicity labels Table 3: F1 scores across datasets. constr = construc-
tiveness labels; tox = toxicity labels We can see from Table 3 that the simple logistic
regression model (LR) results are quite low, but
have a drop for Dataset 2 that is comparable to that
of the BERT models. There are no gains whatso-
ever for the LR model from including more context,
in the form of knowing the prior messages in the
conversation, or more information, such as the con-
structiveness and toxicity labels. This might result
from the absence of word sequence modelling in
LR, which may hinder its ability to capture contex-
tual dependencies between words. PA = 1 −
p
(T −0.0)2 + (S −1.0)2 + (C −1.0)2 (1)
PU = 1 −
p
(T −1.0)2 + (S −0.0)2 + (C −1.0)2 (2)
PS = 1 −
p
(T −0.0)2 + (S −0.0)2 + (C −0.0)2
(3) PS = 1 −
p
(T −0.0)2 + (S −0.0)2 + (C −0.0)2
(3) where T is the Toxicity Likelihood, S is the
Constructiveness Likelihood, and C is the Conflict
Likelihood. The results from BERT are more interesting. First of all, a simple model with just the last mes-
sage shows F1 = 94.58, with a 0.32 drop if topic
words are not present. Including all messages helps
considerably, raising the score to its highest level,
96.06, but with an ever larger drop for Dataset 2, Using these proxy scores, we can compare the ra-
tios of unproductive versus agonistic conversations
across different trending topics over time, shown
in Figure 3. Part of our future work involves a 12200 6
Conclusions and future work Implementing a secondary machine
learning model on a larger dataset with labelled
data from this secondary annotation exercise ap-
pears to be a promising next step in our goal to-
wards modeling agonism. We have made data and code available in a repos-
itory.6 This includes: the dataset of 4,022 conversa-
tions with annotations, the code to collect conver-
sations, the LabelStudio annotation scheme, and
the code for all the experiments described. The
appendices in this paper include detailed informa-
tion about the data collection process, the coding
schema, and multiple examples of annotated con-
versations from the dataset. The repository also
contains links to raw data, a larger dataset of con-
versations we collected but have not annotated (the
entire dataset contains 220,626 conversations). We
will, additionally, make available a demo web appli-
cation linked in the repository to experiment with
the model results. Figure 3: Conversations plotted by conflict, construc-
tiveness, and toxicity likelihoods. We are postulating
the green volume as the ‘zone of agonism’. The next steps in our research program involve
deploying the other dimensions in the data (us vs. them conflict; individual/group conflict, etc.). We
also plan to perform a qualitative analysis of con-
versations with high conflict, high constructiveness,
and low toxicity, which we have defined as a poten-
tial zone of agonism. Further experiments will ex- Figure 3: Conversations plotted by conflict, construc-
tiveness, and toxicity likelihoods. We are postulating
the green volume as the ‘zone of agonism’. Figure 3: Conversations plotted by conflict, construc-
tiveness, and toxicity likelihoods. We are postulating
the green volume as the ‘zone of agonism’. 12201 that we are at the early stages of a long and complex
research process. tend this model to other topics in our larger dataset,
to test whether it can be used for semi-automatic an-
notation. We also plan to gather additional topics
that generated discussion by querying keywords
from monthly snapshots of the Wikipedia Por-
tal:Current_events and the Top_25_Report through
the Wayback Machine. Beyond identifying individual comments as con-
structive, productive, or leading to agonism, it is
also important to acknowledge the role of user and
interface design in how comments are produced
and presented (Masullo et al., 2022). We also note more common limitations, includ-
ing the source of the data (Twitter), the language
of study (English), and the lack of demographic
information about the participants in the discus-
sions. Ethics Statement We adhere to the ACL Ethics Policy. In particular,
we strive to contribute to societal well-being and
the public good by studying how conflict evolves in
online conversations. We take the directive to avoid
harms quite seriously. To avoid individual harm
and respect privacy, we anonymize the tweets be-
fore releasing them publicly (although the Twitter
ids will provide a link to the original). We also take
into account duty of care for our researchers and
annotators, and have provided them with opportuni-
ties to debrief and protect their mental health when
they read conflictual and hateful material. We are concerned about the damage to the envi-
ronment caused by training and fine-tuning large
language models. We mitigated, in part, by using
only pre-trained models. The BERT model was
fine-tuned on a data center powered by hydroelec-
tric energy, thus producing fewer CO2 emissions. Precisely because there is so much conflict on-
line, a dataset with about 4,000 conversations is not
a representative sample. The method of collection,
where we started with topics likely to generate con-
flict (both hateful and agonistic), may also result in
biased data. One alternative we could contemplate
is to draw from datasets that have been labelled for
toxicity, as those are more likely to contain conflict. Such data, however, does not necessarily contain
agonistic conflict. Furthermore, as we mention in
the conclusion section, we have not yet reached
the stage of identifying agonism automatically. We
hope that, by detecting conflict overall, we can ex-
tract many instances automatically, leading us to
a method for distinguishing antagonistic conflict
from agonism. The main limitation of our study is Although our current system is far from perfect
in detecting conflict, and we have not yet produced
a method to detect agonistic conflict, one risk of
such systems lies in their misuse by employers,
governments, or other social agents to quell agonis-
tic and productive conversations. For example, an
employer may wish to suppress agonistic discourse
that could lead to employee unionization. Limitations Research on conflict consistently draws attention to
its complex nature. Peace studies scholar Giorgio
Gallo (2013) states that conflict can be character-
ized by multiple, diverse, at times hidden, unde-
fined, and evolving objectives. Thus, translating
such a complex phenomenon into a set of labels
output by a machine learning system is reductionist
at best. Gallo notes that most research on conflict
tends to isolate it from its context, thus oversim-
plifying the inquiry. We note that ours is one such
simplification. The nature of online conflict, with
long conversations unbounded by time and space
limitations, unlike face-to-face communication and
debate, renders more fine-grained and contextual-
ized approaches impossible. We nevertheless at-
tempt to incorporate context beyond the words in
the individual messages or message threads, by ex-
amining features of the conversation, the number of
participants, and the topology of the conversation. 6
Conclusions and future work We do not include information about lan-
guage varieties and rhetorical strategies that may
be characteristic of some online groups and linguis-
tic communities and not in the mainstream. We
do not know whether the conversations are repre-
sentative of some mythical mainstream culture or
of demographic groups with their own norms of
debate and argumentation. Defining and identifying agonism in conversa-
tions is a difficult task. We also acknowledge the
difficulty of fostering the kinds of spaces that are
conducive to agonistic debate, both online and of-
fline, which is why an interdisciplinary approach
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Cristian
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CSCW, page Article 163, Glasgow, UK. Reid Swanson, Brian Ecker, and Marilyn Walker. 2015.
Argument mining: Extracting arguments from online
dialogue. In Proceedings of the 16th Annual Meet-
ing of the Special Interest Group on Discourse and (1) #billc11, #cancon, #CRTC, #onlinecensorship, #online-
harmsreduction, #StopAsianHate, #stopC11, bean dad,
bell hooks, biden loans, bill gates divorce, canadian truck-
ers, capitol insurrection, china gamer ban, coastal gas
link, convoy, defund the police, depp heard, el salvador
bitcoin, elon twitter, evergrande, gamestop, groomers,
hbo test, india pakistan missile, ivermectin, libsoftiktok,
metaverse, ocean fire, online harms, pandora papers, re-
aldonaldtrump, rittenhouse, robb elementary, roe, rogers,
sci_hub, social distancing, suez canal, taliban, tigray, tmx
pipeline, west elm caleb, will smith slap, vaccine B
Appendix: Topic distribution over time • defund-the-police: police, defund
• defund-the-police-realdonaldtrump: police, realdon-
aldtrump, defund
• depp-heard: depp, heard, amber, johnny
• elon-twitter: twitter, elon, musk, elonmusk
• groomer: govrondesantis, groomers, travlingsnowman
• india-pakistan-missile: pakistan, india, indian
• libsoftiktok: libsoftiktok, taylorlorenz
• metaverse: metaverse, crypto, bsc
• protests-social-distancing: protests, distancing
• queen-elizabeth: us
• roe-v-wade: roe, abortion, wade
• russia-ukraine: ukraine, russia
• salman-rushdie-attack: rushdie, salman
• social-distancing: distancing
• tigray-ethiopia: tigray, ethiopia, tplf
• vaccine: vaccine, covid Figure 4 shows the number of conversations related
to each topic over time. We can see that certain
topics exhibit a more enduring presence in the dis-
course (Social Distancing), while others appear to
be more transient, capturing attention for only a
day or two (Will Smith Slap, Rogers Outage). Each
topic was sampled from its peak of discussion. We
have observed that topics with high peaks (Will
Smith Slap or India Pakistan Missile) tend to have
a higher level of conflict than topics that last over
longer periods of time. A
Appendix: Search keywords The following are the list of keywords used for the
search described in Section 3.1. The following are the list of keywords used for the
search described in Section 3.1. Christian Stab and Iryna Gurevych. 2017. Parsing argu-
mentation structures in persuasive essays. Computa-
tional Linguistics, 43(3):619–659. Jonathan Stray. 2022. Designing recommender systems
to depolarize. First Monday, 27(5):https://doi. org/10.5210/fm.v27i5.12604. Reid Swanson, Brian Ecker, and Marilyn Walker. 2015. Argument mining: Extracting arguments from online
dialogue. In Proceedings of the 16th Annual Meet-
ing of the Special Interest Group on Discourse and 12205 12205 E.1
Is there conflict? A yes/no answer, based on the definition of conflict
provided (see Section 2.1). Annotators were in-
structed to label only the last message (e.g., the last
tweet in Figure 5), but use the context (the previous
tweets) if necessary. A ‘yes’ answer triggers all the
decisions below. H =
12
N(N + 1)
X
j
R2
j
nj
−3(N + 1)
(4) (4) E.2
Internal/external If the annotators decided that there was conflict,
they had to label the conflict as internal to the con-
versation (‘when it involves people/entities directly
engaged in the conversation’) or external. For ex-
ample, if people are discussing Black Lives Matter
as an organization, then the conflict is external. But
if they discuss people in the conversation involved
in BLM, then it’s internal. A tweet can be both
internal and external, so both labels are allowed. This label had a relatively high level of agreement
(see Table 1), so we feel this was a valid distinction. Examples (2) and (3) show instances of each. This calculation obtained a p-value < .005. We
can confidently say that topics are clearly differ-
ent in their rate of conflict, with discussions about
Biden’s loan forgiveness program being 100% con-
flictual, and conversations about Facebook’s Meta-
verse being low in conflict (8% of the conversations
in that topic had conflict). E
Appendix: Coding schema Content warning: Examples in this section con-
tain offensive language. Table 5 presents summary statistics for the top top-
ics (a total of 2,718 conversations, out of the 4,022
in the dataset). The first column shows the total
number of conversations in that topic. The next few
columns display the percentage of conversations
in that topic with the label (e.g., 62% of the Social
Distancing conversations contained conflict, and
11% had sarcasm). We see that most topics con-
tained some form of conflict (making the dataset
unbalanced), but that presence of other dimensions
of conflict varies widely. Using the percentage of
conflict per topic in Table 5, we can test for statisti-
cally significant differences in conflict rates across
topics. Assuming non-normality and non-equal
variances, we can use the Kruskal-Wallis H test
defined as follows: Annotators were presented with a LabelStudio
interface that provided: the tweet to annotate, the
previous 3-7 messages in the conversation, and the
labels to choose from, as seen in Figure 5. Labels
were always for the last tweet in the conversation,
but in the context of the entire thread. Here, we
elaborate on the descriptions for each annotation
decision, from the schema in Figure 1. (2)
[Internal] You are going to arrst me for standing 4’
away from someone? Go for it
(3)
[External] Honestly at this point second wave is gonna
have to happen for it to get through their THICK SKULLS
that they completely and utterly fucked this up E.3
Who/what is the target? (6)
[Policy/institutions] That assumption is based on
a busy car park. With miles of beach I am sure it’s easy
to stay far enough away from others. Visiting Tesco’s is
100x more dangerous The target of the conflict can be more than one, so
multiple labels are possible here. Some examples
are presented in (4)-(6). • Individual The following is the list of keywords removed from
each topic as described in Section 3.1. The following is the list of keywords removed from
each topic as described in Section 3.1. • Jan-6th-insurrection: capitol, insurrection, trump (2)
[Internal] You are going to arrst me for standing 4’
away from someone? Go for it • TMX-pipeline/coastal-gaslink: pipeline, tmx, trudeau
• biden-loans: loans student biden • TMX-pipeline/coastal-gaslink: pipeline, tmx, trudeau • biden-loans: loans, student, biden (3)
[External] Honestly at this point second wave is gonna
have to happen for it to get through their THICK SKULLS
that they completely and utterly fucked this up • canada-day: canada • canadian-truckers: truckers, canadian, convoy • canadian-truckers, protests: truckers, protests, canadian,
canada 12206 2020-03-01
2020-09-01
2021-03-01
2021-09-01
2022-03-01
2022-09-01
2023-03-01
India Pakistan Missile
Rogers Outage
Vaccine
Tigray Ethiopia
Roe v Wade
Metaverse
Defund the Police
Biden Loans
Libsoftiktok
Canadian Truckers
Depp Heard
Elon Twitter
Will Smith Slap
Social Distancing
Figure 4: Volume of messages for some of the top topics over time Figure 4: Volume of messages for some of the top topics over time Topic
N Conflict Sarcasm Metaphors Directives Target
Social Distancing
615
0.62
0.11
0.36
0.29
0.40
Will Smith Slap
320
0.97
0.20
0.76
0.05
0.96
Elon Twitter
268
0.90
0.23
0.67
0.14
0.91
Depp Heard
233
0.97
0.11
0.70
0.08
0.95
Canadian Truckers
230
0.97
0.10
0.67
0.15
0.53
Libsoftiktok
191
0.97
0.18
0.55
0.09
0.80
Biden Loans
190
1.00
0.13
0.72
0.23
0.78
Defund the Police
157
0.94
0.06
0.41
0.42
0.33
Metaverse
131
0.08
0.18
0.50
0.05
0.59
Roe v Wade
91
0.95
0.10
0.38
0.19
0.43
Tigray Ethiopia
90
1.00
0.06
0.68
0.40
0.31
Vaccine
76
0.89
0.20
0.24
0.30
0.45
Rogers Outage
69
0.48
0.15
0.20
0.30
0.41
India Pakistan Missile
57
1.00
0.21
0.61
0.12
0.46
Total/average
2,718
0.84
0.12
0.49
0.16
0.56
Table 5: Top topics and percentage of conversations in those topics that were given select annotation labels Topic
N Conflict Sarcasm Metaphors Directives Target Table 5: Top topics and percentage of conversations in those topics that were given select annotation labels E.4
Authority evoked • Group This question asked whether the position, stance, or
claim is recognized, consistent with, legitimated, or
supported by some form of authority. Annotators
were instructed to consider how different forms
of authority are invoked by the participants, again
focusing on the last tweet in the conversation. • Policy/institutions • Systemic issue or cause • Systemic issue or cause
• Events y
• Events • Events • Other (specify) (4)
[Individual] Cummings should be sacked! And prose-
cuted in criminal law. • Personal experience. This could be an individ-
ual encounter, an understanding, or an insight,
usually derived from proximity and epistemic
positions. (5)
[Group] Its unreasonable and unrationale to say that we
can’t let emotions take over. Imagine being a black person
in America right now. We should be angry, we shouldn’t
be pushing down or hiding our feelings. We want justice,
everything else is not important right now. 12207 Figure 5: Annotation interface This label had a very low level of agreement with
a kappa value of 0.02. We believe the explanation
of this distinction, as shown above, was unclear to
annotators, perhaps also because these oppositional
relations are not often explicitly stated. Some ex-
amples are provided below. Note the dual label in
(10). (10)
[Us above them], [Us/them disjuntive] idk why
y’all continue to defend people who prove themselves
over and over again the rich will always find ways to
hide their money and avoid paying their dues, if we
continue to let them Figure 5: Annotation interface Figure 5: Annotation interface Figure 5: Annotation interface (11)
[Us/them
disjunctive] Please sign our petition
to fire RCMP Commissioner Brenda Lucki here, and
maybe we can finally get some accountability for an
organization that has gone completely off the rails. Keep
fighting for what is right, (11)
[Us/them
disjunctive] Please sign our petition
to fire RCMP Commissioner Brenda Lucki here, and
maybe we can finally get some accountability for an
organization that has gone completely off the rails. Keep
fighting for what is right, • Common sense. Authority is evoked by refer-
ring to common sense knowledge, some sense
of what is shared in the specific or cultural
context (regardless whether common sense is
actually true). E.5
Groups being discussed This is a binary question, asking whether the con-
versation involves clearly identified groups or fac-
tions, presented as an attempt to render conflict as
isolated incompatibility between groups. If the an-
swer is ‘yes’, this triggers another set of questions,
about the relationships among those groups and
how they are being discussed: (14)
[Sarcasm] So doxxing people who disagree with you
is OK, but not doxxing people who agree with you. Got
it. E.6
Rhetorical strategy • Factual/distant expertise or institution. Par-
ticipants support their statements by referring
to experts, facts, or (statistics provided by)
institutions. A ‘yes’ answer here means that the annotator saw
persuasion or appeals to sensibilities and mean-
ings that were grounded in linguistic techniques,
language moves, or other linguistic mechanisms. If the answer was ‘yes’, then they were asked to
specify what type of strategy was deployed: • Authority is rejected. Participants reject or
challenge authorities invoked by others. • Other (specify) • Explicit directives and calls to action (7)
[Personal experience] Pretty sure for my son to go
to boarding school he needed proof of vaccinations (7)
[Personal experience] Pretty sure for my son to go
to boarding school he needed proof of vaccinations • Associations, metaphors, or analogies • Sarcasm • Sarcasm (8)
[Common sense] The mothers who raise these men play
a role. They, too, are responsible. Many of them support
the Taliban. It is naive to believe otherwise • Other (specify) (12)
[Explicit directives], [Sarcasm] Yo ACLU and
Amnesty International, calm down. Why sudden panic
about “free speech” when Elon wanna buy Twitter? (9)
[Factual/distant expertise or institution] All
indications are that the practice of sex with social dis-
tancing will have a profound effect on population control. Perhaps even larger that what ‘the pill’ had in its day
when it first came out. (13)
[Associations, metaphors, or analogies] So
seems like evergrande stroke a deal which is good for
now (kicking can down the road) and news just came out
of China about reducing coal. Now just the Fed gotta
worry about. Besides the fact that charts are looking
ugly. E.7
Meta questions Finally, regardless whether the answer to the con-
flict question was yes or no, annotators were asked
three further questions, about context and about
their own reaction. • Us above them • Us below them • Us/them conjunctive. The two or more groups
being discussed or involved in the conversa-
tion are presented as being allied, connected,
or somehow related. The first question was whether more context was
needed, beyond the thread. As we see in Figure
5, annotators were asked to label the last message
and were allowed to read the previous messages
on the screen. But they also had the possibility
to click on the tweet and look at the context on • Us/them disjuntive. The two or more groups
are presented as not allied, connected, or re-
lated. 12208 12208 the Twitter platform, including any media. If they
clicked, they were asked to answer ‘yes’ to this
question. Further, they could specify what kind of
context was needed: • Cultural. The annotator needed to know more
about the issue at stake. This could be current
news topics, subcultural trends online, or other
aspects of the topic. Figure 6: Conversation, with labels shown in Figure 7 • Conversational. The conversation was miss-
ing some elements, perhaps earlier than the
messages included in the annotation platform,
which made it difficult to interpret. • Missing content. Tweets in the thread had
been deleted. • Media. Videos or images in the thread were
not available, but seemingly necessary for in-
terpretation. Figure 6: Conversation, with labels shown in Figure 7 The second meta question asked about the emo-
tional reaction of the annotator, simply asking
‘How did you feel when reading the conversation?’
Some suggestions were provided: • Shock • Sadness • Confusion • Indifference Figure 7: Full annotation for conversation in Figure 6 • Entertained • Hopeful The third and final question was about level of
confidence (‘How confident are you about your
analysis?’). Annotators were provided a 1-5 scale,
with 1 being not confident at all (more context was
needed, tweets had been removed, tweet was inde-
cipherable), and 5 being a high level of confidence
or that the interpretation was straightforward. E.8
Full annotation Figure 6 displays a message (last message) and its
context, to show the richness and complexity of the
annotation scheme. Labels for the annotation of
this example are given in Figure 7. Figure 7: Full annotation for conversation in Figure 6 12209
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Argumentation-Based Health Information Systems: A Design Methodology
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IEEE intelligent systems
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Argumentation-Based Health Information
Systems: A Design Methodology Helena Lindgren, Timotheus Kampik
, Esteban Guerrero Rosero
, Madeleine Blusi
, and Juan Carlos Nieve
Umea
University, 90187 Umea
, Sweden In this article, we present a design methodology for argumentation-based health
information systems. With a focus on the application of formal argumentation, the
methodology aims at eliciting requirements in regard to argumentation reasoning
behavior, knowledge and user models, and business logic on levels below and above
the argumentation layer. We highlight specific considerations that need to be made
dependent on the system type, i.e., for clinical decision-support systems, patient-facing
systems, and administration systems. In addition, we outline challenges in regard to
the design of argumentation-based intelligent systems for healthcare, considering the
state of the art of argumentation research, health information systems, and software
design methods. For each challenge, we outline a mitigation strategy. F F
ormal argumentation has emerged as a promis-
ing method for automated reasoning. While
a large body of works exists on theoretical
aspects of formal argumentation,1 the application of
the method to real-world use cases is—despite some
success stories—still at an early stage. Hence, it is
important to advance research that closes the gap
between the theoretical knowledge the community is
accumulating and real-world applications. clinical pathways recommends the intake of a, based
on treatment plan d. In contrast, a medical specialist
recommends the intake b, based on treatment plan c. Because stimulate medication and antidepressants
counteract each other, only one of the treatment
options can be chosen. The practitioner who is
responsible for treating the patient needs to decide
which advise to follow. Figure 1 shows the argumenta-
tion graph of the example. Formal argumentation approaches are frequently
proposed in the context of health information sys-
tems2 to derive conclusions from conflicting, incon-
sistent,
or
uncertain
information. The
following
(simplified) example highlights the usefulness of
argumentation in healthcare (using the abstract
argumentation approach).3 A patient shows symp-
toms that could either indicate attention-deficit/
hyperactivity disorder (ADHD) (the treatment of
which we denote by argument c) or depression
(whose treatment we denote by d). ADHD can be
treated with stimulant medication (argument b),
depression with antidepressants (argument a). A
decision support system based on standardized In formal argumentation, problems of this type
can be expressed in a mathematical model, which can
be solved using a formal method, e.g., a so-called argu-
mentation semantics. Published by the IEEE Computer Society
March/April 2021 Published by the IEEE Computer Society DESIGN METHODOLOGY DESIGN METHODOLOGY
The design methodology can be divided into following
three phases. The design methodology can be divided into following
three phases. 1) Use case and architecture identification. 2) Iterative system design and implementation. In
the
past,
formal
argumentation
has
been
highlighted as a potential solution to a set of common
challenges that arise when designing clinical decision
support systems.4 Still, and although the formal argu-
mentation community is thriving, no success stories that
report on the wide-spread adoption or large-scale clinical
trial of argumentation-based systems in healthcare exist. Indeed, much of the research on formal argumentation
and healthcare is limited to the definition and (some-
times) the implementation of running examples, without
the involvement of domain experts, the creation of open-
sourced software artifacts, and the empirical evaluation
of the developed prototype applications. 3) Empirical evaluation. The end of each phase (and end of the Phase 2
iteration cycle) represents an inflection point, at which
a preliminary evaluation of the system design is con-
ducted that informs the decision on how to proceed
further. Also, each phase results in the creation of dis-
tinct artifacts, which can, for example, be presented in
a dissemination or handed over to third parties. Before
the first phase, focus groups of stakeholders (poten-
tial users, domain experts, etc.) should be set up that
accompany the design process. This article presents a design methodology for
argumentation-based health information systems that
can facilitate stronger applied research in this domain
in the future. The design methodology is informed by
our own research experiences, and in particular, the
lessons we have learned during the past 15 years of
research at the intersection of formal argumentation
and artificial intelligence for healthcare. Table 1 pro-
vides an overview of some application scenarios of
argumentation
to the
healthcare
domain,
during
which the methodology was defined and refined, and
highlights some design aspects (which are explained
in this article) for each use case. The methodology can Use Case and Architecture
Identification The first phase is concerned with use case identifica-
tion and high-level application architecture definition. Argumentation-Based Health Information
Systems: A Design Methodology Note that in the context of
formal argumentation, arguments can model any type
of knowledge, and are not necessarily based on natu-
ral language. To solve the framework in Figure 1, we
first need to answer some (potentially use case-
specific) questions, for instance: 1) Are some arguments stronger than others, for
example because they are more likely to be true
or come from a more authoritative source? 2) What medical knowledge should inform the inter-
nal structure of an argument, and how should its
relation to other arguments be generated? This work is licensed under a Creative Commons Attribution
4.0 License. For more information, see https://
creativecommons.org/licenses/by/4.0/
Digital Object Identifier 10.1109/MIS.2020.3044944
Date of publication 15 December 2020; date of current
version 14 May 2021. 3) How should cycles of arguments that can arise
from dependencies between different sources of
information (e.g., medical guidelines from differ-
ent organizations, diverging opinions of medical
practitioners) be resolved? Published by the IEEE Computer Society 72 IEEE Intelligent Systems March/April 2021 SPECIAL ISSUE ON ARGUMENTATION-BASED REASONING SPECIAL ISSUE ON ARGUMENTATION-BASED REASONING FIGURE 1. Example of an argument graph. Treatment c implies
abstaining from the intake of medicine a, whereas treatment
d implies not taking b. Hence, only one of the treatment
options can be chosen. be considered a complementary, argumentation-cen-
tered perspective on a software development process
that assumes a somewhat agile, iterative approach to
software development,5 which we consider a reason-
able assumption in a research-intense development
context; however, it can be adjusted to better inte-
grate with other, noniterative software development
approaches. be considered a complementary, argumentation-cen-
tered perspective on a software development process
that assumes a somewhat agile, iterative approach to
software development,5 which we consider a reason-
able assumption in a research-intense development
context; however, it can be adjusted to better inte-
grate with other, noniterative software development
approaches. FIGURE 1. Example of an argument graph. Treatment c implies
abstaining from the intake of medicine a, whereas treatment
d implies not taking b. Hence, only one of the treatment
options can be chosen. FIGURE 1. Example of an argument graph. Treatment c implies Define high-level architecture After the use case has been defined, high-level
requirements should be specified, and the preliminary
architecture should be designed. It is important that
the architecture primarily serves the use case; the
alignment with a basic research purpose should
merely be a desirable side effect. At this point, it is typ-
ically sufficient to model the architecture using gen-
eral
graphical
diagramming
tools;
more
detailed
specifications in standardized modeling notations
such as the Unified Modeling Language or ArchiMate 9
can follow later. Cocreate interactive prototypes
In parallel to building the knowledge model, interac-
tive prototypes are created; again, domain experts
should be involved, and in addition, potential nonex-
pert stakeholders like patients. Knowledge model and
user interface depend on each other. On the one
hand, the user interface provides abstractions of the
knowledge model that compromises between accu-
racy and conciseness. On the other hand, the knowl-
edge model needs to consider user interaction needs. During the initial prototype design phase, knowledge
model and interactive prototype should be only
loosely coupled, to ensure that user interaction needs
are treated as first-class citizens. Tools that allow for
the rapid creation of prototypes can already be used
in this phase. However, the employment of such tools
at a too early stage can hamper creativity, as a rather
strict frame for the system’s user interface (UI) design
is dictated by these platforms. Inflection point. After this phase, it should be clear
whether an argumentation-based system can, indeed,
serve the use case at hand. If this is not the case, a dif-
ferent type of system (for example: a simple rule-
based system or a machine learning classifier) can be
implemented, or—if this is not feasible—the project
can be abandoned. In particular, the application of for-
mal
argumentation
as
an
agreement
technology
implies that the use case requires the management of
potentially inconsistent or uncertain information from
multiple sources in decision processes. Artifacts. Results of this phase are a preliminary feasibility analy-
sis, a high-level architecture and requirements specifi-
cation,
and
an
activity
analysis
specifying
the
work and decision-making processes that are to be
supported. Identify use case Right from the start, the system’s use case should be
defined in close collaboration with relevant medical
experts. Adopting an activity-centric perspective is
important to specify the type of support the system
should provide in the application scenario, as well as
the way the system and its users are supposed to TABLE 1. Medical use case examples, their use case types, knowledge bases, conflict types, and argumentation approaches. Medical Use Case
Use Case Type
Knowledge
Base
Conflict Types
Argumentation
Approach
Reference
Decision-support for dementia
diagnosis and treatment
Clinical decision-
support system
Possibilistic
answer-set
programs
Guideline
inconsistencies
Inquiry dialogues Yan et al.6
Self-management application
for mental/social well-being
Patient-facing
system
Extended logic
programs
Human goal
conflicts
Abstract
argumentation
Guerrero et al. 7
Fall prevention application
for the elderly
Patient-facing
system
Extended logic
programs
Inconsistencies in
human activities
Abstract
argumentation
Guerrero et al. 8
March/April 2021
IEEE Intelligent Systems xamples, their use case types, knowledge bases, conflict types, and argumentation approaches. TABLE 1. Medical use case examples, their use case types, knowledge bases, conflict types, and argumentati 73 IEEE Intelligent Systems March/April 2021 SPECIAL ISSUE ON ARGUMENTATION-BASED REASONING collaborate to work toward the users’ and the health-
care providing organization’s goals. always given. Indeed, in this fact lies strength of the
argumentation-based approach: Inconsistencies bet-
ween ideal standards and local realities can be exp-
licitly modeled and resolved at run-time. Another
important aspect when designing the knowledge
model is the knowledge modeling language. Because
medical professionals are not necessarily well-versed
in knowledge modeling languages like the Web Ontol-
ogy Language, it is important to use a high-level,
potentially informal language to specify the rough
model, and, when agreement on the most important
aspects is reached, iteratively refine details. Elicit arguments The second phase implements a system prototype
that is based on the artifacts that result from the
previous phase. The implementation is conducted iter-
atively in collaboration with relevant stakeholders. After the general knowledge model and UI have been
designed, argument elicitation can begin. As the first
step in this activity, one needs to distinguish between
elicitation at design-time and elicitation at run-time. IEEE Intelligent Systems SPECIAL ISSUE ON ARGUMENTATION-BASED REASONING regards to argument strength and dialogue type, the
structure of the arguments should be defined. The
structure is dependent on the knowledge model, i.e.,
on the nature and structure of the knowledge base(s)
from which conclusions are to be derived. For this,
one can rely—to some extent—on the argument inter-
change format,15 which is an early stage effort to pro-
vide guidelines and best practices for the elicitation of
arguments from knowledge bases, as well as for argu-
ment exchange. In conjunction with the design of
argument structure and argument strength, the argu-
mentation semantics that determines how an argu-
mentation graph is resolved needs to be defined; in
this context, the argumentation principles16 that a
specific semantics fulfills should be considered. In par-
ticular, principles that are aligned with the require-
ments of the application scenario should be identified. To construct arguments and detect conflicts, the
formal logic that formalizes the semantics of the knowl-
edge specification language should provide polynomial-
time inference operators. This means it is feasible to
define efficient algorithms for constructing arguments
and detecting attacks between arguments in a medical
knowledge base. To mine arguments from unstructured
or poorly structured data, machine learning techniques
can be applied.11 During elicitation, it is recommendable
to assign each argument to one or several groups; for
example, one argument can be assigned to the group
end-user preference, whereas another one is assigned
to the groups expert diagnosis and International Classi-
fication of Diseases. No matter whether an argument is
elicited at run-time or design-time, the argument’s
strength needs to be considered. The strength can be
derived from the groups the argument is in, or be
inferred from data. For instance, in case a diagnosis-sup-
port system is implemented, expert opinions might be
considered stronger than end-user self-assessments,
because the latter can be considered as less reliable.6
However, when designing a self-management mobile
application, end-user preferences can be considered
more important than expert opinions in some contexts,
for example with regards to the configuration of daily
schedules and motivational recommendations.7 In for-
mal argumentation, strength can be modeled qualita-
tively, for example by constructing preference orders
over arguments,12 or quantitatively, for example, using
probabilistic approaches.13 An important concern in argumentation seman-
tics is their computational complexity. Design knowledge model 1) At design-time, arguments are manually curated,
mined from an unstructured dataset, or auto-
matically transferred from another already well-
structured
knowledge
base. Arguments
and
argumentation frameworks can be refined and
sanity-checked before deployment, which places
less strict requirements on the algorithms for
argument generation. A
domain-specific
knowledge
model
should
be
devised, again in collaboration with domain experts. The knowledge model should be based on existing
models of the corresponding domain, for example, on
standardized data models like Health Level Seven
(HL7),10 clinical paths that have been specified by the
relevant authorities, or international disease classifi-
cation standards. However, it is important to consider
that local realities might diverge from the standard-
ized specifications. For example, the information
scheme that a specific electronic health record sys-
tem uses might not be standard-compliant, and even
if it is, information-completeness is practically not 2) At run-time, arguments can be derived directly
from user-interactions, or from additional data that
is uploaded to the system; i.e., in this case, algo-
rithms for the autogeneration of arguments need
to be defined and properly tested to ensure they
perform as intended when the system is deployed. 74 March/April 2021 March/April 2021 SPECIAL ISSUE ON ARGUMENTATION-BASED REASONING The deci-
sion problems of the well-accepted argumentation
semantics range from NP-complete to PðpÞ
2 -com-
plete.17 In this context, tradeoffs may need to be
made; for instance, argumentation semantics like
the grounded semantics that are of comparably low
computational complexity do not allow for particu-
larly nuanced conflict resolution (colloquially speak-
ing). To enhance computational performance, some
applications of formal argumentation place condi-
tions on the structure or size of the argumentation
graph and, for example, only construct noncyclic
argumentation graphs.18 Hence, use case-specific
running examples should be constructed to evaluate
if a semantics’ output is reasonable from a compu-
tational complexity and a subject-matter expert
perspective. Also, it is crucial to consider the type(s) of argu-
mentation dialogues the system should support; fol-
lowing are the examples.14 1) Inquiry dialogues: The system uses (multiagent)
argumentation for knowledge-seeking purposes,
for example, to derive new conclusions by elicit-
ing arguments from distributed and potentially
inconsistent knowledge bases. Evaluate qualitatively To assess whether the system is ready to be deployed
for a long-running empirical study, a preliminary, qualita-
tive evaluation should be conducted. In contrast to the
previous stages of the iterative design process, this
evaluation should be conducted using a deployed, run-
ning, and stable system instance. Ideally, a new set of
experts and end-users is involved in the evaluation. This
avoids biased feedback from persons who are already
invested from a design perspective. As a first evaluation
step, the initial knowledge base should be validated. For
example, the system’s outputs can be compared to clini-
cal guidelines and protocols, and be evaluated by medi-
cal experts and patients. Additional feedback can be
solicited on the system itself and the conclusions
derived from it in example scenarios, and by conducting
trial runs of the system in a carefully controlled real-life
environment. Also, observations should be made on
how the use of the system affects decision-making and
human behavior. For example, given a decision support
system, it should be documented to what extent users
follow the system’s recommendations. Inflection point. When the empirical evaluation has
been concluded, further steps can be planned based
on the implications of the results. In particular, follow-
up studies can be conducted, for which the system
might need to be customized to fit a new context (for
example, to the needs of medical practitioners in a dif-
ferent country). In successful cases, the hand-over of
the system to parties that can ensure long-term oper-
ations can be initiated; for instance, local health
authorities or other healthcare-providing organiza-
tions may have an interest in taking over the mainte-
nance and operations of a system that is evidently
useful in a particular medical context. Artifacts. The
resulting artifact of this phase is an analysis document
that contains an empirical evaluation of the system. Figure 2 shows the process diagram of the design
methodology. Inflection point. When an implementation and
design cycle has been absolved, the system designers
should decide whether 1) further iterations are neces-
sary, 2) the system is sufficiently mature for an empirical
study, or 3) the system prototype should be discontin-
ued without an empirical evaluation being feasible. Arti-
facts. The resulting artifacts of this phase are a detailed
system specification, a system prototype, and a prelimi-
nary qualitative evaluation of the system. SPECIAL ISSUE ON ARGUMENTATION-BASED REASONING SPECIAL ISSUE ON ARGUMENTATION-BASED REASONING the existing knowledge base, and what actions
should be triggered by such user feedback? Empirical Evaluation
In the final phase, the system is empirically evaluated
with the objective to provide a strong assessment of
the system’s usefulness in real-world medical applica-
tion scenarios. As a rule of thumb, the level of detail the user inter-
face provides in terms of both data view abstractions and
data input and feedback opportunities should be more
fine-grained for medical expert users than for patients. Evaluate empirically To assess the medical efficacy of the developed sys-
tem, an empirical evaluation in practice is necessary,
if possible in a randomized-controlled trial setting. Ide-
ally, the medical practitioners who participate in the
empirical evaluation of the system are not (at least
not exclusively) the ones who have helped design it. Otherwise, there is a risk that the participating medi-
cal experts are 1) biased toward the efficacy of the sys-
tem and 2) have a level of expertise in working with
the system that is hard to obtain by third parties. Implement system prototype In parallel to the previous four design steps, the system
prototype should be iteratively implemented. As men-
tioned earlier, rapid prototyping tools can facilitate the
implementation. In particular, data schemes that
underlie the system should ideally be (auto)generated
directly from the models that have been defined in col-
laboration with the focus group(s). Given the safety-
critical nature of medical information systems, even for
the prototype implementation, quality assurance best
practices like test-driven development and continuous
integration should be followed. Expert-validated run-
ning examples can serve as test cases. To enable a real-world study, the system needs to be
integrated into the corresponding healthcare process
or clinical pathway. If the study is conducted across
regions with different local routines, the study should
ideally be detached from these local routine variants to
ensure comparability across regions. In the empirical
evaluation,
a
mixed-method
approach
is
recom-
mended. Generally, the system’s recommendations or
decisions can be quantitatively evaluated. Unexpected
system behavior can be analyzed qualitatively to find
the reason for the deviation and to determine whether
the unexpected behavior is indeed undesirable. Design knowledge model UI Design knowledge model UI
In parallel to the argument elicitation step, the
abstraction the user interface provides on system
data and processes, i.e., on argumentation frame-
works, knowledge bases, and reasoning methods,
should be designed. In particular, the following ques-
tions should be answered. 2) Deliberation dialogues: The system facilitates
tradeoffs between the positions of different par-
ties, for example, by consolidating conflicting
opinions of experts or conflicts between medical
guidelines on regional and national level. 1) What abstractions should the user interface pro-
vide on the knowledge base and how detailed
should these abstractions be? 3) Persuasion dialogues: The system uses formal
argumentation to persuade a user, for example,
by exchanging arguments with a patient to moti-
vate them to work toward a specific goal. 2) Are there data that should under no circumstan-
ces be exposed to a user, for example, for data-
privacy reasons? The selection of dialogue type is done based on
the activity analysis that specifies phases and deci-
sion points in the clinical pathway and decision pro-
cesses. Alongside with making design decisions with 3) When should the user be able to add additional
knowledge as a means of providing feedback,
how should the knowledge be integrated into IEEE Intelligent Systems 75 IEEE Intelligent Systems Clinical Decision-Support Systems Clinical Decision-Support Systems Clinical Decision-Support Systems In recent years, it has been acknowledged that the han-
dling of inconsistent knowledge, for example, of diverg-
ing expert opinions, is crucial in many medical decision-
support scenarios. However, existing, industry-scale
products do not provide first-class abstractions for
managing inconsistent or conflicting knowledge. Argu-
mentation-based clinical decision-support systems can
help facilitate decision-making by enabling the manage-
ment of these inconsistencies that are common in med-
ical decision-making, and indeed often part of the
medical process by design, e.g., when the opinions of
several medical experts are solicited with regards to a
specific case. An instance of an argumentation-based
decision support system is a dementia diagnosis and
management support application as presented by Yan
et al.6 In a decision-support context, it is important that
arguments from different types of sources are marked
according to the corresponding source type’s strength. For
instance,
the
argument
strength
assignment
can reflect that the opinion of a single practitioner can-
not invalidate well-established domain knowledge as
specified
in
national
or
international
guidelines. A potential hierarchy of arguments could be as follows
(assuming a total ordering), but is most certainly
scenario-dependent. FIGURE 2. Design methodology process diagram. 1) Arguments derived from global guidelines and
standards
take
precedence
over
all
other
arguments. 2) Local guidelines define aspects that have not
been specified in sufficient detail by global
guidelines and standards, but arguments derived
from local guidelines are weaker than arguments
derived from global guidelines. 3) Arguments derived from practitioner opinions
can inform decision-making on individual cases
but are weaker than local and global guidelines. From a formal perspective, a plethora of potential
argumentation strength design mechanism exist19; so
far, there are no best practices as to when to choose
which mechanism. March/April 2021 APPLICATION SUBDOMAINS The context in which a health information system is
applied should self-evidently inform its design. A par-
ticularly important distinction is whether the system
is primarily used by medical experts, by patients, or by
administration staff. 76 IEEE Intelligent Systems March/April 2021 SPECIAL ISSUE ON ARGUMENTATION-BASED REASONING CHALLENGES AND RESEARCH
STRATEGIES The design methodology may be considered an ideal-
ized approach that cannot always be complied with in
reality, for example, because of constraints in time,
budget, or expertise. Indeed, when examining existing
literature on the topic, we observe a set of shortcom-
ings, from which we derive new research strategies. Lack of integration with theoretical
foundations A large part of the theoretical research results on for-
mal argumentation has not been examined from an
application perspective. This applies in particular to
the broad range of works that exist on argument
strength19 and argumentation principles.16 When deciding on the knowledge model, argument
structure and strength, as well as on the argumenta-
tion reasoner, works that provide concise overviews
of the corresponding basic research foundations
should be considered in an as systematic manner as
possible; also, the practical considerations that guide
this process step in particular instances should inform
theoretical research going forward and not only vice
versa. Patient-Facing Systems When implementing patient-facing health information
systems, it is crucial that tradeoffs are made between
the advice and recommendations that the system
provides based on existing medical knowledge and
patient data, and the patient’s self-assessment and
personal preferences. To facilitate these compro-
mises, formal argumentation methods can be consid-
ered a natural fit. A particularity of patient-facing FIGURE 2. Design methodology process diagram. systems is that the recommendations provided and
the actions executed by these systems must not only
fulfill the quality standards of the healthcare domain,
but also allow for informed dissent if the assumptions systems is that the recommendations provided and
the actions executed by these systems must not only
fulfill the quality standards of the healthcare domain,
but also allow for informed dissent if the assumptions 77 IEEE Intelligent Systems March/April 2021 SPECIAL ISSUE ON ARGUMENTATION-BASED REASONING the system makes are not aligned with a patient’s per-
sonal preferences or life circumstances. An instance
of this system type is an argumentation-enabled
behavior change management personal assistant.7 If
the assistant recommends a user to do more physical
exercise, the user should have the possibility to pro-
vide the feedback to the system that in the current sit-
uation, family responsibilities do not allow to act
according to the recommendation. The possibility for
dissent is not only important from the perspective of
medical efficacy, but also for the sake of personal
autonomy: A user should be empowered to make
tradeoffs between an optimally healthy lifestyle and
other aspects of life, and not have a system dictate all
details. From the perspective of argumentation, this
means that users should be able to add new argu-
ments to the system that defeat the conclusions
the system provides; i.e., the system should be able to
provide alternative conclusions based on the new
arguments
while
maintaining
the
evidence-based
knowledge foundation. This behavior allows for verifi-
able compliance with guidelines and regulations,
which is, for example, a requirement for regulatory
approvals like CE-marking. In this regard, the testabil-
ity and verifiability of argumentation-based systems is
a key advantage, for example, in comparison to
machine learning-based systems. Besides the aforementioned peculiarities, the imple-
mentation of argumentation-based healthcare admin-
istration systems can have similar implications as
the implementation of systems for medical experts in
that conflicts between global (e.g., governmental)
guidelines, local implementation details, and diverging
expert assessments need to be made. Systems y
A third type of healthcare information system in which
formal argumentation can be applied are administra-
tive systems, for example, enterprise resource plan-
ning systems that are used in the healthcare domain. To our knowledge, little research exists on the applica-
tion of argumentation-based methods for this health-
care information system type; one exception is an
argumentation-based scheduling tool for nurses.20 A
possible reason for the sparsity of related research
is that administrative systems are tightly integrated
into the information system landscape of an organiza-
tion; this increases the engineering effort and organi-
zational overhead of implementing prototypes that
are sufficiently mature for pilot studies. However, from
a use-case perspective, the management of conflict-
ing information may be considered useful from an
administrative planning perspective; for example, in
the wake of the COVID-19 crisis, decisions on how to
distribute medical equipment to different administra-
tive regions could be informed by infection spread
prognoses that are provided by disagreeing epidemiol-
ogy experts or based on conflicting base scenarios. IEEE Intelligent Systems Short-lived software artifacts Short-lived software artifacts Short-lived software artifacts CONCLUSION 7. E. Guerrero, J. C. Nieves, and H. Lindgren, “An
activity-centric argumentation framework for
assistive technology aimed at improving health,”
Argument Comput., vol. 7, no. 1, pp. 5–33, 2016. The presented design methodology is a point of de-
parture
toward
a
more
structured
and
rigorous
application of argumentation-based approaches for
intelligent health information systems. The methodol-
ogy is an important tool to bridge the divide between
the communities that advance the theoretical and
engineering foundations of formal argumentation, and
researchers who study the application of intelligent
systems in the healthcare domain. We expect that the
methodology will further mature as the body of
research that studies the application of formal argu-
mentation to health information systems growths. Also, the presented methodology can be transformed
into a domain-independent model, or be adjusted to
other system types. 8. E. Guerrero,
J. C. Nieves,
M. Sandlund,
and
H. Lindgren, “Activity qualifiers using an argument-
based construction,” Knowl. Inf. Syst., vol. 54, no. 3,
pp. 633–658, 2018. 9. A. Q. Gill, “Agile enterprise architecture modelling:
Evaluating the applicability and integration of six
modelling standards,” Inf. Softw. Technol., vol. 67,
pp. 196–206, 2015. 10. B. G. M. E. Blobel, K. Engel, and P. Pharow, “ Semantic
interoperability—Hl7 Version 3 compared to
advanced architecture standards,” Methods Inf. Med., vol. 45, no. 4, pp. 343–353, 2006. 11. M. Lippi and P. Torroni, “Argument mining: A
machine learning perspective,” in Theory and
Applications of Formal Argumentation, E. Black,
S. Modgil, and N. Oren, Eds. Cham, Switzerland:
Springer, 2015, pp. 163–176. Lack of strong empirical research Research on argumentation-based health information
systems lacks strong empirical evaluation results. This is in contrast to research on traditional decision-
support systems, but can potentially be explained
by the relative novelty of the argumentation-based
approach. Another possible reason for the lack of
strong empirical evaluations of argumentation-based
health information systems is the fact that the artifi-
cial intelligence researchers who are most engaged
with the design and implementation of these systems
typically lack experience in running long-term empiri-
cal assessments of complex sociotechnical systems. Hence, before the last stage of the design process, a
handover of the developed system prototypes from
artificial intelligence researchers to research groups
that focus on the evaluation of information systems
can be considered recommendable. 78 IEEE Intelligent Systems March/April 2021 SPECIAL ISSUE ON ARGUMENTATION-BASED REASONING REFERENCES A related problem is the lack of openly shared and
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s10458-018-9396-x ACKNOWLEDGEMENTS The authors thank the anonymous referees. This work
was supported by the Wallenberg AI, Autonomous
Systems and Software Program (WASP), funded by the
Knut and Alice Wallenberg Foundation and by the
European Union’s Horizon 2020 programme, under
Grant 825619. 12. L. Amgoud
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2012, pp. 1–16. received the Nordea Scientific Award 2018. She serves in
committees of several large-scale research programs on AI. Contact her at helena@cs.umu.se. TIMOTHEUS KAMPIK is currently working toward the Ph.D. degree with Umea
University, Umea
, Sweden. His research
interests are the intersection of non-monotonic reasoning
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University, Umea
, Sweden. Her research interests include intelli-
gent systems and patient-centered healthcare. She received
the Ph.D. degree from Mid Sweden University, €Ostersund, Swe-
den, in 2014. ACKNOWLEDGEMENTS Contact her at madeleine.blusi@umu.se. 19. M. Beirlaen, J. Heyninck, P. Pardo, and C. Straûer,
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Umea
University, Umea
, Sweden. He received the Ph.D. degree
from Universitat Politecnica de Catalunya—Barcelona Tech
(UPC), Barcelona, Spain, in 2008. His research interests include
the theory and application of nonmonotonic reasoning and mul-
tiagent systems. Contact him at juan.carlos.nieves@umu.se. HELENA LINDGREN is currently a Professor with Umea
HELENA LINDGREN is currently a Professor with Umea
HELENA LINDGREN is currently a Professor with Umea
University, Umea
, Sweden. She received the Ph.D. degree
from Umea
University in 2007. She has combined clinical
expertise with artificial intelligence research, for which she University, Umea
, Sweden. She received the Ph.D. degree
from Umea
University in 2007. She has combined clinical
expertise with artificial intelligence research, for which she 80 March/April 2021 IEEE Intelligent Systems March/April 2021
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Correspondence:
Laura Augusta Barufaldi
Edifício Premium Térreo, sala 15
SAF Sul, Trecho 02 Lotes 05/06
Bloco “F” Torre 1
70070-600 Brasília, DF, Brasil
E-mail: laurabarufaldi@yahoo.com.br http://www.rsp.fsp.usp.br/ Supplement ERICA
Original Article Supplement ERICA
Original Article Rev Saúde Pública 2016;50(suppl 1):6s ERICA: prevalence of healthy eating habits
among Brazilian adolescents Laura Augusta BarufaldiI, Gabriela de Azevedo AbreuII, Juliana Souza OliveiraIII, Debora França
dos SantosII, Elizabeth FujimoriIV, Sandra Mary Lima VasconcelosV, Francisco de Assis Guedes de
VasconcelosVI, Bruno Mendes TavaresVII I Departamento de Vigilância de Doenças e Agravos Não Transmissíveis e Promoção da Saúde. Secretaria de
Vigilância em Saúde. Ministério da Saúde. Brasília, DF, Brasil g
II Instituto Medicina Social. Universidade do Estado do Rio de Janeiro. Rio de Janeiro, RJ, Brasil
III Núcleo de Nutrição. Centro Acadêmico de Vitória. Universidade Federal de Pernambuco. Vitória de Santo
Antão, PE, Brasil II Instituto Medicina Social. Universidade do Estado do Rio de Janeiro. Rio de Janeiro, RJ, Brasil
III Núcleo de Nutrição. Centro Acadêmico de Vitória. Universidade Federal de Pernambuco. Vitória de San
Antão, PE, Brasil ,
,
IV Escola de Enfermagem. Universidade de São Paulo. São Paulo, SP, Brasil VI Departamento de Nutrição. Universidade Federal de Santa Catarina. Florianópolis, SC, Brasil
VII Instituto de Saúde e Biotecnologia Universidade Federal do Amazonas Coari AM Brasil VI Departamento de Nutrição. Universidade Federal de Santa Catarina. Florianópolis, SC, Brasil
VII Instituto de Saúde e Biotecnologia. Universidade Federal do Amazonas. Coari, AM, Brasil VI Departamento de Nutrição. Universidade Federal de Santa Catarina. Florianópolis, SC, Brasil
VII Instituto de Saúde e Biotecnologia. Universidade Federal do Amazonas. Coari, AM, Brasil How to cite: Barufaldi LA, Abreu
GZ, Oliveira JS, Santos DF,
Fujimori E, Vasconcelos SML et al.
ERICA: prevalence of healthy
eating habits among Brazilian
adolescents. Rev Saude Publica.
2016;50(suppl 1):6s. INTRODUCTION Adolescence is a period of intense change, in which individuals suffer influences from
socioeconomic conditions, family habits, values, and social and cultural rules. Habits and
knowledges acquired during this period influence many aspects of adult life related to food,
health, and psychosocial development preferences, among others. Thus, healthy eating
habits during adolescence are crucial to a healthy productive and reproductive life and for
the prevention of noncommunicable chronic diseases in adult lifea. For Matias et al.14, eating habits comprise a set of actions related to food, starting with the
decision, availability, preparation, utensils, eating-schedule and meals division, and finishing
with consumption. Having meals with family members, consuming breakfast, and drinking
the recommended amount of water are considered healthy habits. Studies suggest a positive association between having meals with family members and
ingesting healthy foods, and inverse association between such habit and obesity occurence2,11. The National Survey of School Health (PeNSE)12, performed with Brazilian students of the
ninth grade of elementary school, evaluated these children’s habits of having meals in the
presence of their mothers or legal guardians, among other variables of food consumption. Two-thirds of the students frequently had at least one of the main meals in the presence of
the mother or legal guardian (for five or more days a week), even though about a quarter
of them rarely or had never done this12. According to the Dietary Guidelines for the Brazilian Population, breakfast is one of the
three main meals of the dayb. Compared to snacks, breakfast provides a greater intake of
vitamins and minerals and a lower intake of fat and cholesterol22. Studies suggest a positive
relationship between breakfast consumption and a healthy lifestyle19. Scientific evidences relate the frequent consumption of breakfast to lower risks of overweight
and abdominal obesity16. On the other hand, the absence of this meal may contribute to
calcium deficiency, considering that, generally, breakfast concentrates the highest daily
intake of milk and dairy products, sources of the mineral24. Studies conducted in different countries indicate that the proportion of adolescents that
consume breakfast ranges from 45.0% to 95.0%24. With the increase in the number of
individuals living alone and the lack of time to have meals, the way of life of contemporary
society have been associated to the decline of breakfast consumption19. Proper water
intake is vital, preventing dehydration and its adverse effects, such as headaches, urinary
lithiasis, and prejudices in cognition, among others18. a World Health Organization.
Nutrition in adolescence: issues
and challenges for the health
sector: issues in adolescent health
and development. Geneva;
2005 [cited 2015 Jul 31]. (WHO
discussion paper on adolescence).
Available from: http://apps.who.
int/iris/bitstream/10665/43342/1/
9241593660_eng.pdf
b Ministério da Saúde,
Secretaria de Atenção à Saúde,
Departamento de Atenção
Básica. Guia alimentar para
a população brasileira. 2.ed.
Brasília (DF); 2014. INTRODUCTION Drinking water before meals and
in the place of sugary drinks decrease the consumption of energy and prevent obesity
and dental caries18,23. Despite the importance of healthy eating habits, studies approaching adolescents are still
scarce, which justifies the importance and relevance of this article, whose data represent
adolescent students nationally. The aim of this study is to describe the prevalence of healthy
eating habits in adolescents according to sex, age, education level of the mother, school type,
session of study, and geographic region. ABSTRACT OBJECTIVE: To describe the prevalence of eating habits considered healthy in adolescents
according to sex, age, education level of the mother, school type, session of study, and
geographic region. METHODS: The assessed data come from the Study of Cardiovascular Risks in Adolescents
(ERICA), a cross-sectional, national and school-based study. Adolescents of 1,247 schools of
124 Brazilian municipalities were evaluated using a self-administered questionnaire with a
section on aspects related to eating behaviors. The following eating behaviors were considered
healthy: consuming breakfast, drinking water, and having meals accompanied by parents or
legal guardians. All prevalence estimates were presented proportionally, with their respective
95% confidence intervals. The Chi-square test was used to evaluate the differences in healthy
eating habits prevalences according to other variables. The module survey of the Stata program
version 13.0 was used to analyze complex data. RESULTS: We evaluated 74,589 adolescents (72.9% of the eligible students). Of these, 55.2% were
female, average age being 14.6 years (SD = 1.6). Among Brazilian adolescents, approximately half
of them showed healthy eating habits when consuming breakfast, drinking five or more glasses
of water a day, and having meals with parents or legal guardians. All analyzed healthy eating
habits showed statistically significant differences by sex, age, type of school, session of study, or
geographic region. Received: 9 Set 2015
Approved: 4 Nov 2015 How to cite: Barufaldi LA, Abreu
GZ, Oliveira JS, Santos DF,
Fujimori E, Vasconcelos SML et al. ERICA: prevalence of healthy
eating habits among Brazilian
adolescents. Rev Saude Publica. 2016;50(suppl 1):6s. CONCLUSIONS: We suggest that specific actions of intersectoral approach are implemented for
the dissemination of the benefits of healthy eating habits. Older female adolescents (15 to 17 years
old) who studied in public schools, resided in the Southeast region, and whose mothers had
lower education levels, should be the focus of these actions since they present lower frequencies
concerning the evaluated healthy habits. Copyright: This is an open-access
article distributed under the
terms of the Creative Commons
Attribution License, which permits
unrestricted use, distribution, and
reproduction in any medium,
provided that the original author
and source are credited. DESCRIPTORS: Adolescent. Food Habits. Food Preferences. Health Behavior. Cross-Sectional
Studies. 1s
DOI:10.1590/S01518-8787.2016050006678 1s 1
DOI:10.1590/S01518-8787.2016050006678 1s Healthy eating habits among adolescents
Barufaldi LA et al INTRODUCTION METHODS This is an analysis of data from the Study of Cardiovascular Risks in Adolescents (ERICA). The ERICA is a cross-sectional, national and school-based study that aimed to estimate the
prevalence of diabetes mellitus, obesity, cardiovascular risk factors, and inflammatory and
insulin resistance markers in adolescents aged from 12 to 17 years, enrolled in public and
private schools of Brazilian municipalities with more than 100,000 inhabitants. 2s Healthy eating habits among adolescents
Barufaldi LA et al Data collection took place between March 2013 and December 2014. About 74,589 adolescents
of 1,247 schools from 124 Brazilian municipalities were evaluated. The population under
research was stratified in 32 strata comprising 27 capitals and five sets of municipalities with
more than 100,000 inhabitants in each of the five geographical macro-regions of the Country. For each geographic stratum, schools were selected with probability proportional to size
and inversely proportional to the distance from the capital. The sample is representative
for medium and large municipalities (> 100,000 inhabitants) at national and regional level,
and also for all Brazilian capitals. More details on the sampling process can be found in the
publication by Vasconcellos et al.25 Data collection took place between March 2013 and December 2014. About 74,589 adolescents
of 1,247 schools from 124 Brazilian municipalities were evaluated. The population under
research was stratified in 32 strata comprising 27 capitals and five sets of municipalities with
more than 100,000 inhabitants in each of the five geographical macro-regions of the Country. For each geographic stratum, schools were selected with probability proportional to size
and inversely proportional to the distance from the capital. The sample is representative
for medium and large municipalities (> 100,000 inhabitants) at national and regional level,
and also for all Brazilian capitals. More details on the sampling process can be found in the
publication by Vasconcellos et al.25 All students of the selected classes that signed the assent form were interviewed and
examined. Adolescents that did not belong to the age group of 12 to 17 years, pregnant
girls, and individuals with physical or mental disabilities (temporary or permanent) were
considered not eligible and were excluded from the analysis. This study was approved by the
Research Ethics Committee of the central coordinating institution of study (IESC/UFRJ) and
of each Brazilian State. Bloch et al.3 presents details concerning the protocol of this study. METHODS The self-administered questionnaire used was applied with the use of an electronic data
collector, the personal digital assistant (PDA). It contained approximately 100 questions
divided into 11 sections: sociodemographic aspects, occupational activities, physical practices,
eating habits, smoking habits, use of alcoholic beverages, reproductive health, oral health,
referred morbidity, sleeping hours, and common mental disorders. The following eating habits were considered healthy: consuming breakfast, drinking water,
and having meals with parents or legal guardians. The section about eating habits included
questions on breakfast and on the company of parents or legal guardians during meals as
lunch and dinner, with the following answer options: “no”, “sometimes”, “almost every day”
and “every day”. For the analysis, we grouped the responses to “almost every day” and “every
day”, thus obtaining a variable with the options: “I do not consume it”; “sometimes I consume
it” and “I consume it almost/every day”. We created the variable “meals with parents or legal guardians” by combining the variables
“I never have lunch or dinner with my parents or legal guardians” presenting the following
answer options: “I never have meals with my parents or legal guardians”; “sometimes I have
one or two meals with my parents or legal guardians” and “I often (or always) have at least
one meal with my parents or legal guardians”. Another habit evaluated in the same questionnaire was daily water intake, by considering
as answer options: “I do not drink water”, “I drink from one to two glasses”, “I drink from
three to four glasses” and “I drink at least five glasses per day”. The prevalence of healthy habits related to food was analyzed according to the following
variables: geographic region (North, Northeast, Midwest, Southeast, and South), sex (male or
female), age (in years, analyzed categorically: from 12 and 14, and from 15 to 17), education
level of the mother (analyzed categorically: incomplete high school, complete high school or
more), session of study (morning or afternoon sessions), and type of school (public or private). Data analyses were performed in the Strata software, version 13.0, using the module survey for the
analysis of complex sample data, in which prevalence estimates were presented proportionally
(%), with their respective 95% confidence intervals (95%CI). The Chi-square test was used to
evaluate the differences in healthy eating habits prevalences according to other variables. 3s
DOI:10.1590/S01518-8787.2016050006678 RESULTS Of the 102,327 eligible students, we evaluated about 74,589, which represent 72.9% of the
eligible students enrolled in schools and classes previously selected during the sampling
process. The study coverage was higher in females (75.8%) than in males (69.6%), and also Of the 102,327 eligible students, we evaluated about 74,589, which represent 72.9% of the
eligible students enrolled in schools and classes previously selected during the sampling
process. The study coverage was higher in females (75.8%) than in males (69.6%), and also 3s Healthy eating habits among adolescents
Barufaldi LA et al higher in younger adolescents (77.6%) when compared to those of a higher age group (66.4%). The geographic region where coverage was the highest was the South (81.0%), while the
region with the smallest coverage was the Midwest (68.3%). Of the evaluated adolescents,
55.2% were female with average age being 14.6 years (SD = 1.6). Regarding the type of school,
78.7% were public and 71.5% of the students studied in the morning session. About 31.0%
of the adolescents evaluated resided in the Northeast, 22.9% in the Southeast, 20.2% in the
North, 13.0% in the Midwest, and 12.8% in the South. More than two-thirds of adolescents (68.0%) “often (or always)” had meals with their parents
or legal guardians. However, almost 25.0% had meals in the company of their parents or legal
guardians only “sometimes”, while 7.4% declared to “never” do it (Table 1). The prevalence
of such habit presented statistically significant difference for all the variables studied,
except for study session. We observed higher prevalences of consumption of meals in the
presence of parents or legal guardians among younger male adolescents that studied in
private schools, resided in the South and Midwest regions, and whose mothers had higher
education level (Table 1). Table 1. Prevalence of meals with their parents or legal guardians in Brazilian adolescents aged between
12 and 17 years, considering complex sampling. ERICA, 2013-2014. * Statistically significant difference – Chi-square test. RESULTS (N = 74,589)
Variable
Prevalences
Never
Sometimes
Often/always
%
95%CI
%
95%CI
%
95%CI
Sex*
Female
7.9
7.3-8.5
26.7
25.6-27.8
65.4
64.2-66.6
Male
6.9
6.3-7.6
22.4
21.5-23.5
70.6
69.3-71.9
Age (years)*
12-14
5.7
5.1-6.4
21.6
20.7-22.5
72.7
71.3-74.0
15-17
9.3
8.6-9.9
27.9
26.8-28.9
62.9
61.6-64.1
Education level of the mother
Incomplete high
school
7.5
6.6-8.5
25.5
24.1-27.0
67.0
64.9-69.0
Complete high school
or more
7.2
6.4-7.9
22.4
21.2-23.7
70.5
68.8-72.0
Type of school*
Public
7.5
7.0-8.1
25.3
24.5-26.1
67.2
66.1-68.2
Private
6.7
5.3-8.4
21.1
19.2-23.2
72.2
69.1-75.1
Session of study
Morning
7.4
6.8-8.1
24.6
23.7-25.7
67.9
66.5-69.3
Afternoon
7.3
6.6-8.1
24.3
22.9-25.8
68.3
66.6-70.0
Geographical region*
North
6.9
6.4-7.4
25.3
24.3-26.3
67.8
66.7-68.9
Northeast
8.6
7.7-9.5
29.2
27.8-30.6
62.3
60.6-63.9
Midwest
7.0
6.1-8.1
21.2
19.8-22.5
71.8
70.2-73.1
Southeast
7.4
6.6-8.3
24.8
23.5-26.2
67.8
65.9-69.6
South
5.8
4.9-6.7
16.9
15.6-18.2
77.3
75.9-78.6
Total
7.4
6.9-7.9
24.6
23.8-25.3
68.0
67.0-69.0
* Statistically significant difference – Chi-square test. Table 1. Prevalence of meals with their parents or legal guardians in Brazilian adolescents aged between
12 and 17 years, considering complex sampling. ERICA, 2013-2014. (N = 74,589) Healthy eating habits among adolescents
Barufaldi LA et al As for the consumption of breakfast, nearly half of the adolescents (48.5%) reported
consuming breakfast frequently (or always), but more than one-fifth (21.9%) usually do not
eat this meal (Table 2). We observed a statistically significant difference in the prevalence
of breakfast consumption for all the variables analyzed: the greatest prevalences were
among younger male adolescents (12 to 14 years), who studied in private schools during
the afternoon session, resided in the North and Northeast regions, and whose mothers
had higher education (Table 2). Regarding water intake, about half of the adolescents (48.2%) reported drinking five or
more glasses of water per day, while 18.9% only drink from one to two glasses, and 1.6%
reported not drinking water at all (Table 3). Prevalences of such habit presented significant
differences according to sex, age, and geographic region. Younger adolescent boys residing
in the Northern region presented the habit of consuming more water daily. Table 2. Prevalence of breakfast consumption in Brazilian adolescents aged between 12 and 17 years,
considering complex sampling. ERICA, 2013-2014. RESULTS (N = 74,589)
Variable
Prevalence
Never
Sometimes
Often/Always
%
95%CI
%
95%CI
%
95%CI
Sex*
Female
25.8
24.5-27.2
31.8
30.7-33.0
42.3
40.6-44.0
Male
18.2
17.0-19.4
27.2
26.2-28.3
54.6
53.1-56.1
Age (years)*
12-14
20.1
18.8-21.6
28.3
27.0-29.5
51.6
50.0-53.1
15-17
24.0
22.5-25.7
30.9
29.6-32.3
45.0
42.7-47.4
Education level of the mother
Incomplete high school
21.9
20.6-23.3
30.3
28.8-31.8
47.8
45.7-49.9
Complete high school or
more
20.9
19.8-21.9
27.7
26.4-29.0
51.5
50.1-52.8
Type of school*
Public
21.9
20.7-23.1
30.9
30.0-31.9
47.2
45.5-48.9
Private
22.5
20.4-24.9
22.8
20.7-25.1
54.6
51.9-57.3
Session of study
Morning
26.4
25.3-27.7
30.3
29.0-31.7
43.2
41.3-45.1
Afternoon
12.2
11.2-13.3
27.7
26.6-28.9
60.0
58.2-61.8
Geographical region*
North
13.7
12.6-15.0
25.5
24.2-26.8
60.8
58.6-62.9
Northeast
18.2
16.3-20.2
27.2
25.7-28.6
54.7
52.5-56.8
Midwest
22.3
20.2-24.6
35.2
32.9-37.5
42.6
40.1-45.1
Southeast
23.2
21.4-25.2
30.0
28.5-31.6
46.7
44.2-49.2
South
29.1
26.9-31.4
30.8
28.6-33.0
40.1
36.7-43.6
Total
21.9
20.9-23.1
29.5
28.6-30.4
48.5
47.0-49.9
* Statistically significant difference – Chi-square Test. Table 2. Prevalence of breakfast consumption in Brazilian adolescents aged between 12 and 17 years,
considering complex sampling. ERICA, 2013-2014. (N = 74,589) Healthy eating habits among adolescents
Barufaldi LA et al Table 3. Prevalence of daily water intake in Brazilian adolescents aged between 12 and 17 years, considering complex sampling. ERICA,
2013-2014. RESULTS (N = 74,589)
Variable
Prevalence
I do not drink water
1 to 2 glasses
3 to 4 glasses
5 or more glasses
%
95%CI
%
95%CI
%
95%CI
%
95%CI
Sex*
Female
2.3
1.9-2.6
24.0
22.8-25.3
33.1
31.9-34.2
40.6
39.2-42.0
Male
0.9
0.7-1.0
13.9
12.8-15.0
29.5
28.2-30.8
55.7
53.9-57.5
Age (years)*
12-14
1.4
1.1-1.6
17.8
16.9-18.6
30.7
29.7-31.7
50.1
48.9-51.4
15-17
1.8
1.5-2.1
20.2
18.9-21.6
31.9
30.6-33.3
46.0
44.2-47.9
Education level of the mother
Incomplete high school
1.5
1.2-1.8
18.9
17.4-20.5
32.0
30.7-33.3
47.6
45.9-49.5
Complete high school or more
1.4
1.2-1.8
17.2
16.1-18.3
30.9
30.5-33.2
49.5
47.8-51.2
Type of school*
Public
1.6
1.4-1.8
19.3
18.3-20.3
30.8
29.8-31.8
48.4
47.0-49.7
Private
1.5
0.99-2.2
17.4
15.6-19.4
33.7
31.7-35.8
47.4
44.1-50.7
Session of study
Morning
1.7
1.4-1.9
19.2
18.2-20.3
31.7
30.6-32.9
47.4
45.9-48.8
Afternoon
1.4
1.1-1.7
18.3
16.9-19.8
30.3
28.8-31.7
50.0
47.8-52.2
Geographical region*
North
0.3
0.2-0.4
8.9
8.3-9.7
25.1
24.1-26.1
65.7
64.6-66.8
Northeast
0.5
0.3-0.6
12.7
11.2-14.3
29.8
27.5-32.2
57.0
53.7-60.3
Midwest
0.9
0.6-1.2
1.9
14.1-17.3
31.0
29.7-32.4
52.5
50.4-54.6
Southeast
1.8
1.5-2.1
21.2
19.8-22.6
33.1
31.7-34.5
43.9
42.1-45.8
South
4.1
3.2-5.2
30.0
27.8-32.3
30.7
28.9-32.6
35.1
33.5-36.8
Total
1.6
1.4-1.8
18.9
18.1-19.8
31.3
30.4-32.2
48.2
47.0-49.4
ll
fi
d ff
h Table 3. Prevalence of daily water intake in Brazilian adolescents aged between 12 and 17 years, considering complex sampling. ERICA,
2013-2014 (N = 74,589) Table 3. Prevalence of daily water intake in Brazilian adolescents aged between 12 and 17 years, considering complex sampling. ERICA,
2013-2014. (N = 74,589) DISCUSSION Among Brazilian adolescents, approximately half of them showed healthy eating habits when
it comes to having meals with their parents or legal guardians, consuming breakfast, and
drinking five or more glasses of water per day. However, a significant percentage reported
never (or only sometimes) having meals with their parents, never consuming breakfast, and
not drinking water or just drinking from one to two glasses daily. All the analyzed healthy eating habits showed statistically significant differences according
to sex, age, type of school, session of study, or geographic region. The results showed that
younger male adolescents who studied in private schools, resided in the South and Midwest
regions, and whose mothers had higher education, had more meals with their parents or
legal guardians. We observed that the greatest prevalences of breakfast consumption, by
its turn, were among younger male adolescents who studied in private schools, during the
afternoon session, resided in the North and Northeast regions of Brazil, and whose mothers 6s
DOI:10.1590/S01518-8787.2016050006678 Healthy eating habits among adolescents
Barufaldi LA et al had higher education level. Water intake was higher among younger male adolescents who
resided in the North region. Having family meals is an important aspect of the familiar environment that promotes healthy
eating habits in adolescence and its maintenance in adulthood. It also contributes to the
reduction of unhealthy eating practices, influencing positively increased consumption of
fruits, vegetables, and dairy products and decreased consumption of sugary drinks7. Despite
not having a consensus on the subject, some records consider family meals as contributors to
the prevention of obesity. Results of the meta-analyses showed that children and adolescents
that have regular family meals were less likely to be overweight7. In addition to healthy eating
habits and obesity prevention, family meals can help to reduce violence, sexual activity,
mental health problems, and self-injury among children and adolescents6,21. They can also
promote positive family interactions, including greater communication, socialization, and
transmission of values and culture10. The frequency of meals with parents or legal guardians
observed in this study (68.0%) is similar to that found by PeNSE12, which found that 62.6% of
adolescents had (at least) lunch or dinner with the mother or legal guardian. DISCUSSION The association
with the variable sex also showed to be similar to the results from PeNSE, which observed
a higher prevalence of this habit among males, but without distinction between public and
private school students12, differently to what we observed in this study. The greatest prevalence of breakfast by male adolescents reaffirms the results found in other
studies9,20. To Keski-Rahkonen et al.9, the smallest prevalence among girls may be due to body
dissatisfaction and the attempt to lose weight; these authors found associations between
having breakfast and having breakfast accompanied by the parents and not having this meal
with low education levels at age 16, smoking habits, frequent use of alcohol beverages, little
exercise and high body mass indices. The inverse relation we found in this study between breakfast and age is similar to the observed
in other studies. Breakfast consumption seems to increase with age when it comes to adults
(between 18 and 60 years)4 and decrease with age in children and adolescents (between four
and 18 years)19. The main excuses that adolescents who do not have breakfast give are: “lack
of time”, “lack of hunger”, “intention of dieting” and “preference for sleeping”24. Adolescents
from private schools and children of mothers with higher education (complete high school or
more) consume breakfast and meals with parents or legal guardians with greater frequency. This indicates that socioeconomic status may be related to these eating habits, i.e., adolescents
from higher socioeconomic levels present healthier behaviors. Although water balance involves complex mechanisms, an adequate supply of water represents
a health practice as important as the adoption of healthy eating (being also considered
part of it)8. Moreover, drinking water can help controlling weight15,23 and the consumption
of sugary drinks; consequently, it prevents problems associated with the consumption of
these drinks, such as dental caries1, obesity5, and type 2 diabetes13. Park et al.17 observed
that 54.0% of high school students from the United States reported drinking water less than
three times a day. The authors also found a significant association between low intakes of
pure water and the following factors: lower consumption of milk, fruits (including 100%
fruit juices), and vegetables; higher consumption of soft drinks, other sugary drinks, and
fast food; and physical inactivity. Despite quantifying water intake differently, the results of
this study suggest that Brazilian adolescents present a healthier level of water intake than
American adolescents. 7s
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e comportamento alimentar entre adolescentes brasileiros: Pesquisa Nacional de
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approach are necessary. They should be implemented to spread the benefits of having meals Healthy eating habits among adolescents
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DOI:10.1590/S01518-8787.2016050006678 8s Healthy eating habits among adolescents
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with higher body adiposity and abdominal obesity in Malaysian school-aged adolescents. PLoS
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with higher body adiposity and abdominal obesity in Malaysian school-aged adolescents. PLoS
ONE. 2013;8(3): e59297. DOI:10.1371/journal.pone.0059297 17. Park S, Blanck HM, Sherry B, Brener N, O’Toole T. Factors associated with low water intake
among US high school students - National Youth Physical Activity and Nutrition Study, 2010. J
Acad Nutr Diet. 2012;112(9):1421-7. DOI:10.1016/j.jand.2012.04.014 18. Popkin B, D’Anci K, Rosenberg I. Water, hydration and health. Nutr Rev. 2010;68(8):439-58. DOI:10.1111/j.1753-4887.2010.00304.x 19. Rampersaud GC, Pereira MA, Girard BL, Adams J, Metzl JD. Breakfast habits, nutritional
status, bodyweight, and academic performance in children and adolescents. J Am Diet Assoc. 2005;105(5):743-60;quiz 61-2. DOI:10.1016/j.jada.2005.02.007 20. Rampersaud GC. Benefits of breakfast for children and adolescents: update
and recommendations for practitioners. Am J Lifestyle Med. 2009;3(2):86-103. DOI:10.1177/1559827608327219 21. Skeer MR, Ballard EL. Are family meals as good for youth as we think they are? A review of
the literature on family meals as they pertain to adolescent risk prevention. J Youth Adolesc. 2013;42(7):943-63. DOI:10.1007/s10964-013-9963-z 22. Sugiyama S, Okuda M, Sasaki S, Kunitsugu I, Hobara T. Breakfast habits among
adolescents and their association with daily energy and fish, vegetable, and fruit intake:
a community-based cross-sectional study. Environ Health Prev Med. 2012;17(5):408-14. DOI:10.1007/s12199-012-0270-1 23. Tate DF, Turner-McGrievy G, Lyons E, Stevens J, Erickson K, Polzien K et al. Replacing caloric
beverages with water or diet beverages for weight loss in adults: main results of the Choose
Healthy Options Consciously Everyday (CHOICE) randomized clinical trial. Am J Clin Nutr. 2012;95(3):555-63. DOI:10.3945/ajcn.111.026278 24. Trancoso SC, Cavalli SB, Proença RPC. Café da manhã: caracterização, consumo e importância
para a saúde. Rev Nutr. 2010;23(5):859-69. DOI:10.1590/S1415-52732010000500016 25. Vasconcellos MTL, Silva PLN, Szklo M, Kuschnir MCC, Klein CH, Abreu GA et al. REFERENCES Desenho
da amostra do Estudo do Risco Cardiovascular em Adolescentes (ERICA). Cad Saude Publica. 2015;31(5):921-30. DOI:10.1590/0102-311X00043214 Funding: Department of Science and Technology of the Secretary of Science, Technology and Strategic Inputs
of the Brazilian Ministry of Health (Decit/SCTIE/MS); Health Sector Fund (CT-Saúde) of the Brazilian Ministry
of Science, Technology and Innovation (MCTI) (FINEP Protocol: 01090421 and CNPq 565037/2010-2 and
405.009/2012-7). The author Santos DF is a fellow of CNPq (Process 444138 / 2014-5). Author’s Contribution: LAB participated in the design and planning of the study, in the analysis and interpretation
of data, and in the drafting and approval of the final version of the manuscript. GAA participated in the design
and planning of the study and of the drafting and approval of the final version of the unpublished study. JSO,
DFS, EF, SMLV, FAGV, and BMT joined the drafting and the approval of the unpublished study. Acknowledgements: To the Brazilian Institute of Geography and Statistics (IBGE) for the disposal of electronic
data collectors, the PDA (personal digital assistant), and to the Fundação Universitária José Bonifácio (FUJB)
for managing the resources of this study. Conflict of Interest: The authors declare no conflict of interest.
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Barcoding Chrysomelidae: a resource for taxonomy and biodiversity conservation in the Mediterranean Region
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http://zoobank.org/4D7CCA18-26C4-47B0-9239-42C5F75E5F42 Citation: Magoga G, Sassi D, Daccordi M, Leonardi C, Mirzaei M, Regalin R, Lozzia G, Montagna M (2016)
Barcoding Chrysomelidae: a resource for taxonomy and biodiversity conservation in the Mediterranean Region. In: Jolivet P, Santiago-Blay J, Schmitt M (Eds) Research on Chrysomelidae 6. ZooKeys 597: 27–38. doi: 10.3897/
zookeys.597.7241 *
these authors contributed equally Copyright Giulia Magoga et al. 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. onservation...
27
Launched to accelerate biodiversity research
A peer-reviewed open-access journal onservation... 27
Launched to accelerate biodiversity research
A peer-reviewed open-access journal Barcoding Chr
ZooKeys 597: 27–38 (2016)
doi: 10.3897/zookeys.597.7241
http://zookeys.pensoft.net Barcoding Chry
ZooKeys 597: 27–38 (2016)
doi: 10.3897/zookeys.597.7241
http://zookeys.pensoft.net esource for taxonomy a
RESEARCH ARTICLE Barcoding Chrysomelidae: a resource for ta
and biodiversity conservation in
the Mediterranean Region Giulia Magoga1,*, Davide Sassi2, Mauro Daccordi3, Carlo Leonardi4,
Mostafa Mirzaei5, Renato Regalin6, Giuseppe Lozzia7, Matteo Montagna7,* Giulia Magoga1,*, Davide Sassi2, Mauro Daccordi3, Carlo Leonardi4,
Mostafa Mirzaei5, Renato Regalin6, Giuseppe Lozzia7, Matteo Montagna7,* 1 Via Ronche di Sopra 21, 31046 Oderzo, Italy 2 Centro di Entomologia Alpina–Università degli Studi di
Milano, Via Celoria 2, 20133 Milano, Italy 3 Museo Civico di Storia Naturale di Verona, lungadige Porta
Vittoria 9, 37129 Verona, Italy 4 Museo di Storia Naturale di Milano, Corso Venezia 55, 20121 Milano, Italy
5 Department of Plant Protection, College of Agriculture and Natural Resources–University of Tehran, Karaj,
Iran 6 Dipartimento di Scienze per gli Alimenti, la Nutrizione e l’Ambiente–Università degli Studi di Milano,
Via Celoria 2, 20133 Milano, Italy 7 Dipartimento di Scienze Agrarie e Ambientali–Università degli Studi di
Milano, Via Celoria 2, 20133 Milano, Italy Corresponding authors: Matteo Montagna (matteo.montagna@unimi.it) Corresponding authors: Matteo Montagna (matteo.montagna@unimi.it) Corresponding authors: Matteo Montagna (matteo.montagna@unimi.it) cademic editor: J. Santiago-Blay | Received 20 November 2015 | Accepted 30 January 2016 | Published 9 June Academic editor: J. Santiago-Blay | Received 20 November 2015 | Accepted 30 January 2016 | Published 9 Ju http://zoobank.org/4D7CCA18-26C4-47B0-9239-42C5F75E5F42 Keywords
f b
l y
eaf beetles, molecular taxonomy, DNA barcoding, Cytochrome c oxidase subunit 1, C-bar project Leaf beetles, molecular taxonomy, DNA barcoding, Cytochrome c oxidase subunit 1, C-bar project Abstracth The Mediterranean Region is one of the world’s biodiversity hot-spots, which is also characterized by high
level of endemism. Approximately 2100 species of leaf beetle (Coleoptera; Chrysomelidae) are known
from this area, a number that increases year after year and represents 5/6% of the known species. These
features, associated with the urgent need to develop a DNA-based species identification approach for a
broad spectrum of leaf beetle species, prompted us to develop a database of nucleotide sequences, with a
solid taxonomic background, for all the Chrysomelidae Latreille, 1802 sensu latu inhabiting the Mediter
ranean region. The Mediterranean Chrysomelidae Barcoding project, which has started in 2009, involves
more than fifty entomologists and molecular biologists from different European countries. Numerous
collecting campaigns have been organized during the first seven years of the project, which led to the col Giulia Magoga et al. / ZooKeys 597: 27–38 (2016) 28 lection of more than 5000 leaf beetle specimens. In addition, during these collecting campaigns two new
allochthonous species for Europe, namely Ophraella communa LeSage, 1986 and Colasposoma dauricum
Mannerheim, 1849, were intercepted and some species new to science were discovered (e.g., Pachybrachis
sassii Montagna, 2011 and Pachybrachis holerorum Montagna et al., 2013). DNA was extracted from
1006 specimens (~13% of the species inhabiting the Mediterranean region) and a total of 910 cox1 gene
sequences were obtained (PCR amplification efficiency of 93.8%). Here we report the list of the bar
coded subfamilies, genera and the number of species for which cox1 gene sequences were obtained; the
metadata associated with each specimen and a list of problematic species for which marker amplification
failed. In addition, the nucleotide divergence within and between species and genera was estimated and
values of intraspecific nucleotide divergence greater than the average have been discussed. Cryptocephalus
quadripunctatus G. A. Olivier, 1808, Cryptocephalus rugicollis G. A. Olivier, 1791 and Exosoma lusitanicum
Linnaeus, 1767) are representatives of these cases. Introduction In the last decades we have witnessed what has been defined as the “taxonomy impedi
ment” (Rodman and Cody 2003) indicating the crisis in taxonomic studies due primar
ily to a shortage of time and taxonomists (Wheeler 2004, Wheeler et al. 2004, Wilson
2004), a situation that is made even more critical due to the decrease in the funding of
natural history studies. The causes of the taxonomy crisis are many and complex, and a
comprehensive analysis of this situation is beyond our purpose (see as example Boero
2001, Tautz et al. 2003). In our view, the causes can be described by the sentence …a
lack of prestige and resources that is crippling the continuing cataloguing of biodiversity (God
fray 2002). If we consider the increased rate of species extinction (Thomas et al. 2004)
amplified by climate change and habitat erosion due to exploitation by human beings the
situation is worsened. A DNA-based strategy, which plays a central role in modern taxo
nomic studies, has been proposed by different authors as a methodology to overcome the
identified problems (Tautz et al. 2002, Tautz et al. 2003, Hebert et al. 2004, Goldstein
and DeSalle 2010) whilst maintaining the importance of a traditional approach mainly
based on morphology. Interestingly, in a survey conducted among Coleopteran taxono
mists, taxonomic initiatives based on DNA have been regarded of potential utility in
solving the “taxonomy impediment”, even if a few consider it absolutely useless (Löbl
2005). Currently, in the scientific world, an agreement on the correct approach to be
adopted has not yet been reached. The “gold standard” for species identification studies
based on molecular markers (e.g. mitochondrial cytochrome oxidase subunit I–cox1, or
the nuclear small ribosomal subunit–SSU 18S rRNA) is to develop sequence databases
used as a reference, beginning with DNA extracted from type and type series specimens
preserved in Museum dry collections. The main problem with this strategy is related
to the conservation status of the old dry specimens; 18th and 19th century specimens
have fragmented DNA (not easily amplified through standard PCR approaches targeting Barcoding Chrysomelidae: a resource for taxonomy and biodiversity conservation... 29 fragments of 500-700 bp) and are often infested by fungal hypha, which contaminate
the insect’s genomic DNA. Even with the advent of high-throughput sequencing tech
nologies to solve the problem of fragmented sequences, the contamination due to fungal
DNA remains. Introduction Developing strategies for the acquisition and storage of molecular data to
address molecular taxonomy purposes, we face another problem, which affects the DNA
sequences deposited in publicly available databases, i.e. the accuracy of specimen identi
fication. In light of these issues, an alternative strategy has been adopted in the Mediter
ranean Chrysomelidae Barcoding project (C-Bar). The aim of the C-Bar project is to
develop a reference database of cox1 gene sequences for all the Chrysomelidae (excluding
Bruchinae Latreille, 1802), the Megalopodidae Latreille, 1802 and the Orsodacnidae
Thomson, 1859 (hereafter indicated as Chrysomelidae or leaf beetles sensu latu – s. l.)
inhabiting the Mediterranean region. The study area of C-Bar includes all the states that
possess coastline on the Mediterranean Sea or territories characterized by Mediterranean-
type habitat plus Romania and Switzerland (Figure 1). Starting from the Catalogue of
Palaearctic Coleoptera (Löbl and Smetana 2010), about 2100 species of Chrysomelidae
s. l. (corresponding to an estimated 5/6% of all described species) are present in this
area. The Mediterranean Region is one of the world’s biodiversity “hot-spots” (Myers et
al. 2000, Cuttelod et al. 2008), which is characterized by exceptional concentrations of
species with high levels of endemism that inhabit one of the most populated areas. The
assumption of high levels of endemic species inhabiting the Mediterranean Region is
also valid for leaf beetles (Biondi et al. 2013, Sassi 2006). Although the Mediterranean
region has been the subject of investigation by generations of entomologists, knowledge
of Chrysomelidae inhabiting this area is far from being fully known. The number of leaf
beetle species new to science described from the Mediterranean region in the last dec
ades, associated with the fact that they are widespread among different genera, confirms
the need to increase the effort in biodiversity-based studies (e.g. Cryptocephalus O.F. Muller, 1764, Chrysolina Motschulsky, 1860, Gonioctena Motschulsky, 1860, Longitar
sus Berthold, 1827, Psylliodes Berthold, 1827, Colaspidea Laporte de Castelnau, 1833;
Bastazo 1997, Biondi 1997, Sassi 2001, Leonardi 2007, Daccordi and Ruffo 2005, Bavi
era 2007, Vela and Bastazo 2012, Zoia 2014). In this project are involved taxonomists, specialized in different leaf beetle clades,
in order to guarantee the accurate specimen identification. Introduction In our view, the adoption of
this strategy is a way to bring together traditional (intended as based on morphology)
and molecular taxonomy in order to tentatively overcome the “taxonomy impedi
ment” (Rodman and Cody 2003).h The purpose of this paper is to report the preliminary results achieved during the
first seven years of the project in order to show the potential of a cooperation between
molecular biologists and traditional taxonomists. In particular, we report: i) the meth
od adopted and issues arisen in the development of the sequence dataset; ii) the list
of subfamilies, genera and the number of species for which cox1 gene sequences were
obtained; iii) the metadata associated with the processed organisms; iv) mean values of
intraspecific and interspecific nucleotide divergence v) the new species described and
the important faunistic findings. Giulia Magoga et al. / ZooKeys 597: 27–38 (2016) 30 Figure 1. Area investigated by the Chrysomelidae Barcoding project. The countries in which were per
formed the collecting campaigns are reported in dark grey. The percentage of the total processed speci
mens is reported for each country. Figure 1. Area investigated by the Chrysomelidae Barcoding project. The countries in which were per
formed the collecting campaigns are reported in dark grey. The percentage of the total processed speci
mens is reported for each country. Specimen collection and identification More than 50 entomologists, from different European Countries, have joined the C-
Bar project and have actively participated in samples collection. During the first seven
years of the project (from 2009 to 2015) numerous collecting campaigns were organ
ized from March to September of each year. The specimens were collected using dif
ferent methods: from the vegetation by sweep net or by beating sheet, and directly by
hand in specific habitats (e.g. under stones or digging the host plant roots). All the
collected specimens were placed in 5 ml vials filled with absolute ethanol in order to
preserve the genomic DNA. Within an hour of specimen collection, the mixture in the
vials was replaced with fresh absolute ethanol in order to obtain better sample dehydra
tion and preservation for long-term storage. Each vial was preserved at -20°C and was
labeled by a unique identifier plus other metadata related to the sampling locality (i.e. Country, Province, Region, exact site, latitude, longitude and elevation), the date of
collection, the collector/s and other ecological information related to the specimens. Specimen manipulation and dissection (when necessary) were completed with the
auxiliary use of a stereomicroscope. Images of the specimen habitus were acquired by
a reflex camera (Canon EOS 450D, macro objective 60 mm or 100 mm with a set of Barcoding Chrysomelidae: a resource for taxonomy and biodiversity conservation... 31 macro extension tubes) or with Axiocam 506 mounted on Zeiss Axio Zoom V16. The
specimens were morphologically identified by Italian taxonomists expert in different
leaf beetle clades (most of them are listed among the authors of the present article). The nomenclature adopted in the C-bar project follows the work of Bouchard et al. (2011) at the levels of family and subfamily, while at the levels of genus and species was
adopted the recently published Catalogue of Palaearctic Coleoptera–Chrysomeloidea
(Löbl and Smetana 2010). DNA extraction, PCRs and sequence quality control Giulia Magoga et al. / ZooKeys 597: 27–38 (2016) 32 DNA extraction, PCRs and sequence quality control DNA extraction was performed in two different ways since it took place in different
laboratories (Biodiversity Institute of Ontario, University of Guelph and Department
of Agricultural and Environmental Sciences, Università degli Studi di Milano): for
950 samples the DNA was extracted from one hind leg while for the 56 remaining
samples the DNA was extracted from the whole specimen, after the removal of the
abdomen. The latter procedure ensures to keep specimen morphology intact. In both
cases, DNA was purified using the Qiagen DNeasy Blood and Tissue Kit (Qiagen,
Hilden, Germany). Here we describe the adopted non-destructive procedure: the
specimen was taken off from absolute ethanol and dried in single 1.5 ml vials for 45
minutes at 30°C; after the removal of the abdomen with the use of sterile pins and
tweezers the specimen was placed in 180 µL of ATL lysis buffer (Qiagen) with 200
ng/mL proteinase K (Sigma Aldrich, St. Louis, MO, USA) at 56°C for 12 hours. The
following steps of the DNA extraction were performed according to the manufac
turer’s instructions of Qiagen DNeasy Blood and Tissue Kit. After DNA extraction,
the specimens were dry mounted on pins together with genitalia and kept for future
reference. A quote of the extracted DNA was preserved in the C-bar DNA library at
-80°C for long term storage and a rate was preserved at -20° in order to perform the
following amplifications. A fragment of 658 bp at the 5’-end of the mitochondrial
cytochrome c oxidase subunit 1 gene (cox1) was amplified with primers LCO1490
5’-GGT CAA CAA ATC ATA AAG ATA TTG G / HCO2198 5’-TAA ACT TCA
GGG TGA CCA AAA AAT CA (Folmer et al. 1994). When this pair of primers
resulted in unsuccessful amplification of the target marker, other primers amplify
ing the same gene region were used, i.e. LepF1 5’-ATT CAA CCA ATC ATA AAG
ATA TTG G / LepR1 5’-TAA ACT TCT GGA TGT CCA AAA AAT CA (Hebert
et al. 2004). Successful amplifications were determined by gel electrophoresis. PCR
products were directly sequenced on both strands using the marker-specific primers
from ABI technology (Applied Biosystems, Foster City, CA, USA). The obtained
sequences were edited using Geneious R8 (Biomatters Ltd., Auckland, New Zealand)
and primers, pseudogenes and contaminations removed. Finally, they were deposited
in the Bold Systems (Ratnasingham and Hebert 2007) and in the European Nucleo
tide Archive (Montagna et al. under revision). Intraspecific and intrageneric nucleotide divergence The obtained cox1 gene sequences were aligned at codon level using MUSCLE (Edgar
2004) with default parameters. A pairwise nucleotide distance matrix was estimated
starting from the aligned sequences implementing the Kimura-two-parameter (K2P)
model (Kimura 1980), considered as an adequate evolutionary nucleotide model when
p-distances between sequences are low (Nei and Kumar 2000). The nucleotide distance
matrix was used for the calculation of the mean intraspecific and interspecific nu
cleotide distances and for the calculation of mean intrageneric distance; these analyses
were performed using the R package Spider (Brown et al. 2012). We also calculated
nucleotide intraspecific distances for some species with a wide range of distribution. Results and discussion Until now, C-Bar collecting campaigns have investigated some areas of Bulgaria, France,
Greece, Italy, Morocco, Romania, Spain, Switzerland, Turkey and Tunisia (Figure 1). The sampling efforts that have been accomplished until now led to the collection of
more than 5000 Chrysomelidae specimens. During the identification process, some
specimens of previously unknown species were recognized, these samples were used
for the description of the following species: Pachybrachis sassii (Montagna 2011) from
the Giglio Island in the Tuscan Archipelago; Pachybrachis holerorum (Montagna et al. 2013) from the Northern Apennines and Oulema mauroi Bezděk & Baselga, 2015,
from Northen Italy. Other samples collected during the C-Bar collecting campaigns
were used in a revision of Colaspidea genus that led to the description of seven new
species (Zoia 2014). All these new taxa were formally described by a traditional mor
phological approach, in some cases molecular data were added to confirm the existence
of the new species. Besides the discovery of new taxa, two allochthonous species new to
Europe, namely Ophraella communa (Boriani et al. 2013) and Colasposoma dauricum
(Montagna et al. in press), were intercepted. O. communa is a leaf beetle of Nearctic
origin accidentally introduced in 1996 in Taiwan (Wang and Chiang 1998) and Japan
(Takizawa et al. 1999); the species rapidly spread in East Asia and few years ago we in
tercepted it in the Northern part of Italy (Boriani et al. 2013). C. dauricum is a species
originally present in the North and Central-East of Asia, it has never been observed
out of its original range until our interception in 2011 in Piedmont (North of Italy). Among the collected samples, the DNA was extracted from 1006 specimens and
PCRs targeting a fragment of the cox1 gene performed. PCRs with the selected primer
pairs lead to successful amplification in 93.8% of the cases (62 specimens failed the
amplification). Among the specimens for which the amplification failed, 43 specimens
belong to the subfamily Cryptocephalinae Gyllenhaal, 1813: 18 species of Crypto
cephalus (40 specimens); interestingly cox1 sequences have never been obtained for
Cryptocephalus therondi Franz, 1949, Cryptocephalus cantabricus Franz, 1958 and Cryp
tocephalus etruscus Sassi, 1995. We can hypothesize the presence of mutations in the Barcoding Chrysomelidae: a resource for taxonomy and biodiversity conservation... 33 annealing region of the used primers. Results and discussion Sequences obtained from Clytra laeviuscula Rat
zeburg, 1837, Clytra quadripunctata Linnaeus, 1758, Cryptocephalus cristula Dufour,
1843, Cryptocephalus octoguttatus Linnaeus, 1767, Lachnaia tristigma Lacordaire, 1848
and Oomorphus concolor Sturm, 1807 did not possess an open reading frame and were
thus considered as nuclear pseudogenes. Twenty-seven sequences were discarded be
cause of contamination from exogenous DNA. A total of 910 cox1 sequences (267
species corresponding to ~13% of those inhabiting the Mediterranean region) were
obtained, the size of the sequences was > 400 bp in ~99% of the cases. q
p
We observed that only two species, namely Cryptocephalus violaceus Laicharting,
1781 and Cryptocephalus duplicatus Suffrian, 1845, sharing the same haplotype can
not be discriminated through DNA barcoding. In this and in similar cases a barcod
ing failure can be confirmed only ensuring the correct identification of the samples by
expert taxonomists. Therefore 99.3% of the species (265) for which we obtained cox1
sequences possessed unique haplotypes, allowing their molecular identification. The
mean intraspecific nucleotide distance value is of 2%, while the mean interspecific
and intrageneric distances result of, respectively, 25.2% and of 19.8%. The obtained
intraspecific value are higher than that inferred in a previous study on Coleoptera
(Pentinsaari et al. 2014). This results might be the effect of geographical distances
among localities of collection of co-specific specimens; a possible alternative explana
tion is the presence of cryptic species. Among the species showing high intraspecific
nucleotide distance noteworthy are the cases of Cryptocephalus rugicollis (2.8% [0%,
5.5%]), Exosoma lusitanicum (6.7% [0.2%, 9.2%]) and Cryptocephalus quadripuncta
tus that shows a mean intraspecific distance (3% [0%, 4.9%]). To test the formulated
hypotheses further analyses, including the use of other mitochondrial and nuclear
markers as well as a wider sample of specimens, are required. Among the nine subfamilies for which cox1 sequences were obtained (Table 1),
Cryptocephalinae and Galerucinae Latreille, 1802 were better represented. In the first
subfamily are listed 111 species (83 species of Cryptocephalini Gyllenhaal, 1813 and 28
of Clytrini Lacordaire, 1848, 426 specimens in total) while the second counts 88 species
(24 species of Galerucini Latreille, 1802 and 64 of Alticini Spinola, 1844, 274 specimens
in total). Results and discussion The unbalanced sampling towards Cryptocephalini, which in some way might
affect the obtained results, could be explained by the fact that most of the C-bar speci
mens have been collected by Sassi and Montagna, which mainly work on this clade and
are likely to have developed collecting strategies that increase their sampling (Figure 1).hi The metadata related to the specimens (i.e., specimen identification, collection
identifier, collecting date, state, province, exact site of collection, latitude, longi
tude, elevation and collector/s) from which cox1 gene sequences were obtained, are
available in a web site dedicated to the project (http://www.c-bar.org). Regarding
the specimens collected within Italian administrative boundaries the metadata as
sociated with the specimens are also available in the Biodiversity Database and GIS
platform of the Italian National Network of Biodiversity. These faunistic data are
useful because increase the awareness of species presence and distribution in the
sampled area. Giulia Magoga et al. / ZooKeys 597: 27–38 (2016) 34 Table 1. List of the barcoded subfamilies and genera with the number of species and specimens belonging
to each taxon. to each taxon. Results and discussion Subfamily
Genus
Ns
a
bNspec Nb
Zeugophorinae Böving and Craighead, 1931
Zeugophora Kunze, 1818
1
1
1
Orsodacninae Thomson, 1859
Orsodacne Latreille, 1802
3
7
2.3
Donacinae Kirby, 1837
Donacia Fabricius, 1775
2
6
3
Criocerinae Latreille, 1804
Crioceris Muller, 1764
3
18
3
Lilioceris Reitter, 1912
1
Lema Fabricius, 1798
1
Oulema Gozis, 1886
1
Cassidinae Gyllenhal, 1813
Cassida Linnaeus, 1758
14
61
3.4
Hypocassida Weise, 1893
2
Hispa Linnaeus, 1767
1
Dicladispa Gestro, 1897
1
Chrysomelinae Latreille, 1802
Chrysolina Motschulsky, 1860
13
117
3.4
Chrysomela Linnaeus, 1758
3
Entomoscelis Chevrolat, 1836
1
Gastrophysa Chevrolat, 1836
1
Gonioctena Motschulsky, 1860
3
Oreina Chevrolat, 1836
6
Plagiosterna Motschulsky, 1860
1
Phratora Chevrolat, 1836
1
Plagiodera Chevrolat, 1836
1
Prasocuris Latreille, 1802
1
Timarcha Latreille, 1829
3
Galerucinae Latreille, 1802
Agelastica Chevrolat, 1836
1
274
3.1
Arima Chapuis, 1875
1
Calomicrus Stephens, 1831
3
Exosoma Jacoby, 1903
2
Diabrotica Chevrolat, 1836
1
Galeruca Geoffroy, 1762
5
Galerucella Crotch, 1873
3
Lochmaea Weise, 1883
2
Luperus Geoffroy, 1762
6
Nymphius Weise, 1900
2
Sermylassa Reitter, 1913
1
Altica Muller, 1764
4
Aphthona Chevrolat, 1842
6
Argopus Fischer von Waldheim, 1824
1
Arrhenocoela Foudras, 1860
1
Chaetocnema Stephens, 1831
2
Crepidodera Chevrolat, 1836
5
Derocrepis Weise, 1886
2
Dibolia Latreille, 1829
2
Epitrix Foudras, 1860
1
Hermaeophaga Foudras, 1860
1
Hippuriphila Foudras, 1860
1 Galerucinae Latreille, 1802 Barcoding Chrysomelidae: a resource for taxonomy and biodiversity conservation... 35 Subfamily
Genus
Ns
a
bNspec Nb
Longitarsus Berthold, 1827
9
Lythraria Bedel, 1897
1
Neocrepidodera Heikertinger, 1911
6
Phyllotreta Chevrolat, 1836
4
Podagrica Chevrolat, 1836
1
Psylliodes Berthold, 1827
12
Sphaeroderma Stephens, 1831
2
Cryptocephalinae Gyllenhal, 1813
Cryptocephalus Geoffroy, 1762
73
426
3.8
Pachybarchis Chevrolat, 1836
8
Stylosomus Suffrian, 1848
2
Clytra Laicharting, 1781
4
Coptocephala Chevrolat, 1836
3
Labidostomis Chevrolat, 1836
10
Lachnaia Chevrolat, 1836
3
Macrolenes Chevrolat, 1836
1
Smaragdina Chevrolat, 1836
7
Tituboea Lacordaire, 1848
1
Eumolpinae Hope, 1840
Chrysochus Chevrolat, 1836
1
5
1.7
Colaspidea Laporte de Castelnau, 1833
1
Macrocoma Chapuis, 1874
1
aNs indicates the number of barcoded species; bNspec and N indicates respectively the total number and the
average number of barcoded specimens belonging to each subfamily aNs indicates the number of barcoded species; bNspec and N indicates respectively the total number and the
average number of barcoded specimens belonging to each subfamily Conclusion In this paper, we report that C-Bar project, besides having produced useful data for
molecular taxonomy (cox1 sequences were obtained for about 13% of the species in
habiting the investigated area), has obtained important results also from the viewpoint
of the classical taxonomy leading to the morphological description of same new species
of Chrysomelidae. A further important achievement has been the interception of al
lochthonous species. These results have been obtained only thanks to the cooperation
amongst the taxonomists specialized in different leaf beetle clades, which have ensured
the correct identification of samples, the people involved in the extensive collecting
campaigns and the molecular biologists. g
g
The promising preliminary results that have been obtained encourage us to contin
ue with this project since they strongly confirm the urgent need to increase the efforts in
faunistic studies to uncover the real biodiversity of leaf beetles inhabiting the Mediter
ranean region. For these reasons, we are confident that the aim of C-bar project of de
veloping a repository of cox1 sequences for the majority of the species of Chrysomelidae
s. l. inhabiting the Mediterranean region may be achieved in the near future.i In conclusion, as demonstrated by the relevant results obtained during the first
years of the project, we believe that DNA barcoding projects, when developed with
the participation of taxonomists and molecular biologists, represent an opportunity Giulia Magoga et al. / ZooKeys 597: 27–38 (2016) 36 to bring together two different worlds and may be considered the driving force able
to revive interest in what can be regarded as the milestone of biological studies that is
a-taxonomy, helping to fill the “taxonomy impediment”. Acknowledgements The Authors would like to thank Dr. Stefano Zoia for the work performed and pre
cious suggestions. In addition, special thanks go to all those involved in the initiative
and have helped in sample collection across the Mediterranean region (http://www.c-
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Cultural background modulates how we look at other persons’ gaze
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International journal of behavioral development
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Introduction can we study the effect of postnatal environment on the develop-
ment of face gaze in humans? One of the most promising ways is
a cross-cultural comparison, because different cultural norms
would systematically modulate how the people in each culture
would learn to process and interact with others in face-to-face
communication. A brief look at another person’s face would tell you a lot about who
they are (e.g., identity, age, gender, ethnicity, health and attractive-
ness). Faces are also the ‘‘window to the soul’’, because facial
expressions signal their emotional states, and gaze direction would
tell you what they see and what they know. Having such signifi-
cance in human social communication, it is not surprising that faces
attract attention from very early in infancy. Even newborns prefer-
entially orient to faces (Farroni et al., 2005; Johnson, Dziurawiec,
Ellis, & Morton, 1991), especially those with direct gaze (Farroni,
Csibra, Simion, & Johnson, 2002). Eye-tracking studies have
demonstrated that infants start to show adult-like face-scanning
behaviour, such as preferential fixations on the eyes and mouth
(Yarbus, 1967), from as early as 6 weeks after birth (Hunnius &
Geuze, 2004). Atypical patterns of face scanning behaviour
can be found in neurodevelopmental disorders, such as autism
spectrum disorders (ASD), whereby individuals show profound
difficulties in social interaction and communication (Senju &
Johnson, 2009). Although the mechanisms underlying atypical
face-scanning behaviour in ASD is still unclear, it highlights the
potential relationship between face-scanning behaviour and the
development of social skills. Two independent lines of research contrasted face gaze between
Western European/NorthAmericancultureand EasternAsian culture,
and found clear differences in the face gaze between the two cultures. First, a series of studies (McCarthy, Lee, Itakura, & Muir, 2006, 2008)
reported that Canadian participants maintain longer eye contact with
an interviewer than Japanese participants when they answer cogni-
tively demanding questions. In these studies, the gaze direction of the
participants were analysed from the video recording. These studies
clearly show the differential face gaze in realistic face-to-face interac-
tion, which isconsistent with the cultural normsthat gaze avoidance is
perceivedasinsincereinWestern culture,butthesamebehaviourdoes
not have such a negative value in Eastern culture; it can even signal
respectinsomecontexts(Argyle, Henderson,Bond,Iizuka,&Contar-
ello, 1986). However, the video recording does not have a sufficient
spatial resolution to examine which part of the face (eyes, nose or
mouth) the participants look at. Special issue International Journal of
Behavioral Development
37(2) 131–136
ª The Author(s) 2012
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DOI: 10.1177/0165025412465360
ijbd.sagepub.com Keywords y
cross-cultural study, eye contact, eye-tracking, face scanning, gaze processing Atsushi Senju,1 Ange´lina Vernetti,1 Yukiko Kikuchi,2
Hironori Akechi,2 Toshikazu Hasegawa,3 and Mark H. Johnson1 Atsushi Senju,1 Ange´lina Vernetti,1 Yukiko Kikuchi,2
Hironori Akechi,2 Toshikazu Hasegawa,3 and Mark H. Johnson1 Abstract The current study investigated the role of cultural norms on the development of face-scanning. British and Japanese adults’ eye movements
were recorded while they observed avatar faces moving their mouth, and then their eyes toward or away from the participants. British
participants fixated more on the mouth, which contrasts with Japanese participants fixating mainly on the eyes. Moreover, eye fixations of
British participants were less affected by the gaze shift of the avatar than Japanese participants, who shifted their fixation to the correspond-
ing direction of the avatar’s gaze. Results are consistent with the Western cultural norms that value the maintenance of eye contact, and
the Eastern cultural norms that require flexible use of eye contact and gaze aversion. Corresponding author:
Atsushi Senju, Centre for Brain and Cognitive Development, Birkbeck,
University of London, Malet Street, London WC1E 7HX, UK.
Email: a.senju@bbk.ac.uk Introduction All the faces were initially presented with
(e) forward gaze and closed mouth, which was followed by a mouth movement after 1 second, and a gaze shift after another second (i.e., 2 seconds from the
onset of the stimulus). After the gaze shift, the face remained still for another 4 seconds. The orientation of the face was right in half of the stimuli and the left
in the other half. movements were either smile (Figure 1a, 1b) or mouth opening
(Figure
1c,
1d). Eye
movements
were
either
direct
gaze
(Figure 1a, 1c) or averted gaze (Figure 1b, 1d), which involved
rotating both eyes laterally by 25 either towards the centre (direct
gaze) or away from it (averted gaze). The amount of rotation for
direct gaze was selected based on the rating of 10 naı¨ve observers,
who rated the perception of ‘‘directedness’’ of the gaze (Todorovic,
2006). The same amount of rotation in the opposite direction
was used for averted gaze. In total, 32 animations were generated
(4 faces, 2 mouth movements, 2 gaze directions and 2 face
orientations). The faces extended 18.4 13.0 cm on the screen. showed morefixation on the centre of the face(Blais, Jack, Scheepers,
Fiset, & Caldara, 2008; Kelly et al., 2011). It was also suggested that
reduced fixation on the mouth could partly explain cross-cultural dif-
ference in facial expression processing (Jack, Blais, Scheepers,
Schyns, & Caldara, 2009). These studies clearly show the subtle dif-
ferences in face fixations between participants with different cultural
backgrounds,but itisnot clear whether itisspecific tothe contextthey
analyse facial information from static images, or a more general pat-
tern of fixations in a more realistic context where they face dynamic
sequences of facial actions. The current study aimed to bridge these gaps in knowledge by
investigating how cultural background (British or Japanese) affects
the face gaze when they observe dynamic face stimuli. We also
examined whether the gaze direction of face stimuli (looking
toward or away from the observer) would affect cross-cultural dif-
ferences in face gaze. We predicted that British participants would
maintain longer and sustained eye contact and make a triangular
fixation (that is, more fixation on the mouth than Japanese partici-
pants), whereas Japanese participants would show shorter and flex-
ible eye fixations and more central fixations. Introduction Second, another series of studies
recorded eye movements of Western European (British) and Eastern
Asian (mainly Chinese) participants as they processed static images
of faces, and found that Western European participants showed trian-
gular fixation on both eyes and mouth, but Eastern Asian participants An important question about the development of face gaze is the
role of postnatal environment. Several major theories of social
skills development emphasize the role of input from their parents
(or caregiver) as well as those from other members of the society,
which are essential for the infant brain to learn the social world and
become an ‘‘expert’’ (Gauthier & Nelson, 2001; Pascalis et al.,
2005). For example, Sugita (2008) reared infant monkeys with no
exposure to faces, and found that general preference to faces devel-
ops without exposure to faces, but fine discrimination of faces do
not develop. The results highlight the role of innate capacity to
detect face-like shapes, and the role of postnatal learning in shaping
the capacity to recognize individual faces. However, such a control
of postnatal environment is impossible in human studies. So, how 1 Birkbeck, University of London, UK
2 Japan Society for the Promotion of Science, Japan
3 The University of Tokyo, Japan Corresponding author:
Atsushi Senju, Centre for Brain and Cognitive Development, Birkbeck,
University of London, Malet Street, London WC1E 7HX, UK. Email: a.senju@bbk.ac.uk International Journal of Behavioral Development 37(2) 132 Figure 1. Sample of the gaze direction and mouth movement of the stimuli. Note. (a) direct gaze, smile; (b) averted gaze, smile; (c) direct gaze, mouth opening; (d) averted gaze, mouth opening. All the faces were initially presented with
(e) forward gaze and closed mouth, which was followed by a mouth movement after 1 second, and a gaze shift after another second (i.e., 2 seconds from the
onset of the stimulus). After the gaze shift, the face remained still for another 4 seconds. The orientation of the face was right in half of the stimuli and the left
in the other half. Figure 1. Sample of the gaze direction and mouth movement of the stimuli. Note. (a) direct gaze, smile; (b) averted gaze, smile; (c) direct gaze, mouth opening; (d) averted gaze, mouth opening. Introduction We also predicted that
the response to direct and averted gaze would be modulated by the
cultural background because of the different cultural norms on the
use of eye contact, but no further specific predictions have been
made due to the exploratory nature of the manipulation. Two Tobii T120 eye-trackers (Tobii, Stockholm, Sweden),
equipped with an integrated 17-inch display, were used to present
stimuli and record eye-movement in London and in Tokyo. Tobii
Studio software was used to control stimulus presentation and to
analyse the gaze data. Recordings were conducted in a quiet and soundproofed room
within each research institute. Participants were instructed to watch
the movies of the faces. The same experimenter (AS) conducted the
recording in both the UK and Japan, to maintain strictly similar
experimental conditions such as instructions. A 9-point calibration
was conducted using Tobii Studio software before the recording. Recording consisted of two blocks, and each of 32 animations was
presented twice (once in each block), in a randomized order. An
experimenter also sat in the same testing room, out of sight of the
participant, and monitored the recording with Tobii studio
software. Viewing distance was approximately 60–65 cm from
the display. Methods Both groups showed longer
fixations on the front eye in the direct gaze condition than in averted
gaze condition. However, only Japanese participants showed differ-
ential fixations on the back eye, with longer fixation in averted gaze
condition (Figure 3). No other contrasts reached significance,
including any contrast in the bridge area. Further analyses were conducted on the seven 1-second bins of
the data, to explore the time-course of the differential fixations
on the four AOIs showing the gaze and cultural background
interactions; front eye, back eye, mouth and centre. Front eye. No effects reached significance for the first, second
and third bins. In the fourth bin, the point right after the gaze shift
of the stimuli, both groups showed longer fixation in direct gaze
condition than in averted gaze condition. Interestingly, this effect
was exaggerated in Japanese participants, who showed even longer
fixation than British participants in response to direct gaze and even
shorter fixation than British participants in the averted gaze condi-
tion (Figure 4a). The same trend remained in the fifth bin, in which
only Japanese participants looked longer in the direct gaze
condition than in the averted gaze condition. No effect reached
significance from the sixth bin. Figure 2. Examples of the area of interest (AOI); front eye; front eye,
back eye, bridge, centre and mouth. The size and the location of the AOI
were constant across different stimuli. Back eye. In the first, second and third bins, the only significant
effects were the differences between groups, with Japanese partici-
pants fixating longer than British participants. From the fourth bin
onward, however, Japanese participants fixated longer than British
participants only in the averted gaze condition, but not in the direct
gaze condition (Figure 4b). analysed with mixed-design ANOVAs to test the effects of cultural
background (British or Japanese) and the sex (male or female) of the
participants, as well as the ethnicity (Caucasian or Eastern Asian),
gender (male or female), gaze direction (direct or averted), mouth
movement (smile or mouth opening) and the AOI (front eye, back
eye, bridge, centre and mouth) of the stimuli. An initial ANOVA was
conducted on the whole 7-second data, which were then followed up
by the analyses of seven 1-second bins of the data. Methods Nineteen British adults (ten females and nine males, mean age
27.98 years) and 22 Japanese adults (11 females and 11 males,
mean age 27.75 years) participated in the study. (Four participants
were not included because of excessive eye tracker data loss, under
70% samples.) British adults were recruited in central London, and
Japanese adults were recruited in central Tokyo. All the participants
have normal or corrected-to-normal acuity. The gaze data were initially processed with Tobii studio
software to calculate the total visit time. Then, we calculated the
fixation duration for each stimulus, for the following areas of inter-
est (AOIs); front eye, back eye, bridge, centre and mouth. Note that
faces are tilted either to the right or to the left, one of the eyes is
always closer to the observer (i.e., Front Eye) than the other eye
(i.e., Back Eye), the latter which is off to the side (Figure 2). The
AOIs were selected based on the findings of relevant literature in
cross-cultural face scanning studies (Blais et al., 2008; Jack et al.,
2009; Kelly et al., 2011). These fixation duration data were
extracted from the Tobii Studio for statistical analyses. Four computer-generated faces (one Caucasian female, one
Caucasian male, one Eastern Asian female, one Eastern Asian
male) were selected from the library of Poser 7 (Smith Micro
Software, Aliso Viejo, CA), and were used to create 7-second ani-
mations with the same software. All the animations started with a
face presented upright, facing 30 to the left or to the right and gaz-
ing forward, followed by a mouth movement (1 second after the
start) and an eye movement (2 seconds after the start). Mouth The gaze data for different head orientations and the blocks were
averaged together. The visiting duration for each AOI were then
divided with the total visiting duration of the whole face, to calculate
the relative visiting duration. The relative visiting duration was 133 Senju et al. Figure 2. Examples of the area of interest (AOI); front eye; front eye,
back eye, bridge, centre and mouth. The size and the location of the AOI
were constant across different stimuli. showed longer fixation on the centre, which was only significant
in the averted gaze condition (Figure 3). The two groups showed similarities and differences in the
response to different gaze directions. Methods For the significant
interactions, post-hoc analyses were conducted on each contrast with
Wilcoxon sign rank tests with the Bonferroni correction for multiple
testing, to provide robust statistics. Mouth. The group difference in mouth fixation remained from
the first to the fourth bin (Figure 4e), which then became only
significant in averted gaze condition in the fifth bin and
non-significant from the sixth bin. Centre. The group differences were significant in the fourth and
fifth bins, only in averted gaze condition (Figure 4d). Discussion contrast, we even found a longer fixation in British participants
on the centre in averted gaze condition (Figure 4d), which hap-
pened later than the increased mouth fixation (Figure 4e) and is
possibly explained by the residual effects of fixations on the
mouth. It might suggest that the increased central fixation on the
face in the Eastern Asian population is specific to the task which
requires perceptual analyses of the faces, such as the recognition
and categorization tasks used in the previous studies (Blais et al.,
2008; Kelly et al., 2011), and does not happen in passive viewing. Another possibility is that Eastern Asians look more to the eyes
when the face is expressive, but fixate centrally when they
observe faces with neutral expression. Moreover, Japanese partici-
pants did not show shorter eye fixation than British participants,
which is consistent with some studies (e.g., Jack et al., 2009) but
not with others (McCarthy et al., 2006, 2008). Further studies will
be required to test whether the shorter face gaze could be observed
in Eastern Asian participants in more naturalistic settings. contrast, we even found a longer fixation in British participants
on the centre in averted gaze condition (Figure 4d), which hap-
pened later than the increased mouth fixation (Figure 4e) and is
possibly explained by the residual effects of fixations on the
mouth. It might suggest that the increased central fixation on the
face in the Eastern Asian population is specific to the task which
requires perceptual analyses of the faces, such as the recognition
and categorization tasks used in the previous studies (Blais et al.,
2008; Kelly et al., 2011), and does not happen in passive viewing. Another possibility is that Eastern Asians look more to the eyes
when the face is expressive, but fixate centrally when they
observe faces with neutral expression. Moreover, Japanese partici-
pants did not show shorter eye fixation than British participants,
which is consistent with some studies (e.g., Jack et al., 2009) but
not with others (McCarthy et al., 2006, 2008). Further studies will
be required to test whether the shorter face gaze could be observed
in Eastern Asian participants in more naturalistic settings. contrast, the fixation on the back eye was not affected by the gaze
direction in British participants. Discussion As predicted, the interaction between the cultural background, gaze
direction and the AOI was significant, F(4, 148) ¼ 4.684, p < .01,
Zp
2 ¼ 0.11, demonstrating that British and Japanese participants
fixated differently to the face, depending on whether the face was
with direct or averted gaze. The effect was modulated by the sex,
F(4, 148) ¼ 4.142, p < .01, Zp
2 ¼ 0.10 (see also the supplementary
material), but not with other factors such as the mouth movement,
the gender or the ethnicity of the face stimuli. These interactions
remained significant when we excluded British participants of
non-Caucasian ethnic origin (three females), and when we excluded
British participants who had stayed two years in East Asian coun-
tries (two males, one in China and the other in Thailand) and East
Asian participants who had stayed 6 months in the USA (two
males). The current study is the first to investigate how the cultural back-
ground of the observer affects the face gaze when they observe
dynamic faces looking toward or away from the observer. The
results clearly demonstrate the cultural difference between British
and Japanese participants in the way they look at different parts
of the faces, and how they respond to different gaze direction. First, British participants fixated more on the mouth than did
Japanese participants, replicating previous studies using static
images of faces (Blais et al., 2008; Jack et al., 2009; Kelly
et al., 2011). Second, both groups of participants fixated equally
long on the eye closer to the observer, but Japanese fixated longer
on the other eye than British participants. It is consistent with the
finding that Eastern Asian participants fixate longer on the eyes
(Jack et al., 2009) but it is a novel finding because to our knowl-
edge, this study is the first to use head-turned faces. On the other
hand, we did not replicate the longer fixation on the central parts
of the faces in Eastern Asian participants, which is reported in
previous studies (Blais et al., 2008; Kelly et al., 2011). By Follow-up analyses revealed that Japanese participants fixated
longer on the back eye than did British participants, in both direct
gaze and averted gaze conditions. British participants, by contrast,
fixated longer on the mouth than Japanese participants, in both
direct gaze and averted gaze conditions. Discussion British participants also International Journal of Behavioral Development 37(2) 134 0
0.1
0.2
0.3
0.4
0.5
0.6
Front eye
0
0.1
0.2
0.3
0.4
0.5
0.6
Back eye
British / Direct
Japanese / Direct
British / Averted
Japanese / Averted
0
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Bridge
0
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0
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Centre
*
*
*
*
*
*
*
*
Figure 3. Relative visiting duration on each AOI during the entire period of stimulus presentation, for each cultural background of the participants and the
gaze direction of stimuli. Note. * p < .05 (corrected); error bar: standard error. 0
0.1
0.2
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0.6
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0
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*
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*
*
* 0
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0
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British / Direct
Japanese / Direct
British / Averted
Japanese / Averted
*
*
*
*
* Back eye British / Direct
Japanese / Direct
British / Averted
Japanese / Averted Japanese / Averted 0
0.1
0.2
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Bridge
0
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0
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Centre
*
*
*
Figure 3. Relative visiting duration on each AOI during the entire period of stimulus presentation, for each cultural background of the participants and the
gaze direction of stimuli. Note. * p < .05 (corrected); error bar: standard error. 0
0.1
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0
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Centre
*
*
*
Figure 3. Relative visiting duration on each AOI during the entire period of stimulus presentation, for each cultural background of the participants and t Figure 3. Relative visiting duration on each AOI during the entire period of stimulus presentation, for each cultural background of the participants and the
gaze direction of stimuli. N
*
05 (
d)
b
d
d Figure 3. Relative visiting duration on each AOI during the entire period of stimulus presentation, for each cultural background of the participants and the
gaze direction of stimuli. Note. * p < .05 (corrected); error bar: standard error. Discussion The results showed that Japanese
participants shifted their own fixation to the corresponding direc-
tion of the observed gaze shift (i.e., to the front, medial eye in
response to direct gaze and to the back, lateral eye in response to
averted gaze), as if they ‘‘followed’’ the direction of the face gaze. British participants did not show such a change of fixation follow-
ing observed gaze shift. It might suggest that the eye fixation of
British participants reflects the cultural expectation to maintain eye
contact, but the eye fixation of Japanese participants reflects the
cultural norm to conform to others’ behaviour. Note that it is not
a general difference in the sensitivity to facial motion, because
mouth movements did not exaggerate or diminish the cultural
differences in mouth fixation. The effect was more prominent in
males than in females (see the Supplementary material), suggesting
that the effect of cultural norm on face and gaze processing
manifests more strongly in males. Further studies will be required
to see how the differences in gender-related cultural norm interact
with face-scanning behaviour. Interestingly, these two groups responded differently to the gaze
shift of the stimuli. Second-by-second analyses revealed that both
groups of participants fixated longer on the front eye immediately
(that is, around 1 second) after they saw the gaze shift toward them,
but such a change was more exaggerated and lasted longer with
Japanese participants than British participants. Moreover, the initial
cultural difference in the fixation on the back eye was overridden by
the effect of gaze shift, in which Japanese participants fixated lon-
ger on the back eye only when they saw an averted gaze. By The current study clearly demonstrates the cultural differences in
face gaze in adults, but it cannot tell us how it develops. For example,
Kelly et al. (2011) demonstrated that 70% of British-born Chinese
adults show a face fixation pattern similar to Eastern culture,
whereas 30% of them show a Western pattern of face fixation. Discussion This
study suggests that cultural diversity in face fixation is more
strongly affected by early familial environment, but could also be affected by societal environment (e g
peers) in some individuals
stimuli) which contrasts with previous infant research (Liu et al
0
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7
0
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*
*
*
*
*
*
*
*
*
*
*
*
*
*
0
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(b)
(a)
(d)
(c)
0
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*
*
*
*
*
*
*
*
(e)
British / Direct
Japanese / Direct
British / Averted
Japanese / Averted
Figure 4. Relative visiting duration on each AOI for each 1-second bin of stimulus presentation, for each cultural background of the participants and the
gaze direction of stimuli. Note. (a) front eye, (b) back eye, (c) bridge, (d) centre and (e) mouth. The mouth movement occurred during the second bin, and the eye movement
occurred during the third bin. * p < .05 (corrected); error bar: standard error. Senju et al. 135 135 Senju et al. 0
0.1
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*
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(a)
(d)
(c)
0
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0 4
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0.6
(e)
Senju et al. 135 0
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0
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*
*
*
*
*
*
*
*
*
*
*
*
*
*
0
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0.6
(b)
(a)
(d)
(c)
0
0.1
0.2
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0.4
0.5
0.6 Figure 4. Discussion Relative visiting duration on each AOI for each 1-second bin of stimulus presentation, for each cultural background of the participants and the
gaze direction of stimuli. Note. (a) front eye, (b) back eye, (c) bridge, (d) centre and (e) mouth. The mouth movement occurred during the second bin, and the eye movement
occurred during the third bin. * p < .05 (corrected); error bar: standard error. Figure 4. Relative visiting duration on each AOI for each 1-second bin of stimulus presentation, for each cultural background of the participants and the
gaze direction of stimuli. Note. (a) front eye, (b) back eye, (c) bridge, (d) centre and (e) mouth. The mouth movement occurred during the second bin, and the eye movement
occurred during the third bin. * p < .05 (corrected); error bar: standard error. stimuli), which contrasts with previous infant research (Liu et al.,
2011; Wheeler et al., 2011). Future developmental studies will be
essential to assess the role of face familiarity on face scanning
throughout the course of development. We also need to examine
how the gender difference found in the current study (that is, larger
cross-cultural differences in male than in female participants)
develops, by testing younger populations. affected by societal environment (e.g., peers) in some individuals. Further studies will be necessary to study the time-course of the
emergence of cultural diversity in face gaze early in the develop-
ment. Moreover, as in previous adult studies (Blais et al., 2008;
Jack et al., 2009; Kelly et al., 2011), we did not replicate the other
race effect on face scanning (that is, the significant interaction
between the ethnicity of the participants and the ethnicity of the International Journal of Behavioral Development 37(2) 136 Hunnius, S., & Geuze, R. H. (2004). Developmental changes in visual
scanning of dynamic faces and abstract stimuli in infants: A
longitudinal study. Infancy, 6, 231–255. To summarize, the current study revealed that the cultural
background of the participant affects how they look at another
person’s eyes and mouth, and how they modulate eye fixation in
response to the gaze direction of others. These differences are
consistent with the culturally-relevant strategy of perceptual
analyses, as well as the cultural norms on the use of eye contact
in face-to-face communication. Discussion These results highlight the new
frontier of the research about how cultural norms can affect
behavioural, cognitive and neural development, which would pro-
vide a great opportunity to study the effect of postnatal environment
on human behavioural and cognitive development. Jack, R. E., Blais, C., Scheepers, C., Schyns, P. G., & Caldara, R. (2009). Cultural confusions show that facial expressions are not uni-
versal. Current Biology, 19, 1543–1548. Johnson, M. H., Dziurawiec, S., Ellis, H., & Morton, J. (1991). New-
borns’ preferential tracking of face-like stimuli and its subsequent
decline. Cognition, 40, 1–19. Kelly, D. J., Jack, R.E., Miellet, S., De Luca, E., Foreman, K., &
Caldara, R. (2011). Social experience does not abolish cultural
diversity in eye movements. Frontiers in Psychology, 2: 95. doi:
10.3389/fpsyg.2011.00095 Acknowledgement Liu, S., Quinn, P. C., Wheeler, A., Xiao, N., Ge, L., & Lee, K. (2011). Similarity and difference in the processing of same- and other-race
faces as revealed by eye tracking in 4- to 9-month-olds. Journal of
Experimental Child Psychology, 108, 180–189. We thank Megan Schulossmacher for the help in collecting the data
and Gizelle Anzures for her comments on an earlier version of the
draft. McCarthy, A., Lee, K., Itakura, S., & Muir, D. W. (2006). Cultural
display
rules
drive
eye
gaze
during
thinking. Journal
of
Cross-Cultural Psychology, 37, 717–722. Funding This work has been supported by the UK Medical Research Council
[grant number G1100252; G0701484]; the UK Economic and Social
Research Council [grant number RES-063-27-0207]; the Great
Britain Sasakawa Foundation [grant number #3495]; and the Japan
Society for the Promotion of Science [grant number B16330192]. McCarthy, A., Lee, K., Itakura, S., & Muir, D. W. (2008). Gaze display
when thinking depends on culture and context. Journal of
Cross-Cultural Psychology, 39, 716–729. Pascalis, O., Scott, L. S., Kelly, D. J., Shannon, R. W., Nicholson, E.,
Coleman, M., & Nelson, C. A. (2005). Plasticity of face processing
in infancy. Proceedings of National Academy of Science of the
United States of America, 102, 5297–5300. References Argyle, M., Henderson, M., Bond, M., Iizuka, Y., & Contarello, A. (1986). Cross-cultural variations in relationship rules. International
Journal of Psychology, 21, 287–315. Senju, A., & Johnson, M. H. (2009). Atypical eye contact in autism:
Models, mechanisms and development. Neuroscience & Biobeha-
vioral Reviews, 33, 1204–1214. Blais, C., Jack, R. E., Scheepers, C., Fiset, D., & Caldara, R. (2008). Culture shapes how we look at faces. PLoS ONE, 3, e3022. Sugita, Y. (2008). Face perception in monkeys reared with no exposure
to faces. Proceedings of the National Academy of Sciences of the
United States of America, 105, 394–398. Farroni, T., Csibra, G., Simion, F., & Johnson, M. H. (2002). Eye contact
detection in humans from birth. Proceedings of the National Acad-
emy of Science of the United States of America, 99, 9602–9605. Todorovic, D. (2006). Geometrical basis of perception of gaze
direction. Vision Research, 46, 3549–3562. Farroni, T., Johnson, M. H., Menon, E., Zulian, L., Faraguna, D., &
Csibra, G. (2005). Newborns’ preference for face-relevant stimuli:
Effects of contrast polarity. Proceedings of National Academy of
Science of the United States of America, 102, 17245–17250. Wheeler, A., Anzures, G., Quinn, P. C., Pascalis, O., Omrin, D. S., &
Lee, K. (2011). Caucasian infants scan own- and other-race faces
differently. PLoS ONE, 6(4), e18621. Yarbus, A. (1967). Eye movement and vision. New York, NY:
Plenum. Gauthier, I., & Nelson, C. A. (2001). The development of face exper-
tise. Current Opinion in Neurobiology, 11, 219–224.
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Perception of Nigerian Dùndún Talking Drum Performances as Speech-Like vs. Music-Like: The Role of Familiarity and Acoustic Cues
|
Frontiers in psychology
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cc-by
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ORIGINAL RESEARCH
published: 20 May 2021
doi: 10.3389/fpsyg.2021.652673 ORIGINAL RESEARCH
published: 20 May 2021
doi: 10.3389/fpsyg.2021.652673 ORIGINAL RESEARCH
published: 20 May 2021
doi: 10.3389/fpsyg.2021.652673 Perception of Nigerian Dùndún
Talking Drum Performances as
Speech-Like vs. Music-Like: The
Role of Familiarity and Acoustic Cues
Cecilia Durojaye1,2*†, Lauren Fink1,3†, Tina Roeske1, Melanie Wald-Fuhrmann1,3 and
Pauline Larrouy Maestri3 4 Cecilia Durojaye1,2*†, Lauren Fink1,3†, Tina Roeske1, Melanie Wald-Fuhrmann1,3 and
Pauline Larrouy-Maestri3,4 1 Department of Music, Max Planck Institute for Empirical Aesthetics, Frankfurt am Main, Germany, 2 Department
of Psychology, Arizona State University, Tempe, AZ, United States, 3 Max Planck-NYU, Center for Language, Music,
and Emotion, Frankfurt am Main, Germany, 4 Neuroscience Department, Max Planck Institute for Empirical Aesthetics,
Frankfurt am Main, Germany Keywords: speech surrogacy, Yorùbá, categorization, amplitude modulation spectrum, pitch, intensity, timbre,
timing Edited by:
Laura McPherson,
Dartmouth College, United States It seems trivial to identify sound sequences as music or speech, particularly when the
sequences come from different sound sources, such as an orchestra and a human
voice. Can we also easily distinguish these categories when the sequence comes from
the same sound source? On the basis of which acoustic features? We investigated
these questions by examining listeners’ classification of sound sequences performed
by an instrument intertwining both speech and music: the dùndún talking drum. The
dùndún is commonly used in south-west Nigeria as a musical instrument but is also
perfectly fit for linguistic usage in what has been described as speech surrogates in
Africa. One hundred seven participants from diverse geographical locations (15 different
mother tongues represented) took part in an online experiment. Fifty-one participants
reported being familiar with the dùndún talking drum, 55% of those being speakers of
Yorùbá. During the experiment, participants listened to 30 dùndún samples of about
7s long, performed either as music or Yorùbá speech surrogate (n = 15 each) by a
professional musician, and were asked to classify each sample as music or speech-
like. The classification task revealed the ability of the listeners to identify the samples as
intended by the performer, particularly when they were familiar with the dùndún, though
even unfamiliar participants performed above chance. A logistic regression predicting
participants’ classification of the samples from several acoustic features confirmed the
perceptual relevance of intensity, pitch, timbre, and timing measures and their interaction
with listener familiarity. In all, this study provides empirical evidence supporting the
discriminating role of acoustic features and the modulatory role of familiarity in teasing
apart speech and music. Reviewed by:
Elizabeth Margulis,
Princeton University, United States
Aaron Carter-Enyi,
Morehouse College, United States *Correspondence:
Cecilia Durojaye
cdurojay@asu.edu *Correspondence:
Cecilia Durojaye
cdurojay@asu.edu *Correspondence:
Cecilia Durojaye
cdurojay@asu.edu
†These authors have contributed
equally to this work †These authors have contributed
equally to this work Specialty section:
This article was submitted to
Language Sciences,
a section of the journal
Frontiers in Psychology Received: 12 January 2021
Accepted: 21 April 2021
Published: 20 May 2021 Citation:
Durojaye C, Fink L, Roeske T,
Wald-Fuhrmann M and
Larrouy-Maestri P (2021) Perception
of Nigerian Dùndún Talking Drum
Performances as Speech-Like vs.
Music-Like: The Role of Familiarity
and Acoustic Cues.
Front. Psychol. 12:652673.
doi: 10.3389/fpsyg.2021.652673 INTRODUCTION et al., 2013) are altered as a function of listener familiarity with
a stimulus type. Regarding pitch, Weidema et al. (2016) showed
that the same contours are perceived differently depending on the
context in which they are embedded, with better discrimination
in music than speech context. These studies highlight that the role
of specific acoustic features in shaping perceptual categorization
may, in part, be driven by previous exposure. When we turn on the radio it seems trivial to determine
whether what we are hearing is music or someone speaking. Sound sequences can generally be described in terms of
pitch, timbre, and timing (e.g., Kraus et al., 2009); acoustical
differences have been shown between sequences associated with
music and language domains. For instance, speech typically
comprises many gliding tones and more variation in pitch
trajectory than (Western) music (Patel, 2008), with a temporal
modulation spectrum peaking around 5 Hz, approximating the
syllable rate (Ding et al., 2017). On the other hand, music is
characterized by discrete pitches sustained for longer durations
(Zatorre and Baum, 2012), and a temporal modulation spectrum
peaking around 2 Hz, approximating the average beat rate
(Ding et al., 2017). However, note that such studies often
examine material that has different sound sources, such as the
human voice vs. musical instruments, which might enhance the
distinctive characteristics of the sequences associated with each of
the two categories. Acoustically, sound sequences from music and language
domains can be defined in several ways, with summary statistics
(i.e., mean), as reported earlier, but also in terms of changes over
time. In the case of pitch, for example, speech and music are
rarely monotonous. In speech, the intonation or pitch direction
(e.g., utterances interpreted as statements or questions, Bolinger,
1986; Ladd, 2008), the pitch accent (e.g., Ladd et al., 1999),
or the prosodic patterns (e.g., Bänziger and Scherer, 2005;
Kraljic and Brennan, 2005; Dilley and McAuley, 2008), play a
considerable role in carrying paralinguistic information, such as
the emotional state (Banse and Scherer, 1996) or the intention
(Hellbernd and Sammler, 2016) of the speaker. Additionally,
pitch contrasts and thus changes of pitch over time additionally
carry lexical information in tonal languages (Yip, 2002; Carter-
Ényì and Carter-Ényì, 2016). In music, changes of pitch over
time also provide crucial information that allow listeners to
recognize, evaluate, and enjoy a musical performance. INTRODUCTION There exist
different musical (cultural) systems that define pitch movements,
with specific scales and rules (Krumhansl, 1979; Lerdahl and
Jackendoff, 1983; Cross, 2001; Ringer, 2002; Thompson, 2013),
but some features such as the presence of small intervals
or descending melodies are present in different cultures (e.g.,
Huron, 2001) and have been described as statistical universals
for music (Savage et al., 2015). Besides the relevance of pitch
changes over time (in addition to mean pitch), a large range
of literature in the music and language domains supports the
perceptual relevance of changes in other dimensions such as
timbre, intensity, or duration. It is thus important to explore
acoustic features that take into account dynamics (rather than
means) not only with regard to pitch, but also more broadly in
the dimensions of timing, timbre, and intensity. When coming from the same source (e.g., the vocal
instrument), music and language categories can show a certain
overlap. For instance, certain types of speech are considered
more musical than others (e.g., child-directed speech, rhymes,
poetry), while certain types of vocal music are considered speech-
like (e.g., rap). To better understand the ambiguity of vocal
stimuli, Merrill and Larrouy-Maestri (2017) presented several
versions of Arnold Schoenberg’s Pierrot lunaire—a piece notable
for its use of sprechstimme or “speech-song” (Stadlen, 1981)—to
vocal experts and found a large variety in the description of the
material, from very spoken-like to very sung-like. Interestingly,
the same exact material can be interpreted as either song or
speech. A phenomenon called the speech-to-song illusion has
been reported by Deutsch et al. (2008, 2011): A spoken sentence
can be perceived as sung after several repetitions. In other words,
the same acoustic contents can be perceived as belonging to
two distinct categories, which makes it difficult to identify clear
boundaries between speech and music, even in a culture in which
the idea of contrasting categories is widely accepted (see Brown,
2000 for a gradual view of music-language). Further, it has been
shown that categorization, in the context of the speech-to-song
illusion task, changes when the material is difficult to pronounce
(Margulis et al., 2015) or to understand (Jaisin et al., 2016), which
supports the role of listeners’ familiarity or prior knowledge in
stimulus categorization (Vanden Bosch der Nederlanden et al.,
2015) and suggests a potential downside of using vocal stimuli,
which are highly familiar to listeners. Citation: Citation:
Durojaye C, Fink L, Roeske T,
Wald-Fuhrmann M and
Larrouy-Maestri P (2021) Perception
of Nigerian Dùndún Talking Drum
Performances as Speech-Like vs. Music-Like: The Role of Familiarity
and Acoustic Cues. Front. Psychol. 12:652673. doi: 10.3389/fpsyg.2021.652673 Citation:
Durojaye C, Fink L, Roeske T,
Wald-Fuhrmann M and
Larrouy-Maestri P (2021) Perception
of Nigerian Dùndún Talking Drum
Performances as Speech-Like vs. Music-Like: The Role of Familiarity
and Acoustic Cues. Front. Psychol. 12:652673. doi: 10.3389/fpsyg.2021.652673 May 2021 | Volume 12 | Article 652673 1 Frontiers in Psychology | www.frontiersin.org Talking Drum Perception Durojaye et al. Frontiers in Psychology | www.frontiersin.org INTRODUCTION In this study, we further examine speech-music specificities by
employing an instrument intertwining language and music: the
dùndún talking drum. The dùndún is commonly used in south-
west Nigeria as a musical instrument. The dùndún of the Yorùbá
is played by people of all ages, though mostly men, and in a variety
of sacred and secular cultural contexts (Durojaye, 2020). It is
used to play musical rhythms without semantic information but
also to communicate announcements, warnings, prayer, jokes,
proverbs, or poetry (Sotunsa, 2009). While a dùndún ensemble
consists of drums of varying sizes and functions, we focus here
on the ìyá ìlù dùndún—the lead drum in the ensemble—usually
performing the role of “talker” during performance, imitating
Yorùbá, which is a tonal language, in what has been described as
speech surrogacy (Durojaye et al., in review; McPherson, 2018). Villepastour, in her analysis of bàtá drums—a very close relative
of the dùndún—argues for the interdependence of speech tone
and music and highlights the significance of relative pitch and
rhythm in the surrogacy system of the dùndún (Villepastour,
2010, 2014). In the present study, we focus on the perceptual categorization
of non-vocal material into speech-like vs. music-like, as
well as the role of familiarity in shaping these categories. Familiarity/culture effects on the perception of speech and music
are well-described (Palmer and Krumhansl, 1987; Morrison and
Demorest, 2009; Perrachione et al., 2011; Bregman and Creel,
2014; Sharma et al., 2020). With respect to timbre, familiar
sound sources are recognized more quickly than unfamiliar ones
(Siedenburg and McAdams, 2016). In the time domain, both
rhythm (Hannon et al., 2012) and meter perception (Kalender May 2021 | Volume 12 | Article 652673 2 Durojaye et al. Talking Drum Perception FIGURE 1 | Transcription of a Yorùbá proverb played on the dùndún (stimulus 6S in our corpus). The dots placed on the three horizontal lines represent a schematic
depiction of the pitch changes (high, medium, or low) as the phrase written underneath would be spoken in the Yorùbá language. INTRODUCTION The phrase translates to “Public or
private, there is no place the God cannot see, public or private.” Though the phrase would typically be spoken without the repetition at the end, when played on the
dùndún the proverb is elaborated by repetition of the first phrase (gray shaded areas), as repetition is often a means to provide context and remove ambiguity in
meaning (Stern, 1957; Arewa and Adekola, 1980). FIGURE 1 | Transcription of a Yorùbá proverb played on the dùndún (stimulus 6S in our corpus). The dots placed on the three horizontal lines represent a schematic
depiction of the pitch changes (high, medium, or low) as the phrase written underneath would be spoken in the Yorùbá language. The phrase translates to “Public or
private, there is no place the God cannot see, public or private.” Though the phrase would typically be spoken without the repetition at the end, when played on the
dùndún the proverb is elaborated by repetition of the first phrase (gray shaded areas), as repetition is often a means to provide context and remove ambiguity in
meaning (Stern, 1957; Arewa and Adekola, 1980). MATERIALS AND METHODS The Yorùbá language uses three relative tone levels: Low
(grave accent), Middle (usually left unmarked), and High
(acute accent). The tone levels are vital to distinguish the
meaning of words (Carter-Ényì and Carter-Ényì, 2016). Like
the language, the drum also consistently employs three relative
tone levels. The dùndún is a waisted (hourglass shaped), double-
headed membrane drum, with gut or leather cords securing
the skins around the wooden frame of the drum. The cords
are manipulated with one hand, while the other strikes the top
membrane with a curved stick. Pressure on the cords changes
the pitch of the drum, allowing for a full octave range and
effects like glissandi (Blades, 1992; Euba, 1990). For the drum
to produce the lowest pitch, minimal pressure is applied on
the cords; the more the pressure, the higher the frequency. Thus, the drum can manipulate tone levels and contours, as in
Yorùbá language. This imitation is confirmed by recent acoustical
analyses of mono or disyllabic words performed on the drum
which demonstrated that the three Yorùbá tones (Low, Middle,
and High) are produced on a global level with three measurably
different fundamental frequencies (Akinbo, 2019). The technique
of representing syllables can take many forms such as (1) using
one drum stroke for each syllable (as for a single tone level and
vowel elisions); (2) many strokes for one syllable; (3) one drum
stroke for two or more syllables; (4) one drum stroke for a syllable
with many speech tone levels as would be the case for some glides,
or assimilations (see also, Euba, 1990; Villepastour, 2010, for bàtá
drums). A transcription of dùndún “talking” is provided as an
example in Figure 1. The experimental procedure was in accordance with guidelines
ethically approved by the Ethics Council of the Max Planck
Society. Participants
provided
informed
consent
before
proceeding with the study. Participants One hundred and seven participants (36 self-reported as females,
71 as males, aged from 18 to 75 years old, M = 39.22,
SD = 15.06) were recruited via the research participant database
of the Max Planck Institute for Empirical Aesthetics and
via personal contacts. From various geographical locations
(15 different mother tongues were represented), fifty-one
participants reported being familiar with the dùndún talking
drum (i.e., they knew about the dùndún prior to the survey). Of these 51 participants, 28 (55%) were speakers of Yorùbá. Participants declared to have normal hearing ability and
reported various musical training levels. Participants received no
financial compensation. Frontiers in Psychology | www.frontiersin.org Material Thirty-six dùndún samples were created from performances
by one professional dùndún musician from Ibadan, South-
West Nigeria. The performer (male) is a native Yorùbá and
fluent English speaker with more than 25 years of experience
playing the drum. Performances were recorded at a local music
studio with a SHURE SM57 dynamic microphone directed at
the face of the drum, at a 3-inch distance, sampling at 44.1
kHz. Note that clicks and environmental noise can be heard in
some recordings. In the experiment outlined below, we seek to identify acoustic
features associated with the perception of speech-like vs. music-
like dùndún performances and the potential role of familiarity
in such classification. To do so, we first compared speech-like
vs. music-like dùndún performances with regard to different
acoustic features related to pitch, intensity, timbre, and timing. Second, we presented the same samples to familiar and unfamiliar
listeners and examined their ability to classify the performances
as intended by the performer, as well as their confidence in the
classification. Finally, a statistical model was created to quantify
the role of listener familiarity and acoustic features of dùndún
performance, in predicting listeners’ perception of dùndún as
speech-like vs. music-like. Half of the performances were music-like material consisting
of Yorùbá àlùjó rhythms (literally dance drumming); the other
half were speech-like material, composed of Yorùbá proverbs
and oríkì (poetry). The performer was first asked to use the
drum to say different phrases (“talk”), after which he was
asked to “drum” (the equivalent of music). All instructions
were given in the Yorùbá language. Note that in Yorùbá
dùndún performances, when drummers say they “talk” with
the drum, they refer to the performance of oríkì, proverbs,
or the signal mode of the drum. Similarly, when they talk May 2021 | Volume 12 | Article 652673 Frontiers in Psychology | www.frontiersin.org 3 Durojaye et al. Talking Drum Perception had failed, we manually outlined note onsets with custom-
written MATLAB code. about “drumming” or “playing music” (for those who use
the English term), they are making a reference to àlùjó. These categories were confirmed by the performer after the
task. Also note that dance rhythms, proverbs, and poetry
are used for any occasion (e.g., weddings, burials, religious
events). In the current performances, the “talking” contents
covered various themes, such as a praise to a deity, prayers,
vilification, and admonition. Procedure The task was implemented as an online experiment in
Unipark Enterprise Feedback Suite (QuestBack GmbH, Cologne,
Germany). Prior to the classification task, a brief presentation of
the dùndún was given (origin, uses, description, picture) without
sound examples. The aim was to provide a basic knowledge
for those who reported being unfamiliar with the drum or
the potential use of this instrument as speech surrogates. To
determine participants’ familiarity with the dùndún, we asked
if they knew about the dùndún prior to the survey. Participants
were instructed to listen to each excerpt and to indicate whether
it was best described as “speech-like” or “music-like.” For each
excerpt, the forced choice identification was followed by a
confidence rating on a 4 point-scale (1 = not confident, 4 = very
confident). The order of stimuli and response pattern (speech-
like button as the first or the second option) was randomized for
each participant. (p
y
g
p
)
Besides these time-related measures, we selected various
features typically used to describe auditory signals: pitch,
intensity, and timbre measures. At the note level and between
consecutive notes, for each stimulus, we calculated: pitch height,
intensity, and Wiener entropy (timbre, the maximum value is
a pure sine tone). Scaling was performed within recording,
on the millisecond-wise acoustic features between the 0.5 and
99.5th percentiles (instead of between minimum and maximum)
to exclude outliers. Amplitude envelope and Wiener entropy
were extracted from the audio in 10 ms time windows and
steps of 1 ms using the MATLAB package Sound Analysis for
MATLAB (by Sigal Saar). The pitch function from MATLAB
Audio Toolbox (The MathWorks Inc., 2020) was used for
pitch extraction. For pitch, intensity, and timbre measures
(as well as IOI and ratio), we also calculated the probability
densities for each group of stimuli (music-like and speech-like). Additionally, we computed the average of each feature, across
each stimulus, resulting in the following final measures: mean
pitch, mean intensity, and mean timbre; as well as average
of absolute differences between consecutive tones (leading
to mean intensity change, mean timbre change, and mean
pitch change measures). Note that scaled values (0–1) were
used to compute the measures capturing changes between
consecutive notes. Acoustic Measures of Interest First, we computed the amplitude modulation spectra of the
recordings, following the procedure of Ding et al. (2017),
with MATLAB code kindly provided by Nai Ding. In brief,
we extracted the sound envelope in narrow frequency bands
(corresponding to frequency bands used by the human cochlea),
then, following a re-scaling procedure, we calculated the root
mean square of the Discrete Fourier Transform of each
frequency band and binned over frequencies. High frequencies
in the amplitude modulation spectrum correspond to fast
modulations of intensity, low frequencies to slow modulations
(for details, see Ding et al., 2017). We then calculated the
peak frequency in the spectrum (i.e., the frequency exhibiting
the greatest amplitude modulation), for each recording. To
further analyze differences in timing, we also computed the
inter-onset interval (IOI, in ms) between notes, the two-interval
ratio: interval1/(interval1+interval2), and the pulse clarity. The
first measure corresponds to the timing at the note level and
the second to the change in timing between consecutive time
intervals. A short interval preceding a long interval would result
in a ratio < 0.5, a short interval following a longer one has a
ratio > 0.5, and an isochronous rhythm of two similar intervals
has a ratio of 0.5. Pulse clarity, a measure that estimates the
temporal regularity of events in the signal (Lartillot et al., 2008),
was calculated using all recommended default parameters of the
mirpulseclarity function from the music information retrieval
toolbox v.1.7.2 (i.e., using a frame length of 5 s, a hop factor of
10%, and the maximum value of the autocorrelation curve to
define clarity). Pulse clarity ranges between 0 (no clear pulse) and
1 (perfectly regular pulse). To confirm that the performances clearly represented the
category of speech or music, all recordings were presented to
three independent professional dùndún drummers located in
Nigeria and South Africa. The judges were asked to categorize
the performances according to whether they represented speech
or music. Like the performer, they used the terms àlùjó, oríkì,
owe (proverbs), in their responses. The 30 samples on which the
judges unanimously agreed to represent speech (n = 15) or music
(n = 15) were selected. The duration of the samples ranged from
5 to 10 s (M = 7.37 s, SD = 1.1 s). All recordings can be accessed
at:
https://edmond.mpdl.mpg.de/imeji/collection/ovmWl7
rLtIiGSv1v. Acoustic Analyses The
analysis
of
acoustic
features
was
carried
out
in
MATLAB
2018b
(The
MathWorks,
Inc.,
Natick,
Massachusetts, United States). The
analysis
of
acoustic
features
was
carried
out
in
MATLAB
2018b
(The
MathWorks,
Inc.,
Natick,
Massachusetts, United States). Acoustic Properties of Dùndún
Performances We
first
investigated
whether
the
amplitude
modulation
spectrum (AMS) systematically differs between speech-like and
music-like dùndún stimuli, as the AMS has previously been
shown to distinguish between speech (around 4–6 Hz) and
different kinds of Western music (around 2 Hz). As illustrated
in Figure 2A and confirmed via t-test, however, the AMS of the
two types of stimuli did not significantly differ [t(27.93) = 1.60,
p = 0.120, d = 0.59]; both peaked around 5 Hz, which corresponds
to the previously established amplitude modulation rate typical
of speech (Ding et al., 2017; Figure 2A, bottom panel). In terms
of pulse clarity, we find a significant difference between the
two stimulus categories, with a greater pulse clarity (i.e., greater
temporal regularity) in the music-like stimuli, t(26.14) = 4.03,
p < 0.001, d = 1.47 (Figure 2B). accuracy =
TP + TN
TP + TN + FP + FN However, accuracy has been shown to be biased if the classes are
unbalanced (e.g., if there would be more instances of perceived
speech than music). Therefore, as widely used and recommended,
we computed Matthews Correlation Coefficient (MCC): However, accuracy has been shown to be biased if the classes are
unbalanced (e.g., if there would be more instances of perceived
speech than music). Therefore, as widely used and recommended,
we computed Matthews Correlation Coefficient (MCC): MCC =
TP × TN −FP × FN
√(TP + FP) × (TP + FN) × (TN + FP) × (TN + FN) p
g
In addition to the AMS measure, we examined four types of
features (see section “Materials and Methods” for description of
all measures) that could capture differences between music- and
speech-like stimuli: intensity, pitch, timbre, and timing. Figure 3
illustrates each measure at the note level (mean for each stimulus
and their distribution). As reported in Table 1, we observed
significant differences for intensity and timing, with higher
intensity level (Figure 3A) and longer IOI (Figure 3D) in the
speech-like recordings. Figure 4 illustrates each measure at the
consecutive note level (mean of differences between consecutive
notes for each stimulus and their distribution). Besides being
louder, consecutive notes also varied less in intensity in the
speech-like stimuli (Figure 4A), in line with the distribution
depicted in Figure 3A, with a narrower range for the speech-like
stimuli. Also, we observed that near-isochrony is very common in
both speech- and music-like excerpts (Figure 4D). Statistical Analyses Statistical analyses were conducted in R Core Team (2013). To compare acoustic measures between speech- and music-
like dùndún recordings, we performed independent, two-tailed
t-tests. For measures relative to changes between consecutive
notes, absolute (rather than signed) differences were used. At an
alpha of 0.05, the threshold p-value after correcting for multiple
comparisons (10 t-tests) was p = 0.005. Cohen’s d is used to report
effect sizes and was calculated in R using the cohen.d function in
the effsize package (Torchiano, 2020). To predict participants’ perception of the stimuli as music- or
speech-like, a generalized linear mixed effects logistic regression
model was fit via maximum likelihood using the Laplace
approximation method, with bound optimization by quadratic
approximation, implemented using the glmer function from the
lme4 package (Bates et al., 2015). Acoustic variables were centered
and scaled before being entered into the model. Multicollinearity
was checked using variance inflation factors (VIFs). Some
predictors had a VIF > 5 and, therefore, required removal from
the model. Specifically, inter-onset-interval (VIF = 5.90) and Behavioral Analyses amplitude difference between adjacent notes (VIF = 8.27) were
removed. Correlations between means of all acoustic features
are provided in Supplementary Figure 1. Note that inter-onset
interval has a high correlation with intensity [r(28) = 0.63,
p < 0.001] and intensity difference [r(28) = −0.82, p < 0.001],
and that intensity and intensity difference have a high correlation
with each other [r(28) = −0.71, p < 0.001]. Participants’ Classification of Stimuli Participants’ responses on the task can be summarized using a 2
× 2 contingency table, or confusion matrix M: M =
hTP FN
FP TN
i where we arbitrarily define music as positive and speech as
negative, such that true positives (TP) represent music intended
by the performer and classified by the listener, while true
negatives (TN) represent speech intended by the performer and
similarly classified by listener. TPs (music) and TNs (speech)
represent correct classifications. False positives (FP) designate
instances of intended speech perceived as music, while false
negatives (FN) indicate cases of intended music perceived as
speech. Perfect classification is thus defined as 0 FPs and FNs:
h
n+ 0
0 n−
i
. Accuracy on the task is defined as the number of correct
classifications, divided by the total number of observations: where we arbitrarily define music as positive and speech as
negative, such that true positives (TP) represent music intended
by the performer and classified by the listener, while true
negatives (TN) represent speech intended by the performer and
similarly classified by listener. TPs (music) and TNs (speech)
represent correct classifications. False positives (FP) designate
instances of intended speech perceived as music, while false
negatives (FN) indicate cases of intended music perceived as
speech. Perfect classification is thus defined as 0 FPs and FNs:
h
n+ 0
0 n−
i
. Accuracy on the task is defined as the number of correct
classifications, divided by the total number of observations: Acoustic Properties of Dùndún
Performances However, in
music-like ones, intervals tend to speed up (the second interval
in an isochronous pair being a little shorter, moving the near-
isochronous peak slightly right) while slowing down in speech-
like ones (near-isochronous peak moved slightly left). Some very
high and very low ratios (due to a short interval next to a long
one) become apparent in the wider spread of speech-like data,
in the top right scatter plot, and in the peaks of the probability
density plot, marked by arrowheads. MCC is robust to unbalanced datasets, and has been shown to
be a more reliable measure than accuracy (Chicco and Jurman,
2020). It is a measure of classification performance across all
classes that takes into account the size of each class. MCC ranges
between -1 and 1, with 0 indicating chance performance, 1 perfect
performance, and -1 perfect misclassification. As is common
practice with MCC, in the case of 0 in the denominator, we set
the denominator to 1. Frontiers in Psychology | www.frontiersin.org Segmentation Segmentation of single notes was performed semi-automatically
on each recording’s amplitude envelope, using an adaptive
threshold. First, amplitude envelopes were slightly smoothed
with a Hodrick-Prescott (HP) filter (coefficient = 50). Then,
the adaptive segmentation threshold was created by applying a
stronger HP filter to the amplitude envelope (coefficient = #107). The difference between the slightly smoothed amplitude envelope
and the adaptive filter provided robust segmentation in most
recordings. We visually inspected the segmented waveforms
and sonograms while listening to the audio to validate
segmentation. In cases where the automated segmentation May 2021 | Volume 12 | Article 652673 Frontiers in Psychology | www.frontiersin.org 4 Talking Drum Perception Durojaye et al. Listeners’ Classification of Dùndún
Performances As can be seen in Figure 5A, participants clearly separated
the stimuli into two distinct speech vs. music categories
that largely aligned with the intention of the performer. We May 2021 | Volume 12 | Article 652673 5 Talking Drum Perception Durojaye et al. FIGURE 2 | (A) Amplitude modulation spectra of the dùndún performances (top panel). Thin lines are for AMS of individual recordings, thick lines represent the mean
for 15 music (blue) and 15 speech-like (red) stimuli. Bottom panel shows AMS for speech and music corpora from Ding et al. (2017) for comparison. Dashed arrow
illustrates that dùndún recordings peak at higher modulation rates than most Western music, namely rates that are typical for speech (black line) but also occur in
rock music (teal line). The lower panel in A is reprinted from Neuroscience and Biobehavioral Reviews, 81, Nai Ding, Aniruddh D. Patel, Lin Chen, Henry Butler,
Cheng Luo, David Poeppel, Temporal modulations in speech and music, 7, Copyright Elsevier Ltd. (2017), with permission from Elsevier. (B) Violin plots representing
the pulse clarity of the music (blue) and speech-like (red) stimuli. The black horizontal line indicates the median of both distributions. Black dots represent pulse clarity
for individual stimuli. FIGURE 2 | (A) Amplitude modulation spectra of the dùndún performances (top panel). Thin lines are for AMS of individual recordings, thick lines represent the mean
for 15 music (blue) and 15 speech-like (red) stimuli. Bottom panel shows AMS for speech and music corpora from Ding et al. (2017) for comparison. Dashed arrow
illustrates that dùndún recordings peak at higher modulation rates than most Western music, namely rates that are typical for speech (black line) but also occur in
rock music (teal line). The lower panel in A is reprinted from Neuroscience and Biobehavioral Reviews, 81, Nai Ding, Aniruddh D. Patel, Lin Chen, Henry Butler,
Cheng Luo, David Poeppel, Temporal modulations in speech and music, 7, Copyright Elsevier Ltd. (2017), with permission from Elsevier. (B) Violin plots representing
the pulse clarity of the music (blue) and speech-like (red) stimuli. The black horizontal line indicates the median of both distributions. Black dots represent pulse clarity
for individual stimuli. speech). Within the speech category, 13S was most clearly
perceived as speech, while 5S was most often confused for
music. Readers can access all stimuli online (link in section
“Materials and Methods”). Listeners’ Classification of Dùndún
Performances observed that only four participants categorized every sample
as music (solid blue rows near the bottom of the plot),
whereas the large majority showed few confusions. Twelve
participants exhibited perfect classification (top rows). In the
figure, within the speech and music categories, stimuli (columns)
are sorted by the number of errors made per stimulus
(i.e., the left-most column, stimulus 13M, was least often
confused for speech, while 3M was most often confused for A confusion matrix for perceived vs. intended music and
speech-like performances is plotted in Figure 5B. Overall, the
average accuracy of participants on the task was 66%. The
average rate people perceived speech when the performance
was intended to be music was 12%, while the average rate
at which people perceived music when it was intended to be
speech was 29%. Collectively, these latter two rates indicate
that participants were more likely to perceive speech as music
than music as speech. The illustration of confidence ratings
(underlying histograms in Figure 5B, with ratings from 1 to
4) showed similar patterns, with moderately high confidence
even in the case of false classifications. Note, however, that
the listeners who were unfamiliar (gray) with the dùndún
seem to be least confident when they perceive the stimulus
to be speech (right column in confusion matrix). Confidence
means for unfamiliar (gray) and familiar (gold) participants
are displayed in the lower left and right corners of each
quadrant, respectively. TABLE 1 | Output of the independent t-tests carried out for the four types of
acoustic features (Intensity, Pitch, Timbre, and Timing) between music- and
speech-like stimuli. Intensity
Pitch
Timbre
Timing
Mean for note
level
t(18.18) = −5.37 t(20.61) = 2.06 t(18.84) = −1.38 t(16.18) = −8.53
p < 0.001
p = 0.052
p = 0.185
p < 0.001
d = −1.96
d = 0.75
d = −0.50
d = −3.12
Mean change
for consecutive
notes
t(26.32) = 8.95 t(17.53) = −0.75 t(27.60) = 4.09
t(15.74) = 2.17
p < 0.001
p = 0.465
p < 0.001
p = 0.046
d = 3.27
d = −0.27)
d = 1.49
d = 0.79
For the four types of comparisons, measures consisted of the mean of all notes
(Figure 2) and the mean of the absolute difference between all consecutive notes
(Figure 3). Note that the threshold p-value (for an alpha of 0.05) after correcting
for multiple comparisons was p = 0.005. Listeners’ Classification of Dùndún
Performances Thicker frames highlight the measures
significantly differing after correction. TABLE 1 | Output of the independent t-tests carried out for the four types of
acoustic features (Intensity, Pitch, Timbre, and Timing) between music- and
speech-like stimuli. Given the imbalance in perceiving speech vs. music, and
the statistical properties outlined in the methods, our main
metric of interest for participants’ classifications of the stimuli
was the Matthews Correlation Coefficient (MCC). An MCC
of 1 indicates perfect classification, 0 chance, and -1 perfect
misclassification. Participants’ average MCC was 0.61 (±0.33). For the four types of comparisons, measures consisted of the mean of all notes
(Figure 2) and the mean of the absolute difference between all consecutive notes
(Figure 3). Note that the threshold p-value (for an alpha of 0.05) after correcting
for multiple comparisons was p = 0.005. Thicker frames highlight the measures
significantly differing after correction. For the four types of comparisons, measures consisted of the mean of all notes
(Figure 2) and the mean of the absolute difference between all consecutive notes
(Figure 3). Note that the threshold p-value (for an alpha of 0.05) after correcting
for multiple comparisons was p = 0.005. Thicker frames highlight the measures
significantly differing after correction. May 2021 | Volume 12 | Article 652673 Frontiers in Psychology | www.frontiersin.org 6 Talking Drum Perception Durojaye et al. FIGURE 3 | Acoustic measures computed at the note level for music-like (blue) and speech-like (red) dùndún performances. (A) Mean intensity, (B) Mean pitch, and
(C) Mean timbre (Wiener entropy) (D) Mean inter onset intervals (IOI). Left panels show mean note values for 15 speech- and music-like pieces each in gray, with
mean and SEM per piece in color. Right panels show the respective probability densities. Participants who were familiar1 with the dùndún exhibited
a significantly higher MCC, compared to those who were
unfamiliar with the dùndún [Welch’s independent two-tailed,
t-test: t(93 08) = 6.12, p < 0.001, Cohen’s d = 1.16, 1MCC = 0.33]; FIGURE 3 | Acoustic measures computed at the note level for music-like (blue) and speech-like (red) dùndún performances. (A) Mean intensity, (B) Mean pitch, and FIGURE 3 | Acoustic measures computed at the note level for music-like (blue) and speech-like (red) dùndún performances. (A) Mean intensity, (B) Mean pitch, and
(C) Mean timbre (Wiener entropy) (D) Mean inter onset intervals (IOI). Left panels show mean note values for 15 speech- and music-like pieces each in gray, with
mean and SEM per piece in color. Right panels show the respective probability densities. Participants who were familiar1 with the dùndún exhibited
a significantly higher MCC, compared to those who were unfamiliar with the dùndún [Welch’s independent two-tailed,
t-test: t(93.08) = 6.12, p < 0.001, Cohen’s d = 1.16, 1MCC = 0.33];
see Figure 5C; N.B. each participants’ familiarity (gold = familiar;
gray = unfamiliar) is plotted in the left color bar of Figure 5A). Nonetheless, familiarity is not required to perform the task,
as unfamiliar participants still exhibited an average MCC 1We ran an additional t-test comparing the MCC of the subset of familiar
participants who did not speak Yorùbá to those unfamiliar and still found a large,
significant effect of familiarity, t(58.74) = 3.18, p = 0.002, d = 1.16, 1MCC = 0.211. May 2021 | Volume 12 | Article 652673 Frontiers in Psychology | www.frontiersin.org 7 Talking Drum Perception Durojaye et al. FIGURE 4 | Acoustic measures computed at the level of consecutive notes for music-like (blue) and speech-like (red) dùndún performances. All measures are scaled
from 0 to 1. (A) Mean intensity change: The top panel shows, for each pair of consecutive notes, the intensity of the pair’s first note on the x-axis, and of the second
note on the y-axis. The diagonal line indicates where note pairs with two similar note intensities would fall. The bottom panel shows the probability density of intensity
differences. (B,C) are the Mean pitch change and Mean timbre change, illustrated following the same logic as (A). (D) Similar illustration for change in IOIs, as
measured by ratio = interval1 / (interval1+interval2). In the probability density plot (bottom), green boxes and dashed lines mark interval ratios in terms of “interval1:
interval2,” an alternative way to describe interval ratios, commonly used in music theory. FIGURE 5 | Participants’ classification of music vs. speech-like dùndún performances. (A) Each participant’s (vertical axis) judgment of each stimulus (horizontal
axis) as music (blue) or speech (red). Participant performance is sorted descending from least to most errors. Columns are sorted from left to right, within each
category (speech and music), from least to most errors per stimulus. The color bar to the left of the plot indicates whether each participant was familiar (golden) or
unfamiliar (gray) with the dùndún and whether they spoke Yorùbá (black) or not (white). (B) Confusion matrix for perceived vs. intended stimulus classes, with
histograms of participants’ confidence ratings (1–4) for each response type (i.e., quadrant), grouped by familiarity (gold = familiar; gray = unfamiliar). Confidence
rating densities were computed within each response type (quadrant) for each familiarity group separately. Means for unfamiliar (gray) and familiar (gold) groups are
displayed in the lower left and right corners of each quadrant, respectively. (C) Violin plots and underlying data points indicating the Matthews Correlation Coefficient
(MCC) for each participant, separated according to those who were familiar with the dùndún (golden) vs. unfamiliar (gray). The bottom and top horizontal black lines
in each distribution represent the 25th and 75th percentiles, the middle line represents the median. An MCC of 1 indicates perfect classification; 0 represents
chance, and -1 perfect misclassification. of 0.46, well above chance (0), t(55) = 10.10, p < 0.001,
d = 1.35. logistic regression model. The binary dependent variable was
participants’ perception (speech = 0, music = 1). On the stimulus
level, fixed effects included all measures reported in Figures 2–
4, except intensity difference and inter-onset-interval, which had
to be excluded due to high correlation with intensity and each
other (see section “Materials and Methods”). Since we observed
an effect of familiarity on the classification performance index,
with better classification for listeners who were familiar with
the dùndún, we included familiarity as a fixed effect and in Frontiers in Psychology | www frontiersin org
8
May 2021 | Volume 12 | Article 652673 FIGURE 4 | Acoustic measures computed at the level of consecutive notes for music-like (blue) and speech-like (red) dùndún performances. All measures are scaled
from 0 to 1. (A) Mean intensity change: The top panel shows, for each pair of consecutive notes, the intensity of the pair’s first note on the x-axis, and of the second
note on the y-axis. The diagonal line indicates where note pairs with two similar note intensities would fall. The bottom panel shows the probability density of intensity
differences. (B,C) are the Mean pitch change and Mean timbre change, illustrated following the same logic as (A). (D) Similar illustration for change in IOIs, as
measured by ratio = interval1 / (interval1+interval2). In the probability density plot (bottom), green boxes and dashed lines mark interval ratios in terms of “interval1:
interval2,” an alternative way to describe interval ratios, commonly used in music theory. May 2021 | Volume 12 | Article 652673 8 Durojaye et al. Talking Drum Perception FIGURE 5 | Participants’ classification of music vs. speech-like dùndún performances. (A) Each participant’s (vertical axis) judgment of each stimulus (horizontal
axis) as music (blue) or speech (red) Participant performance is sorted descending from least to most errors Columns are sorted from left to right within each Predictors of Listeners’ Perception of
Speech vs. Music In an effort to understand which acoustic features were most
relevant in participants’ perception of the dùndún excerpts
as music vs. speech-like, we built a linear mixed effects May 2021 | Volume 12 | Article 652673 Frontiers in Psychology | www.frontiersin.org 9 Durojaye et al. Talking Drum Perception FIGURE 6 | Odds ratios, with confidence interval (CI), for each fixed effect in a logistic regression model predicting participants’ perception of stimuli as music-like (1)
or speech-like (0). The vertical line at 1 indicates no effect (i.e., any fixed effect predictor whose odds ratio CI overlaps 1 does not significantly predict participants’
perception). Fixed effects with an odds ratio <1 (red) indicate that a high value on that feature leads to the perception of speech. Odds ratios >1 (blue) indicate that a
high value on that feature leads to the perception of music. The significance of each fixed effect is indicated with stars (***p < 0.001, **p < 0.01, and *p < 0.05). Number of observations: 3,210. Familiarity was a binary predictor (0 = no; 1 = yes); confidence ranged from 0 to 3; all other variables were continuous. FIGURE 6 | Odds ratios, with confidence interval (CI), for each fixed effect in a logistic regression model predicting participants’ perception of stimuli as music-like (1)
or speech-like (0). The vertical line at 1 indicates no effect (i.e., any fixed effect predictor whose odds ratio CI overlaps 1 does not significantly predict participants’
perception). Fixed effects with an odds ratio <1 (red) indicate that a high value on that feature leads to the perception of speech. Odds ratios >1 (blue) indicate that a
high value on that feature leads to the perception of music. The significance of each fixed effect is indicated with stars (***p < 0.001, **p < 0.01, and *p < 0.05). Number of observations: 3,210. Familiarity was a binary predictor (0 = no; 1 = yes); confidence ranged from 0 to 3; all other variables were continuous. confident were more likely to classify the stimulus as music. These
confidence/familiarity results are in line with the general trends
presented in Figure 5B. interaction with all acoustic measures. Confidence ratings were
also entered as fixed effects. Random intercepts were included for
participants and stimuli. Figure 6 shows the odds ratios and confidence intervals for
each fixed effect in the model. Predictors of Listeners’ Perception of
Speech vs. Music Fixed effects with an odds ratio
<1 (red) indicate that a high value on that feature leads to the
perception of speech. Odds ratios >1 (blue) indicate that a high
value on that feature leads to the perception of music. Overall,
the model had a prediction accuracy of 85% and an MCC of 0.70. The model explained a significant proportion of variance in the
data, with a marginal R2 of 0.46 (amount of variance explained
by fixed effects alone) and a conditional R2 of 0.65 (amount of
variance explained by fixed and random effects). Frontiers in Psychology | www.frontiersin.org DISCUSSION Note also that, while mean IOI was significantly
different between the two types of stimuli, it unfortunately
could not be included into our statistical model (like the
mean intensity change measure), as it had high correlation
with other features (Supplementary Figure 1). Thus, it could
well be the case that participants are using IOI (as well
as intensity changes) in their classification. This issue could
be addressed by systematically manipulating IOI, as well as
mean intensity and intensity change, to tease apart their
perceptual relevance. Participants
could
accurately
classify
the
dùndún
performances in the category intended by the performer,
with an unsurprising bias toward the music-like category, given
that drums are more commonly associated with music than
speech. Listeners who were familiar with the instrument seem
to have a clearer representation of what is grouped into speech
or music categories, as visible by their better classification
performance. Such results could be driven by the fact that slightly
over half of the familiar participants also reported speaking
Yorùbá, though the familiarity effect remained even when
Yorùbá-speaking participants were removed from the analysis. In any case, if familiarity (broadly defined) or Yorùbá fluency
sharpens the categories, it is not required to perform the task,
as shown by the above chance level MCC and relatively high
confidence for participants who were not familiar with the
instrument. Such results suggest the relevance of commonly
recognized acoustic cues that shape each category but become
fine-tuned through repeated exposure. In the current study, we asked broadly about listeners’
familiarity and thus are not able to disentangle what exactly
underlies the familiarity effect. Teasing apart perceptual vs. cultural vs. linguistic familiarity might all be of interest in future
research. Additionally, manipulation of familiarity, via priming
or additional explicit information about the dùndún, might allow
one to quantify the amount and type of previous exposure
that affects the classification. Further, exploration of listeners’
perception of dùndún performances with less constrained answer
types might reveal other categories that could include both music
and speech-like performances or less strong boundaries between
these two culturally shaped categories (Brown, 2000). Regarding the dynamic aspect of timing, the present study
focused mainly on consecutive notes or intervals, though we did
include a measure of pulse clarity. DISCUSSION In this study, we used an instrument capable of speech surrogacy
to explore the boundaries between speech and music. While
several instruments such as trumpets (Kaminski, 2008), flutes
(Moore and Meyer, 2014), xylophones (Strand, 2009; Zemp and
Soro, 2010; McPherson, 2018), and whistling (Stern, 1957) can
produce speech surrogates, we focussed here on the dùndún
talking drum to examine listeners’ perception of music and
speech and, more specifically, the role of acoustic features in
distinguishing these two categories. To do so, we recorded expert
dùndún performances, which have the advantage of being able to
create both musical and speech-like stimuli without requiring the
human voice (highly familiar to all listeners). Acoustic measures
relative to pitch, timbre, intensity, and timing were used to
describe the stimuli and we measured listeners’ ability to classify
each performance into two pre-defined categories intended by the
performer: speech- or music-like. Greater pulse clarity predicted perception of music. At the
note level, greater mean intensity predicted speech. In terms of
changes between notes, greater changes in pitch predicted speech,
while greater changes in timbre predicted music. However,
mean intensity, pitch and pulse clarity all interacted with
participants’ familiarity (same direction of the overall effects
but enhanced magnitude). Additionally, confidence predicted
the categorization “music-like,” and interacted with familiarity,
such that those familiar and confident were more likely to
classify a given stimulus as speech, while those not familiar and May 2021 | Volume 12 | Article 652673 10 Talking Drum Perception Durojaye et al. Durojaye et al. et al., 2020). In the current study, peaks stand around 5 Hz
for both music and speech-like dùndún. Though some musical
styles have been shown to also peak around 5 Hz (e.g., rock),
this rate is consistent with the modulation rate of speech
(Ding et al., 2017). However, while Ding et al. (2017) analyzed
a variety of Indo-European languages (American and British
English, French, German, Swedish, Dutch, Danish, Norwegian;
exception: Chinese), they did not include any Niger-Congo
languages, such as Yorùbá, and only included Western types
of music, which limits the generalizability of their findings. Here we extend Ding et al.’s AMS analysis to non-Western
stimuli (dùndún) and show that its peak closely resembles
that of speech. Future work might extend the AMS analyses
to spoken Yorùbá and compare with that of the dùndún
to better understand the findings about the temporal aspect
reported here. DISCUSSION Our pulse clarity metric was
related to maxima in the autocorrelation function of our stimuli
(i.e., periodic repetitions), but our measure set could also be
extended to perceived beat and/or meter (e.g., Tomic and Janata,
2008; London et al., 2017) or the detection of repeated patterns
(e.g., via recurrence quantification analysis Fukino et al., 2016 or
multi-fractal analysis Roeske et al., 2018). The analysis/evaluation
of longer stimuli would allow for application of a more extensive
set of timing measures to investigate their role in speech vs. music distinction. Also, though our stimulus set is novel in that
it consists of speech-like and music-like performances on the
dùndún from the same performer, it is also limited in scope. Future studies might consider developing larger corpora with
more examples of speech-like and music-like material from
multiple performers. In addition, future research might also more
closely consider the relationship between measures like IOI,
perceived beat and meter, and AMS. Though IOI, AMS, and
perceived pulse / meter all have previously reported preferred
temporal ranges, which broadly seem to align with each other
(e.g., Fraisse, 1963; Parncutt, 1994; Farbood et al., 2013; Gotham,
2015; Ding et al., 2017), it is likely that IOI, AMS, and perceived
pulse / meter do not form a trivial and/or consistent relationship
to one another across all timescales. y
p
g
The model proposed to examine the predictors of participants’
classification
revealed
that
participants
relied
on
several
features. Pulse clarity, mean note intensity, and mean timbre
change between consecutive notes were significant predictors. Additionally, familiarity seemed to amplify the effects of
pulse clarity and intensity in predicting music vs. speech,
respectively. These perceptual results are in line with those
we find to distinguish acoustically between the two different
stimulus categories. However, we also observed that features
which did not significantly differ between the speech and
music
recordings
nonetheless
contributed
significantly
to
the perceptual model. Specifically, mean pitch did not differ
acoustically between the two types of stimuli performed by the
musician, though it interacted with familiarity in predicting
the perception of music. Similarly, mean pitch change between
consecutive notes did not significantly differ acoustically but
was associated with the perception of the stimuli as speech. Frontiers in Psychology | www.frontiersin.org REFERENCES Durojaye, C. (2020). The dùndún talking drum of the Yorùbá community in
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acoustic cues for speech act perception. J. Mem. Lang. 88, 70–86. doi: 10.1016/j. jml.2016.01.001 Carter-Ényì, A., and Carter-Ényì, Q. (2016). AUTHOR CONTRIBUTIONS different conceptual domains, which paves the way to a more
in-depth understanding of speech/music differences/similarities. Altogether, our findings confirm the relevance of acoustic
features relative to intensity, pitch, timbre, and timing in
distinguishing speech and music, as well as the role of culture
and/or exposure in defining such categories. different conceptual domains, which paves the way to a more
in-depth understanding of speech/music differences/similarities. CD, MW-F, and PL-M designed the study. CD collected the
data. TR conducted acoustic analyses. LF conducted perceptual
analyses and mixed modeling. LF and PL-M drafted the
manuscript. All authors edited and approved the manuscript. ACKNOWLEDGMENTS The
datasets
presented
in
this
study
can
be
found
in
the links below: https://edmond.mpdl.mpg.de/imeji/collection/
ovmWl7rLtIiGSv1v, https://github.com/lkfink/Dundun. We thank David Poeppel for helpful discussion and feedback
on analyses. We thank the performer for providing the
stimuli and the dùndún experts who assisted in classifying the
stimuli for final use. SUPPLEMENTARY MATERIAL The studies involving human participants were reviewed and
approved by Ethics Council of the Max Planck Society. The participants provided their written informed consent to
participate in this study. The Supplementary Material for this article can be found
online
at:
https://www.frontiersin.org/articles/10.3389/fpsyg. 2021.652673/full#supplementary-material The Supplementary Material for this article can be found
online
at:
https://www.frontiersin.org/articles/10.3389/fpsyg. 2021.652673/full#supplementary-material DISCUSSION In the future, the nature of the music and speech categories
(that are slightly modulated by the familiarity of the listeners)
could be clarified by means of psychophysical experiments
that parametrically manipulate the relevant acoustic features
reported here. Previous studies have suggested that surrogate languages or
language-based music, such as talking drums, may enhance
our understanding of music and language (Patel, 2008; Winter,
2014). In the present study, such ecologically valid material
provided the unique opportunity to compare stimuli coming
from the same sound source (and performer) while representing It is interesting to note the considerably faster amplitude
modulations of the dùndún performances, compared to the
AMS previously established for music (around 2 Hz, Ding
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potential conflict of interest. Ringer, A. L. (2002). “Melody: definition and origins,” in The New Grove Dictionary
of Music Online, ed. L. Macy (London: Macmillan Online Publishing). Roeske, T., Kelty-Stephen, D., and Wallot, S. (2018). Multifractal analysis
reveals
music-like
dynamic
structure
in
songbird
rhythms. Sci. Rep. 8:4570. Copyright © 2021 Durojaye, Fink, Roeske, Wald-Fuhrmann and Larrouy-Maestri. This is an open-access article distributed under the terms of the Creative Commons
Attribution License (CC BY). 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. Copyright © 2021 Durojaye, Fink, Roeske, Wald-Fuhrmann and Larrouy-Maestri. 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. Roeske, T., Larrouy-Maestri, P., Sakamoto, Y., and Poeppel, D. (2020). Listening
to birdsong reveals basic features of rate perception and aesthetic judgements. Proc. R. Soc. B. 287:20193010. doi: 10.1098/rspb.2019.3010 Savage, P. E., Brown, S., Sakai, E., and Currie, T. E. (2015). Statistical universals
reveal the structures and functions of human music. Proc. Natl. Acad. Sci. 112,
8987–8992. doi: 10.1073/pnas.1414495112 May 2021 | Volume 12 | Article 652673 Frontiers in Psychology | www.frontiersin.org 13
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Knockout of Babesia bovis rad51 ortholog and its complementation by expression from the BbACc3 artificial chromosome platform
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Knockout of Babesia bovis rad51 ortholog and its
2
complementation by expression from the BbACc3 artificial
3
chromosome platform
4
5
Short title: Knockout of the B. bovis rad51 gene and its phenotype
6
7
Erin A. Mack1¶#, Yu-Ping Xiao1¶, and David R. Allred1,2,3*
8
9
1Department of Infectious Diseases and Immunology, College of Veterinary Medicine,
10
University of Florida, Gainesville, Florida, United States of America
11
2Genetics Institute, University of Florida, Gainesville, Florida, United States of America
12
3Emerging Pathogens Institute, University of Florida, Gainesville, Florida, United States of
13
America
14
15
*Corresponding author
16
E-mail: allredd@ufl.edu
17
18
¶MEA and YP-X contributed equally to this project. 19
20
#Current address: Department of Biological Sciences, University of Idaho, Moscow, Idaho,
21
United States of America
22 1
Knockout of Babesia bovis rad51 ortholog and its 22 . CC-BY 4.0 International license
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Abstract 24
Babesia bovis establishes persistent infections of long duration in cattle, despite the development
25
of effective anti-disease immunity. One mechanism used by the parasite to achieve persistence is
26
rapid antigenic variation of the VESA1 cytoadhesion ligand through segmental gene conversion
27
(SGC), a phenomenon thought to be a form of homologous recombination (HR). To begin
28
investigation of the enzymatic basis for SGC we initially identified and knocked out the Bbrad51
29
gene encoding the B. bovis Rad51 ortholog. BbRad51 was found to be non-essential for in vitro
30
growth of asexual-stage parasites. However, its loss resulted in hypersensitivity to
31
methylmethane sulfonate (MMS) and an apparent defect in HR. This defect rendered attempts to
32
complement the knockout phenotype by reinsertion of the Bbrad51 gene into the genome
33
unsuccessful. To circumvent this difficulty, we constructed an artificial chromosome, BbACc3,
34
into which the complete Bbrad51 locus was inserted, for expression of BbRad51 under
35
regulation by autologous elements. Maintenance of BbACc3 makes use of centromeric
36
sequences from chromosome 3 and telomeric ends from chromosome 1 of the B. bovis C9.1 line. 37
A selection cassette employing human dihydrofolate reductase enables recovery of transformants
38
by selection with pyrimethamine. We demonstrate that the BbACc3 platform is stably
39
maintained once established, assembles nucleosomes to form native chromatin, and expands in
40
telomere length over time. Significantly, the MMS-sensitivity phenotype observed in the absence
41
of Bbrad51 was successfully complemented at essentially normal levels. We provide cautionary
42
evidence, however, that in HR-competent parasites BbACc3 can recombine with native
43
chromosomes, potentially resulting in crossover. We propose that, under certain circumstances
44
this platform can provide a useful alternative for the genetic manipulation of this group of
45
parasites, particularly when regulated gene expression under the control of autologous elements
46
may be important. 49
Keywords 50
artificial chromosome; Babesia bovis; chromatin; gene complementation; homologous
51
recombination; Rad51 52 . CC-BY 4.0 International license
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Introduction 54
Babesiosis is a tick-borne disease caused by apicomplexan parasites of the genus Babesia. 55
Humans are not the natural host for any babesial parasite but may be an incidental host,
56
acquiring zoonotic infections with a variety of different species. Babesia microti is the most
57
common species of Babesia to infect humans, although in western Europe infections commonly
58
occur with Babesia divergens. In the U.S. infections have been observed with Babesia duncani
59
and B. divergens-like organisms, as well as the unspeciated WA1 and MO1 isolates (reviewed in
60
[1]). Many individuals may carry asymptomatic infections [2], including as a result of inadequate
61
drug treatment of acute parasitemia [3, 4], posing a serious risk to the blood supply [5]. In cattle
62
babesiosis may be caused by at least five different species, with Babesia bovis generally
63
considered the most virulent. B. bovis shares many parallels with the human malarial parasite,
64
Plasmodium falciparum, including immune evasion via cytoadhesion and antigenic variation,
65
and the capacity for development of a lethal cerebral disease [6]. 66
67
Bovine babesiosis caused by B. bovis is quite severe. Cattle that survive the acute disease
68
develop a strong anti-disease immunity, but remain persistently infected for periods of at least
69
several years. This parasite makes use of at least two mechanisms to effect persistence: (i)
70
cytoadhesion of infected red blood cells (iRBCs) to capillary and post-capillary venous
71
endothelium, presumably in order to avoid splenic clearance; and (ii) rapid antigenic variation of
72
the cytoadhesion ligand, VESA1, to avoid antibody-mediated forced re-entry into the circulation
73
[7]. Cytoadhesion is therefore a behavior that is immunologically sensitive, and which the
74
parasite correspondingly has evolved means to protect. The development of the ability to
75
abrogate antigenic variation would allow for ready elimination of the parasite; this capability is a
76
desirable goal for parasite control through disruption of an essential aspect of its biology. 77
78
There is a great need to understand the molecular bases of virulence mechanisms in parasites. 79
One informative way to facilitate such studies is through the ability to genetically manipulate the
80
organisms. To date, the tools developed for the genetic manipulation of B. bovis are limited and
81
remain most appropriate for use in characterizing individual targets, although there is a need for
82
high-throughput methodologies [8]. 53
Introduction The expression of exogenous genes has been demonstrated,
83
using babesial promoter sequences to drive transcription of the target sequences. This has been 54
Babesiosis is a tick-borne disease caused by apicomplexan parasites of the genus Babesia. 55
Humans are not the natural host for any babesial parasite but may be an incidental host,
56
acquiring zoonotic infections with a variety of different species. Babesia microti is the most
57
common species of Babesia to infect humans, although in western Europe infections commonly
58
occur with Babesia divergens. In the U.S. infections have been observed with Babesia duncani
59
and B. divergens-like organisms, as well as the unspeciated WA1 and MO1 isolates (reviewed in
60
[1]). Many individuals may carry asymptomatic infections [2], including as a result of inadequate
61
drug treatment of acute parasitemia [3, 4], posing a serious risk to the blood supply [5]. In cattle
62
babesiosis may be caused by at least five different species, with Babesia bovis generally
63
considered the most virulent. B. bovis shares many parallels with the human malarial parasite,
64
Plasmodium falciparum, including immune evasion via cytoadhesion and antigenic variation,
65
and the capacity for development of a lethal cerebral disease [6]. 66 . CC-BY 4.0 International license
a
ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
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doi:
bioRxiv preprint . CC-BY 4.0 International license
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certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
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doi:
bioRxiv preprint 84
achieved transiently through transfection with circular plasmids [9-13]. The longevity of this
85
class of vector has been improved by the inclusion of a centromeric segment, improving the
86
segregation of the circular molecules [12]. Long-term, stable expression has been achieved by
87
single-crossover insertional mutagenesis of chromosomal loci [12, 14]. This approach
88
demonstrates the capacity for creating targeted gene disruptions and the potential for the use of
89
promoter trapping strategies. Significantly, double-crossover knockout of the thioredoxin
90
peroxidase (tpx) gene has been achieved, providing complete gene knockouts [12]. 53
Introduction 91
Complementation of tpx was achieved by re-integration of the gene ectopically into the EF1
92
locus, resulting in high level expression. However, ectopic expression of exogenous genes in this
93
manner may suffer from improper expression due to local chromatin modifications, including
94
silencing or significant overexpression, compromising interpretation of genetic contributions to
95
phenotype. Despite the available tools there remains a need for convenient platforms that are
96
stable, efficiently and accurately segregated during mitosis, allow the transcription of large
97
sequences, and which may be properly regulated relative to metabolic need. 98
99
Antigenic variation in B. bovis is known to heavily utilize segmental gene conversion (SGC) to
100
modify the VESA1 ligand (BAK), and may participate in in situ transcriptional switching (isTS)
101
as well. SGC is thought to be a form of homologous recombination (HR), much the same as gene
102
conversion, a process that is dependent upon Rad51 proteins. To initiate investigation of the
103
enzymatic underpinnings of antigenic variation by SGC in B. bovis, we describe here the
104
knockout of the B. bovis rad51 gene by double crossover replacement [15]. Given the potential
105
for adverse effects if expressed at inappropriate levels, we did not want to overexpress Bbrad51
106
during complementation. Moreover, because of the knockout of the Bbrad51 gene, homologous
107
recombination was lost and complementation of the knockout by reintroduction of the gene into
108
the genome was not possible. We have addressed this issue through artificial chromosome
109
technology, an approach that allows the complementation of disrupted genes by reintroduction of
110
the entire locus, providing gene control through its autologous regulatory elements. An artificial
111
chromosome platform has already been developed for Plasmodium berghei, enabling
112
transformation efficient enough for shotgun screening to recover genes associated with drug
113
resistance [16, 17]. We have created an analogous platform for use with B. bovis through the
114
assembly of an artificial chromosome, BbACc3. BbACc3 is comprised of B. bovis centromeric 84
achieved transiently through transfection with circular plasmids [9-13]. The longevity of this
85
class of vector has been improved by the inclusion of a centromeric segment, improving the
86
segregation of the circular molecules [12]. Long-term, stable expression has been achieved by
87
single-crossover insertional mutagenesis of chromosomal loci [12, 14]. 53
Introduction This approach
88
demonstrates the capacity for creating targeted gene disruptions and the potential for the use of
89
promoter trapping strategies. Significantly, double-crossover knockout of the thioredoxin
90
peroxidase (tpx) gene has been achieved, providing complete gene knockouts [12]. 91
Complementation of tpx was achieved by re-integration of the gene ectopically into the EF1
92
locus, resulting in high level expression. However, ectopic expression of exogenous genes in this
93
manner may suffer from improper expression due to local chromatin modifications, including
94
silencing or significant overexpression, compromising interpretation of genetic contributions to
95
phenotype. Despite the available tools there remains a need for convenient platforms that are
96
stable, efficiently and accurately segregated during mitosis, allow the transcription of large
97
sequences, and which may be properly regulated relative to metabolic need. 98 99
Antigenic variation in B. bovis is known to heavily utilize segmental gene conversion (SGC) to
100
modify the VESA1 ligand (BAK), and may participate in in situ transcriptional switching (isTS)
101
as well. SGC is thought to be a form of homologous recombination (HR), much the same as gene
102
conversion, a process that is dependent upon Rad51 proteins. To initiate investigation of the
103
enzymatic underpinnings of antigenic variation by SGC in B. bovis, we describe here the
104
knockout of the B. bovis rad51 gene by double crossover replacement [15]. Given the potential
105
for adverse effects if expressed at inappropriate levels, we did not want to overexpress Bbrad51
106
during complementation. Moreover, because of the knockout of the Bbrad51 gene, homologous
107
recombination was lost and complementation of the knockout by reintroduction of the gene into
108
the genome was not possible. We have addressed this issue through artificial chromosome
109
technology, an approach that allows the complementation of disrupted genes by reintroduction of
110
the entire locus, providing gene control through its autologous regulatory elements. An artificial
111
chromosome platform has already been developed for Plasmodium berghei, enabling
112
transformation efficient enough for shotgun screening to recover genes associated with drug
113
resistance [16, 17]. We have created an analogous platform for use with B. bovis through the
114
assembly of an artificial chromosome, BbACc3. BbACc3 is comprised of B. bovis centromeric . CC-BY 4.0 International license
a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 53
Introduction It is made available under
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ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
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bioRxiv preprint 115
and telomeric ends in a modified pBluescript-KS(+) backbone. This construct employs the
116
human dihydrofolate reductase (hDHFR) gene as selectable marker, conferring resistance to
117
pyrimethamine. BbACc3 was constructed and is manipulated as the circular plasmid, pBACc3,
118
which is linearized for transfection. Linearization with PmeI removes a spacer sequence and
119
exposes legitimate telomeric ends. Here, we demonstrate that BbACc3 transforms B. bovis to
120
pyrimethamine resistance, is maintained as a distinct linear chromosome that assembles into
121
chromatin, and undergoes expansion of its telomeres. Significantly, the Bbrad51 gene expressed
122
from BbACc3 under regulation by its autologous elements provided normal levels of
123
complementation of the Bbrad51 knockout upon challenge with methylmethane sulfonate
124
(MMS). Although further optimization is desirable, this platform may facilitate long-term
125
complementation of essential genes for studies connecting genotype and phenotype, particularly
126
where improper regulation would be problematic. 127 130
The orthologous Bbrad51 gene was identified. The B. bovis rad51 gene 131
(Bbrad51) was identified by first searching the genus Babesia non-redundant proteome (S1
132
Table). The search was initiated with the Saccharomyces cerevisiae Rad51 sequence
133
(CAA45563) as bait, using BLAST, with default parameters [18]. In this initial search, proteins
134
XP_001609877 (E= 2e-103) and XP_001609660 (E= 4e-15) were identified as candidates. To
135
cast a wider net and ensure capture of all candidates, CAA45563 was again used to initiate an
136
iterative search of the genus Babesia non-redundant protein database, using Psi-BLAST [19, 20]
137
with default parameters [19]. After three iterations the top B. bovis candidate, XP_001609877,
138
had a significance score of E = 3e-156, and three additional proteins of the Rad51/DMC1/RadA
139
superfamily were also identified. Each of these proteins then was used individually in reciprocal
140
iterative searches of the NCBI non-redundant protein database, excluding the genus Babesia,
141
using Delta-BLAST and composition-based statistics to provide their presumptive identities [19,
142
20]. These proteins included the second-best fit, XP_001609660 (E = 3e-86; XRCC3-like),
143
XP_001610815 (E= 8e-16; Rad51 homolog 2-like), and XP_001609995 (E= 0.077; XRCC2-
144
like). Genes encoding putative orthologs of all four proteins are found among various other . CC-BY 4.0 International license
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ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
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certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
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piroplasms (S1 Table; adapted from PiroplasmaDB). For XP_001609877 this search yielded over
146
200 significant hits of E= 0.0, all of which were confirmed or putative eukaryotic Rad51
147
molecules sharing 47-49% identity. Alignment of the four candidates with a number of
148
confirmed or predicted Rad51 proteins demonstrated excellent alignment of all functional
149
domains and motifs (S1 Fig.). An alignment tree was created by the Neighbor-joining method,
150
using Jukes-Cantor protein distance measures and 100 bootstraps to assess robustness. The tree
151
shows that, among the apicomplexan parasites Rad51 proteins cluster in apparent clades (Fig. 130
The orthologous Bbrad51 gene was identified. The B. bovis rad51 gene 152
1A, colored blocks), suggesting shared selection pressures within individual groups. P. knowlesi
153
was the only exception, failing to cluster with other Plasmodium spp. XP_001609877 clusters
154
with other eukaryotic Rad51 proteins, forming an apparent clade with other piroplasmids, yet is
155
clearly quite different. By contrast, the other three B. bovis RecA/RadA/Rad51-related proteins
156
cluster more closely with the Escherichia coli RecA protein included as outlier. An analysis of
157
XP_001609877 for probable structural domains by the Conserved Domains Database yielded
158
specific hits of PTZ00035 ("Rad51 protein, provisional"; residues 6-343; E= 0e+00) and
159
cd01123 ("Rad51_DMC1_radA, P-loop NTPase superfamily"; residues 105-339; E= 3.33e-111),
160
as well as a lower-confidence hit in the N-terminus for pfam14520, a small "helix-hairpin-helix"
161
domain (residues 39-83; E= 2.02e-03). The pfam14520 "helix-hairpin-helix" domain, which is
162
common to Rad51 proteins, was lacking from the other three candidates. Further support for the
163
putative assignment of this protein as a legitimate Rad51 ortholog was obtained by ab initio
164
prediction of its three-dimensional structure, using Robetta software (http://robetta.bakerlab.org/)
165
[21, 22]. The predicted structure models were superimposed onto the crystal structure of the S. 166
cerevisiae Rad51 H352Y mutant protein (PDB accession 3LDA) [23], using the Chimera
167
Matchmaker algorithm [24]. The predicted three-dimensional structure of XP_001609877
168
(model 4) was found to coordinate spatially extremely well with the 3LDA crystal structure,
169
including Walker A and B motifs, residue Q298 regulating access of ATP to the active site [23],
170
and the interaction domain sequences pivotal to filament formation (Fig. 1B). Based on these
171
results we refer to XP_001609877 as BbRad51, and BBOV_II003540 as the Bbrad51 gene
172
encoding this protein. The Bbrad51 gene was annotated with two predicted introns [25], a
173
structural model that was confirmed by RT-PCR amplification, cloning, and sequencing of the
174
full-length transcript. Flanking sequences were recovered through 5'- and 3'-RACE reactions (see
175
Methods). The results exactly matched the published B. bovis T2Bo isolate genome sequence, 145
piroplasms (S1 Table; adapted from PiroplasmaDB). For XP_001609877 this search yielded over
146
200 significant hits of E= 0.0, all of which were confirmed or putative eukaryotic Rad51
147
molecules sharing 47-49% identity. Alignment of the four candidates with a number of
148
confirmed or predicted Rad51 proteins demonstrated excellent alignment of all functional
149
domains and motifs (S1 Fig.). 130
The orthologous Bbrad51 gene was identified. The B. bovis rad51 gene ;
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certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
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bioRxiv preprint 176
including predicted start and stop codons, and intron boundaries (Fig. 2A; [15, 25]). Active
177
transcription of Bbrad51 by asexual stages has been reported previously [26], and was confirmed
178
here by RT-PCR (S2 Fig., panel A). 180
Fig. 1. Relationship of BbRad51 to other Rad51 proteins. A. Alignment tree of the four B. 181
bovis RecA/RadA/Rad51-related proteins (accession numbers in red) with established or
182
predicted Rad51 proteins of other species (Neighbor-joining; Kimura protein distance
183
measure; 100 bootstrap replicates; numbers at nodes are bootstrap values). Direct
184
experimental evidence supports the catalysis of canonical RecA/RadA/Rad51 activities by
185
those proteins whose accession number is indicated in blue. BbRad51 (black arrow) clusters
186
with, but is comparatively dissimilar to, putative Rad51 proteins of other Piroplasmida (pink
187
panel). In contrast, the three remaining B. bovis proteins cluster more closely with the
188
prokaryotic (E. coli) RecA protein included as outlier to the alignment, and with several algal
189
Rad51 proteins. All four B. bovis proteins cluster away from other apicomplexan parasites
190
(Haemosporida, green panel; Coccidia, yellow panel) or higher eukaryotes. Interestingly,
191
Cryptosporidium spp. cluster separately from other Coccidia (blue panel). B. The predicted
192
three-dimensional structure of BbRad51 (blue ribbon), superimposed upon the crystal
193
structure of the S. cerevisiae Rad51 H352Y mutant (3LDA [23], tan ribbon), shows excellent
194
conformational coherence. Indicated on BbRad51 are the Walker A (red) and Walker B
195
(green) motifs, subunit interaction domains (orange), and the position of residue Q298 that
196
acts as “gatekeeper” controlling access of ATP to the active site (purple). Corresponding
197
ScRad51 residues are shown in yellow. ScRad51 has a disordered N-terminal extension that
198
fails to yield usable crystal data [71], resulting in a lack of sequence to align with that region
199
of BbRad51. 201
Fig. 2. Bbrad51 locus and knock-out strategy. A. The wt Bbrad51 locus structure was
202
confirmed to be comprised of three exons/ two introns. 130
The orthologous Bbrad51 gene was identified. The B. bovis rad51 gene An alignment tree was created by the Neighbor-joining method,
150
using Jukes-Cantor protein distance measures and 100 bootstraps to assess robustness. The tree
151
shows that, among the apicomplexan parasites Rad51 proteins cluster in apparent clades (Fig. 152
1A, colored blocks), suggesting shared selection pressures within individual groups. P. knowlesi
153
was the only exception, failing to cluster with other Plasmodium spp. XP_001609877 clusters
154
with other eukaryotic Rad51 proteins, forming an apparent clade with other piroplasmids, yet is
155
clearly quite different. By contrast, the other three B. bovis RecA/RadA/Rad51-related proteins
156
cluster more closely with the Escherichia coli RecA protein included as outlier. An analysis of
157
XP_001609877 for probable structural domains by the Conserved Domains Database yielded
158
specific hits of PTZ00035 ("Rad51 protein, provisional"; residues 6-343; E= 0e+00) and
159
cd01123 ("Rad51_DMC1_radA, P-loop NTPase superfamily"; residues 105-339; E= 3.33e-111),
160
as well as a lower-confidence hit in the N-terminus for pfam14520, a small "helix-hairpin-helix"
161
domain (residues 39-83; E= 2.02e-03). The pfam14520 "helix-hairpin-helix" domain, which is
162
common to Rad51 proteins, was lacking from the other three candidates. Further support for the
163
putative assignment of this protein as a legitimate Rad51 ortholog was obtained by ab initio
164
prediction of its three-dimensional structure, using Robetta software (http://robetta.bakerlab.org/)
165
[21, 22]. The predicted structure models were superimposed onto the crystal structure of the S. 166
cerevisiae Rad51 H352Y mutant protein (PDB accession 3LDA) [23], using the Chimera
167
Matchmaker algorithm [24]. The predicted three-dimensional structure of XP_001609877
168
(model 4) was found to coordinate spatially extremely well with the 3LDA crystal structure,
169
including Walker A and B motifs, residue Q298 regulating access of ATP to the active site [23],
170
and the interaction domain sequences pivotal to filament formation (Fig. 1B). Based on these
171
results we refer to XP_001609877 as BbRad51, and BBOV_II003540 as the Bbrad51 gene
172
encoding this protein. The Bbrad51 gene was annotated with two predicted introns [25], a
173
structural model that was confirmed by RT-PCR amplification, cloning, and sequencing of the
174
full-length transcript Flanking sequences were recovered through 5'- and 3'-RACE reactions (see . CC-BY 4.0 International license
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certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
this version posted April 11, 2019. 130
The orthologous Bbrad51 gene was identified. The B. bovis rad51 gene For brevity, CE11rad51ko1H5 is
222
hereafter referred to as “ko1H5”. The structure of the modified Bbrad51 locus and loss or gain of
223
Bbrad51 or gfp-bsd transcription, respectively, was confirmed by diagnostic PCR, RT-PCR, and
224
Southern blotting, confirming the replacement of Bbrad51 with the gfp-bsd selection cassette
225
from pRad51KO (Fig. 2B; S2 and S3 Figures). PCR-mediated cloning and sequencing of the
226
modified locus confirmed that error-free double-crossover homologous recombination (HR) had
227
occurred. Two additional independent knockout clonal lines, CE11rad51ko2 and
228
CE11rad51ko3 (for brevity, referred to as “ko2” and “ko3”, respectively), were established
229
approximately one year later. These two lines were confirmed by diagnostic PCR and sequencing
230
of the insertion sites (not shown). Because of the lag in obtaining the latter two lines, tests of
231
phenotype initially were performed with ko1H5 and subsequently confirmed with the latter two. 232
As the results were generated in different experiments they are presented separately. 233
234
Bbrad51 knockout had no effects on growth or morphology Knockout 207
reactions and Southern blotting (S2 Table), and the sites to which each anneals, are indicated. 208
B. Diagnostic PCR was used to initially confirm Bbrad51 knock-out in each line, using the
209
primer pairs indicated. Locus structure was fully confirmed for the ko1H5 mutant line (lanes
210
ko1H5), using PCR, Southern blotting and sequencing. Bbrad51 knock-out was supported by
211
diagnostic PCR for CE11rad51ko2 and CE11rad51ko3 (lanes Rad51ko2 and Rad51ko3,
212
respectively). 214
Bbrad51-null B. bovis lines were established. Bbrad51 is a single copy gene in
215
the haploid B. bovis genome [25]. Therefore, a single-locus double-crossover strategy was used
216
to replace the gene with a selection cassette expressing GFP-Blasticidin-s deaminase fusion
217
protein (gfp-bsd; a gift from C.E. Suarez [10, 14]). The transfection plasmid, pRad51KO (Fig. 218
2A), was constructed and used to transform B. bovis CE11 line parasites to blasticidin-s
219
resistance. The transformed culture, and the clonal line CE11rad51ko1, and subclones
220
CE11rad51ko1H5, CE11rad51ko1C3, and CE11rad51ko1H6 derived from it, were screened by
221
PCR (primers used in this study are provided in S2 Table). For brevity, CE11rad51ko1H5 is
222
hereafter referred to as “ko1H5”. 130
The orthologous Bbrad51 gene was identified. The B. bovis rad51 gene The structure of the modified Bbrad51 locus and loss or gain of
223
Bbrad51 or gfp-bsd transcription, respectively, was confirmed by diagnostic PCR, RT-PCR, and
224
Southern blotting, confirming the replacement of Bbrad51 with the gfp-bsd selection cassette
225
from pRad51KO (Fig. 2B; S2 and S3 Figures). PCR-mediated cloning and sequencing of the
226
modified locus confirmed that error-free double-crossover homologous recombination (HR) had
227
occurred. Two additional independent knockout clonal lines, CE11rad51ko2 and
228
CE11rad51ko3 (for brevity, referred to as “ko2” and “ko3”, respectively), were established
229
approximately one year later. These two lines were confirmed by diagnostic PCR and sequencing
230
of the insertion sites (not shown). Because of the lag in obtaining the latter two lines, tests of
231
phenotype initially were performed with ko1H5 and subsequently confirmed with the latter two. 232
As the results were generated in different experiments they are presented separately. 130
The orthologous Bbrad51 gene was identified. The B. bovis rad51 gene The gene was replaced by double
203
homologous crossover, with a selectable gfp-bsd cassette (purple arrow) driven by the B. 204
bovis EF1-B hemi-promoter [10, 14], using flanking sequences from the locus (crossover
205
regions shown as green arrows). The structures of the Bbrad51 wt locus (top), pRAD51KO
206
plasmid (center), and the disrupted Bbrad51 locus (bottom) are shown. Primers used in PCR . CC-BY 4.0 International license
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certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
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ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
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certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
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bioRxiv preprint 207
reactions and Southern blotting (S2 Table), and the sites to which each anneals, are indicated. 208
B. Diagnostic PCR was used to initially confirm Bbrad51 knock-out in each line, using the
209
primer pairs indicated. Locus structure was fully confirmed for the ko1H5 mutant line (lanes
210
ko1H5), using PCR, Southern blotting and sequencing. Bbrad51 knock-out was supported by
211
diagnostic PCR for CE11rad51ko2 and CE11rad51ko3 (lanes Rad51ko2 and Rad51ko3,
212
respectively). 213
214
Bbrad51-null B. bovis lines were established. Bbrad51 is a single copy gene in
215
the haploid B. bovis genome [25]. Therefore, a single-locus double-crossover strategy was used
216
to replace the gene with a selection cassette expressing GFP-Blasticidin-s deaminase fusion
217
protein (gfp-bsd; a gift from C.E. Suarez [10, 14]). The transfection plasmid, pRad51KO (Fig. 218
2A), was constructed and used to transform B. bovis CE11 line parasites to blasticidin-s
219
resistance. The transformed culture, and the clonal line CE11rad51ko1, and subclones
220
CE11rad51ko1H5, CE11rad51ko1C3, and CE11rad51ko1H6 derived from it, were screened by
221
PCR (primers used in this study are provided in S2 Table). 234
Bbrad51 knockout had no effects on growth or morphology. Knockout Mean differences in growth rate of as little as 2% per generation would result in
246
larger differences than were observed. Thus, within the limits of sensitivity of our assay no
247
differences in growth rates were observed between wild type and knockout lines. 248
249
Fig. 3. Growth of wild type and Bbrad51 knockout parasites. Shown is the comparative
250
growth over a 48h period (mean ± 1 s.d.) of A. B. bovis CE11 wt and the Bbrad51 knockout
251
clonal line, ko1H5, and of B. CE11 wt and the CE11rad51ko2 and CE11rad51ko3 lines. The
252
48h period of the assay represents 5-6 cell cycles. The data in A. and B. were generated in
253
independent experiments separated by a year, and so are presented separately. CE11, white
254
bars; ko1H5, hatched bars; CE11rad51ko2, gray bars; CE11rad51ko3, dark gray bars. 255
256
Assessment of MMS-sensitivity. One common means of assessing contributions of a
257
protein to DNA repair is to determine sensitivity to DNA damage deliberately induced by
258
chemical insult. In this study, we employed exposure to methylmethane sulfonate (MMS), a
259
chemical which alkylates guanines to 7-methylguanine and adenines to 3-methyladenine [28]. To
260
apply that approach in this situation, sensitivity of B. bovis CE11 to MMS first was titrated over
261
a range from 0 to 2000 M, employing a strategy of exposure to MMS for 90 minutes, with a
262
buffer washout of the MMS, followed by growth for 72 hours (S4 Fig.). Initially, we used a
263
microscopic approach to follow parasite growth, to avoid any concerns about MMS effects on
264
erythrocytes or residual DNA fluorescence from dead parasites. The results ranged from little or
265
no effect at ≤250 M to complete killing by exposure to 2000 M MMS. B. bovis CE11 and
266
ko1H5 lines then were compared routinely over a range from 125- 1000 M. B. bovis ko1H5 was 249
Fig. 3. Growth of wild type and Bbrad51 knockout parasites. Shown is the comparative
250
growth over a 48h period (mean ± 1 s.d.) of A. B. bovis CE11 wt and the Bbrad51 knockout
251
clonal line, ko1H5, and of B. CE11 wt and the CE11rad51ko2 and CE11rad51ko3 lines. The
252
48h period of the assay represents 5-6 cell cycles. The data in A. and B. 234
Bbrad51 knockout had no effects on growth or morphology. Knockout 235
of Bbrad51 had no observable effects on parasite morphology at the level of light microscopy
236
(not shown). Despite the expected significance of Rad51 function to DNA repair and genome . CC-BY 4.0 International license
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certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
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doi:
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ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
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bioRxiv preprint 237
stability, no significant differences were observed in the growth rates of wild type and Bbrad51
238
knockout lines (Fig. 3). The growth data shown was obtained with a SybrGreen-based assay of
239
cumulative DNA content to avoid the subjectivity associated with microscopic assays, although
240
microscopic assessments of the percentage of parasitized erythrocytes on Giemsa-stained smears
241
of cultured parasites confirmed these results (not shown). Growth was followed for 48 hours
242
only, to avoid any artifactual differences caused by a need for media changes or cell
243
manipulations. However, as these parasites are not developmentally synchronous in their growth,
244
and have an 8-10 hour asexual cell cycle [27], this period represents approximately 5-6 complete
245
cell cycles. Mean differences in growth rate of as little as 2% per generation would result in
246
larger differences than were observed. Thus, within the limits of sensitivity of our assay no
247
differences in growth rates were observed between wild type and knockout lines. 248 239
cumulative DNA content to avoid the subjectivity associated with microscopic assays, although
240
microscopic assessments of the percentage of parasitized erythrocytes on Giemsa-stained smears
241
of cultured parasites confirmed these results (not shown). Growth was followed for 48 hours
242
only, to avoid any artifactual differences caused by a need for media changes or cell
243
manipulations. However, as these parasites are not developmentally synchronous in their growth,
244
and have an 8-10 hour asexual cell cycle [27], this period represents approximately 5-6 complete
245
cell cycles. 234
Bbrad51 knockout had no effects on growth or morphology. Knockout were generated in
253
independent experiments separated by a year, and so are presented separately. CE11, white
254
bars; ko1H5, hatched bars; CE11rad51ko2, gray bars; CE11rad51ko3, dark gray bars. 256
Assessment of MMS-sensitivity. One common means of assessing contributions of a
257
protein to DNA repair is to determine sensitivity to DNA damage deliberately induced by
258
chemical insult. In this study, we employed exposure to methylmethane sulfonate (MMS), a
259
chemical which alkylates guanines to 7-methylguanine and adenines to 3-methyladenine [28]. To
260
apply that approach in this situation, sensitivity of B. bovis CE11 to MMS first was titrated over
261
a range from 0 to 2000 M, employing a strategy of exposure to MMS for 90 minutes, with a
262
buffer washout of the MMS, followed by growth for 72 hours (S4 Fig.). Initially, we used a
263
microscopic approach to follow parasite growth, to avoid any concerns about MMS effects on
264
erythrocytes or residual DNA fluorescence from dead parasites. The results ranged from little or
265
no effect at ≤250 M to complete killing by exposure to 2000 M MMS. B. bovis CE11 and
266
ko1H5 lines then were compared routinely over a range from 125- 1000 M. B. bovis ko1H5 was . CC-BY 4.0 International license
a
ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
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doi:
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a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
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bioRxiv preprint 267
significantly more susceptible to DNA damage at 250 or 500 M MMS than CE11 wild-type
268
parasites (Fig. 4). In the CE11 line there was little or no effect seen at 250 M. B. bovis CE11
269
parasites exposed to 500 M showed a severe reduction in growth rate that quickly increased,
270
approaching that of 0 M controls by 48 hours. In contrast, ko1H5 parasites were significantly
271
reduced in growth at 250 M, and 500 M-treated parasites failed to regain normal growth even
272
by 72 hours post-treatment. 234
Bbrad51 knockout had no effects on growth or morphology. Knockout These results indicate that BbRad51 plays a role in recovery from
273
DNA damage due to nucleotide alkylation [29-32]. 274
275
Fig. 4. Effects of environmental insult on growth of Bbrad51 knockouts. B. bovis CE11
276
wild-type (CE11), ko1H5 (ko1), and CE11 parasites with pBbrad51wt_comp integrated at the
277
Bbrad51 locus (CE11_comp) were tested for relative sensitivity of growth to a 90-minute
278
exposure to varying concentrations of MMS, in a 72h terminal experiment (upper left panel). 279
Growth characteristics over time are shown for CE11 (upper right), ko1H5 (lower left), and
280
CE11_comp (lower right) following MMS exposure at the various concentrations. These
281
results suggest that the Bbrad51 locus is neither affected by nor refractory to manipulation by
282
recombination. 283
284
Re-integration of Bbrad51 gene. To confirm the contribution of BbRad51 to this
285
phenotype, we attempted to complement B. bovis ko1H5 parasites by replacement of the gfp-bsd
286
selection cassette integrated at the Bbrad51 locus with the vector, pBbrad51wt_comp. 287
pBbrad51wt_comp was designed to integrate by double crossover HR (S5 Fig.), replacing the
288
knockout construct with the intact Bbrad51 wt gene, along with a selection cassette expressing
289
the hDHFR gene [33]. CE11 wild-type parasites also were transformed with this vector to
290
control for any effects on parasite viability or sensitivity to MMS caused by manipulation of the
291
Bbrad51 locus. Importantly, successful complementation by double crossover HR would have
292
resulted in identical Bbrad51 locus structures in both CE11 and ko1H5 parasites. CE11 parasites
293
incorporated this vector on the first attempt. A similar vector, differing only by the addition of
294
enhanced green fluorescent protein (EGFP) sequences to the 5’ end of the Bbrad51 open reading
295
frame, also successfully integrated into the CE11 parasite genome without error in two of two
296
attempts, although each crossed over 3’ to the EGFP sequences and failed to incorporate the 284
Re-integration of Bbrad51 gene. To confirm the contribution of BbRad51 to this
285
phenotype, we attempted to complement B. bovis ko1H5 parasites by replacement of the gfp-bsd
286
selection cassette integrated at the Bbrad51 locus with the vector, pBbrad51wt_comp. 287
pBbrad51wt_comp was designed to integrate by double crossover HR (S5 Fig.), replacing the
288
knockout construct with the intact Bbrad51 wt gene, along with a selection cassette expressing
289
the hDHFR gene [33]. 234
Bbrad51 knockout had no effects on growth or morphology. Knockout CE11 wild-type parasites also were transformed with this vector to
290
control for any effects on parasite viability or sensitivity to MMS caused by manipulation of the
291
Bbrad51 locus. Importantly, successful complementation by double crossover HR would have
292
resulted in identical Bbrad51 locus structures in both CE11 and ko1H5 parasites. CE11 parasites
293
incorporated this vector on the first attempt. A similar vector, differing only by the addition of
294
enhanced green fluorescent protein (EGFP) sequences to the 5’ end of the Bbrad51 open reading
295
frame, also successfully integrated into the CE11 parasite genome without error in two of two
296
attempts, although each crossed over 3’ to the EGFP sequences and failed to incorporate the . CC-BY 4.0 International license
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ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
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doi:
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certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
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bioRxiv preprint 297
fluorescent tag. However, HR readily occurred at this locus. In contrast, ko1H5 parasites could not
298
be complemented in this manner in 11 transformation attempts, 7 performed with linearized
299
vector (favoring double crossover homologous recombination) and 4 with circular vector
300
(favoring single-site integration of the entire plasmid). No integration of the transfection plasmid
301
occurred at the Bbrad51 locus through the intended double-crossover HR, or by single-site
302
integration at the Bbrad51 locus or ectopically at any other site. Our inability to achieve
303
integration via double-crossover HR in ko1H5 suggests that HR is significantly impaired in the
304
absence of BbRad51, consistent with BbRad51 playing a role in HR-based DNA repair. When
305
tested for MMS sensitivity, CE11/pBbrad51wt_comp parasites were indistinguishable from
306
CE11 wild-type (Fig. 4), indicating that modification of the Bbrad51 locus in this way had not
307
affected the ability of the parasite to repair DNA damage caused by MMS. 234
Bbrad51 knockout had no effects on growth or morphology. Knockout While we recognize
308
that CE11/pBbrad51wt_comp does not represent the complementation of the defect on a ko1H5
309
background that we had attempted to achieve, we suggest that this represents the probable
310
outcome of successful complementation of the knockout at the Bbrad51 locus. 311 312
Identification of putative B. bovis centromeres. Because we were unable to 313
complement the knockout phenotype through re-integration of the Bbrad51 gene into the
314
genome, we chose instead to attempt its stable expression from an episomal location. Prior work
315
with an artificial chromosome platform in P. berghei [16] suggested this could be a productive
316
approach for expression of exogenous genes in an HR-compromised parasite line. To create one
317
for B. bovis we first had to recover native B. bovis centromeric and telomeric sequences. Putative
318
B. bovis centromeres were identified by focusing on key features common to centromeres in
319
other organisms, including very high percent A+T content, internal repetitive sequences, and a
320
size large enough to mediate attachment of a kinetochore [34, 35]. In this study we searched the
321
genome for A+T-rich regions that are ≥ 2.5 S.D. below the mean G+C content, using a
322
computational window of 1000 bp and length cutoff of ≥ 2 Kbp. Each region so identified was
323
further analyzed by dotplot analysis for internal repetitive structure. Four regions fitting these
324
criteria were identified in the B. bovis C9.1 line nuclear genome [36], one per region
325
corresponding to the four B. bovis T2Bo chromosomes [25]. The internal repeat structures of the
326
B. bovis T2Bo chromosome 2 centromere and of the C9.1 line chromosome 3 centromere were
327
confirmed by dotplot analyses (S6 Fig.). This was done by plotting each sequence against itself, . CC-BY 4.0 International license
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certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
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doi:
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certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
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bioRxiv preprint 328
using a 21 nucleotide sliding window (not shown). Each candidate matched well with the
329
predicted centromeric regions of the B. bovis T2Bo genome [25], and used by Kawazu and
330
coworkers in constructing the circular centromere-containing plasmid vector, pDHFR-gfp-
331
Bbcent2 [12]. We elected to use the putative centromere from the B. 312
Identification of putative B. bovis centromeres. Because we were unable to bovis C9.1 line chromosome
332
3 due to its shorter length of 2.3 Kbp, as compared with 3.5- 5 Kbp of the other three, and to
333
ensure full compatibility with derivatives of the B. bovis Mexico isolate, such as the C9.1 and
334
CE11 lines. 335
336
Stability of BbACc3. Parasites transformed with BbACc3 (Fig. 5) could be selected with
337
10 M pyrimethamine, the approximate IC90 for this drug, after approximately three weeks. 338
Parasite growth was somewhat slower than non-transformed parasites, with parasites typically
339
reaching a maximum PPE of approximately 3%. To assess stability of BbACc3, established
340
parasites that had been maintained under pyrimethamine selection for 49 days were split into
341
two. One culture was maintained in the presence of 2 M pyrimethamine, whereas the second
342
was grown in the absence of drug for 99 days. Each then was grown for 72h in the presence of a
343
range of pyrimethamine concentrations to measure effective IC50 values, along with B. bovis
344
CE11 and the integrated plasmid line, CE11/pBbrad51wt_comp. As seen in Fig. 6, B. bovis
345
CE11 had an IC50 value for pyrimethamine of approximately 2.5 M, whereas the line carrying
346
the integrated pBbrad51wt_comp plasmid was highly drug-resistant with an IC50 of 34 M. B. 347
bovis CE11/BbACc3 that had been maintained under drug selection exhibited an IC50 of
348
approximately 24 M, indicative of high resistance to drug and expression of hDHFR. In
349
contrast, CE11/BbACc3 that had been grown in the absence of drug pressure was
350
indistinguishable from wild type CE11 parasites (IC50 = 2.7 M). These results demonstrate that
351
significant expression of the exogenous gene, hDHFR, occurs from BbACc3, resulting in a ten-
352
fold increase in IC50. The drop to wild type levels of drug resistance in parasites grown in the
353
absence of continued selection suggests that either silencing of the hDHFR marker or loss of
354
BbACc3 occurred. We did not pursue identification of the cause, but it is clear that when
355
maintained under drug pressure BbACc3 was maintained and that the hDHFR marker remained
356
actively expressed. 336
Stability of BbACc3. Parasites transformed with BbACc3 (Fig. 5) could be selected with
337
10 M pyrimethamine, the approximate IC90 for this drug, after approximately three weeks. 312
Identification of putative B. bovis centromeres. Because we were unable to 338
Parasite growth was somewhat slower than non-transformed parasites, with parasites typically
339
reaching a maximum PPE of approximately 3%. To assess stability of BbACc3, established
340
parasites that had been maintained under pyrimethamine selection for 49 days were split into
341
two. One culture was maintained in the presence of 2 M pyrimethamine, whereas the second
342
was grown in the absence of drug for 99 days. Each then was grown for 72h in the presence of a
343
range of pyrimethamine concentrations to measure effective IC50 values, along with B. bovis
344
CE11 and the integrated plasmid line, CE11/pBbrad51wt_comp. As seen in Fig. 6, B. bovis
345
CE11 had an IC50 value for pyrimethamine of approximately 2.5 M, whereas the line carrying
346
the integrated pBbrad51wt_comp plasmid was highly drug-resistant with an IC50 of 34 M. B. 347
bovis CE11/BbACc3 that had been maintained under drug selection exhibited an IC50 of
348
approximately 24 M, indicative of high resistance to drug and expression of hDHFR. In
349
contrast, CE11/BbACc3 that had been grown in the absence of drug pressure was
350
indistinguishable from wild type CE11 parasites (IC50 = 2.7 M). These results demonstrate that
351
significant expression of the exogenous gene, hDHFR, occurs from BbACc3, resulting in a ten-
352
fold increase in IC50. The drop to wild type levels of drug resistance in parasites grown in the
353
absence of continued selection suggests that either silencing of the hDHFR marker or loss of
354
BbACc3 occurred. We did not pursue identification of the cause, but it is clear that when
355
maintained under drug pressure BbACc3 was maintained and that the hDHFR marker remained
356
actively expressed. 357 . CC-BY 4.0 International license
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certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
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Fig. 5. Components and structure of pBbACc3 and pBbACc3_Bbrad51wt. A. 312
Identification of putative B. bovis centromeres. Because we were unable to CC-BY 4.0 International license
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bioRxiv preprint 389
partially-digested genomic DNAs from B. bovis CE11 wild-type (CE11),
390
ko1H5/BbACc3_Bbrad51wt (ko1H5/Br51), and ko1H5 Bbrad51 knockouts (ko1H5). Lanes
391
are: (1) PstI-cut purified DNA, (lane 2) chromatin partially-digested with 0 U, (lane 3) 0.0075
392
U, (lane 4) 0.015 U, (lane 5) 0.03 U, or (lane 6) 0.06 U of MNase prior to isolation of DNA. 393
Asterisks demonstrate correspondence among the nucleosomal patterns generated from the
394
samples, which form a ladder with 156-159 bp spacing between bands, as previously reported
395
[37]. (center panel) Southern blot of the same gel as in the left panel, probed with -lactamase
396
sequences to detect the pBluescript backbone of BbACc3_Bbrad51wt. (right panel) The same
397
blot stripped and re-probed with Bbrad51 coding sequences to detect endogenous Bbrad51
398
(CE11), both BbACc3_Bbrad51wt-provided and non-deleted 3’ sequences (ko1H5/Br51), and
399
remaining non-deleted sequences from the 3’ end of Bbrad51 (ko1H5). B. Southern blot
400
probing of plasmids and genomic DNAs from B. bovis lines with Bbrad51 coding sequences
401
(left panel). The same blot was stripped and re-probed with -lactamase sequences (right
402
panel). Overall telomeric lengthening of BbACc3_Bbrad51wt by 2-3 Kbp can be seen by
403
comparing lanes 4 and 7 (both probes, arrowheads). Telomeric lengthening of 1-1.5 Kbp at
404
each end of BbACc3_Bbrad51wt can be observed with either probe (compare lanes 11 and
405
13; arrowheads). Extreme lengthening of the “left” half of BbACc3 (compare lanes 3 and 6,
406
or 10 and 12, asterisks) when using the -lactamase probe, perhaps due to crossover with a
407
native chromosome. Lanes are: (1) B. bovis CE11 wt gDNA; (2) B. 312
Identification of putative B. bovis centromeres. Because we were unable to The
359
structure of pBbACc3 in circular plasmid form, prior to removal of stuffer DNA. B. 360
Linearized form of BbACc3_Bbrad51wt following removal of stuffer DNA by cleavage with
361
PmeI restriction endonuclease. Rap1 3’UTR, B. bovis rhoptry-associated protein-1 gene 3’
362
untranslated region; bla, -lactamase coding sequences; CDS, coding sequences; Bb Ch3, B. 363
bovis C9.1 line chromosome 3; hDHFR, human dihydrofolate reductase coding sequences. 364
365
Fig. 6. Pyrimethamine-sensitivity in the presence of BbACc3. Sensitivity profiles are
366
shown for B. bovis CE11, and CE11 transformed with BbACc3 or pBbrad51wt_comp. The
367
presence of either the artificial chromosome, BbACc3, or the integrated construct,
368
pBbrad51wt_comp, results in significant resistance to pyrimethamine. The IC50 value for each
369
line was approximately 2.5 M for CE11, 34 M for CE11/pBbrad51wt_comp, 24 M for
370
CE11/BbACc3 (maintained under constant pyrimethamine pressure; BbACc3 (+pyr)), and 2.7
371
M for CE11/BbACc3 three weeks after relief of drug pressure (BbACc3 (no pyr)). 372
373
Adapatation of BbAcC3 derivatives over time. For BbAcC3 and modified
374
derivatives to be used successfully for many purposes would require that this construct behave
375
like a real chromosome, including assembly into chromatin and expansion of telomeric ends for
376
stability. To test for such adaptation over time, we performed partial micrococcal nuclease
377
(MNase) digestion of isolated chromatin. Digestion products were probed by staining with
378
SybrGreen to detect total chromatin, and by Southern blotting to detect BbAcC3_Bbrad51-
379
derived sequences. BbAcC3_Bbrad51wt sequences provided a ladder of digestion products with
380
a nucleosomal periodicity of approximately 157-158 bp, matching that of total native chromatin
381
(Fig. 7A) and previously reported values [37], indicative of assembly on nucleosomes. Southern
382
blot probing of isolated plasmid or genomic DNAs, either uncut or cut with informative
383
restriction endonucleases, revealed the expansion of both ends of established BbAcC3 and
384
BbAcC3_Bbrad51 chromosomes, indicative of telomere lengthening (Fig. 7B). 385
386
Fig. 7. Assembly of BbACc3_Bbrad51wt into chromatin. Isolated parasite nuclei were
387
subjected to digestion with titrated concentrations of MNase, then Southern-blotted and
388
probed with different sequences. A. (left panel) SybrGreen-stained agarose gel of MNase 386
Fig. 7. Assembly of BbACc3_Bbrad51wt into chromatin. Isolated parasite nuclei were
387
subjected to digestion with titrated concentrations of MNase, then Southern-blotted and
388
probed with different sequences. A. (left panel) SybrGreen-stained agarose gel of MNase . 312
Identification of putative B. bovis centromeres. Because we were unable to bovis ko1H5 gDNA; (3)
408
ko1H5/ BbACc3 gDNA (uncut); (4) ko1H5/ BbACc3_Bbrad51 gDNA (uncut); (5) CE11/
409
pBbrad51wt_comp gDNA (uncut); (6) pBbACc3 plasmid (+ PmeI); (7)
410
pBbACc3_Bbrad51wt plasmid (+ PmeI); (8) CE11 gDNA (+ BamHI); (9) ko1H5 gDNA (+
411
BamHI); (10) ko1H5/ BbACc3 gDNA (+ BamHI); (11) ko1H5/ BbACc3_Bbrad51wt gDNA (+
412
BamHI); (12) pBbACc3 plasmid (+ BamHI, PmeI); (13) pBbACc3_Bbrad51wt plasmid (+
413
BamHI, PmeI); (14) CE11 gDNA (+ EcoRI); (15) CE11/ pBbrad51wt comp gDNA (+ 389
partially-digested genomic DNAs from B. bovis CE11 wild-type (CE11),
390
ko1H5/BbACc3_Bbrad51wt (ko1H5/Br51), and ko1H5 Bbrad51 knockouts (ko1H5). Lanes
391
are: (1) PstI-cut purified DNA, (lane 2) chromatin partially-digested with 0 U, (lane 3) 0.0075
392
U, (lane 4) 0.015 U, (lane 5) 0.03 U, or (lane 6) 0.06 U of MNase prior to isolation of DNA. 393
Asterisks demonstrate correspondence among the nucleosomal patterns generated from the
394
samples, which form a ladder with 156-159 bp spacing between bands, as previously reported
395
[37]. (center panel) Southern blot of the same gel as in the left panel, probed with -lactamase
396
sequences to detect the pBluescript backbone of BbACc3_Bbrad51wt. (right panel) The same
397
blot stripped and re-probed with Bbrad51 coding sequences to detect endogenous Bbrad51
398
(CE11), both BbACc3_Bbrad51wt-provided and non-deleted 3’ sequences (ko1H5/Br51), and
399
remaining non-deleted sequences from the 3’ end of Bbrad51 (ko1H5). B. Southern blot
400
probing of plasmids and genomic DNAs from B. bovis lines with Bbrad51 coding sequences
401
(left panel). The same blot was stripped and re-probed with -lactamase sequences (right
402
panel). Overall telomeric lengthening of BbACc3_Bbrad51wt by 2-3 Kbp can be seen by
403
comparing lanes 4 and 7 (both probes, arrowheads). Telomeric lengthening of 1-1.5 Kbp at
404
each end of BbACc3_Bbrad51wt can be observed with either probe (compare lanes 11 and
405
13; arrowheads). Extreme lengthening of the “left” half of BbACc3 (compare lanes 3 and 6,
406
or 10 and 12, asterisks) when using the -lactamase probe, perhaps due to crossover with a
407
native chromosome. Lanes are: (1) B. bovis CE11 wt gDNA; (2) B. bovis ko1H5 gDNA; (3)
408
ko1H5/ BbACc3 gDNA (uncut); (4) ko1H5/ BbACc3_Bbrad51 gDNA (uncut); (5) CE11/
409
pBbrad51wt_comp gDNA (uncut); (6) pBbACc3 plasmid (+ PmeI); (7) 417
Complementation of Bbrad51 knockout phenotype. Because we were It is thus important that we understand well the biology of these
446
parasites, and prepare to defend against their expansion. Key to understanding the biology of 420
CE11 wild type and ko1H5 knockout parasites were transformed by transfection with the artificial
421
chromosome constructs, BbACc3 (as negative control) and BbACc3_Bbrad51wt. 420
CE11 wild type and ko1H5 knockout parasites were transformed by transfection with the artificial
421
chromosome constructs, BbACc3 (as negative control) and BbACc3_Bbrad51wt. 420
CE11 wild type and ko1H5 knockout parasites were transformed by transfection with the artificial
421
chromosome constructs, BbACc3 (as negative control) and BbACc3_Bbrad51wt. 422
pBbACc3_Bbrad51wt is built upon pBbACc3 but also contains the entire uninterrupted Bbrad51
423
locus, including promoter and 3’-termination sequences to facilitate proper regulation (Fig. 5B). 424
ko1H5/ BbAC_Bbrad51wt parasites displayed reduced sensitivity to MMS compared with ko1H5
425
or ko1H5/ BbACc3 parasites, that was not significantly different from wild-type. While not quite
426
reaching wild type levels, this result indicates successful complementation by providing the
427
Bbrad51 locus on the BbACc3 platform. The contribution of ko1H5/ BbBACc3_Bbrad51wt is
428
especially clear when compared with ko1H5/ BbACc3 parasites, which appear to be somewhat
429
less robust overall than ko1H5 (Fig. 8). 431
Fig. 8. Complementation of the MMS-sensitivity phenotype by Bbrad51 expressed from
432
the BbACc3 platform. B. bovis parasites of the CE11 (wild type), ko1H5 (Bbrad51 knockout),
433
ko1H5/BbACc3_Bbrad51wt, and ko1Ht/BbACc3 lines were exposed to varying concentrations
434
of MMS for 90 minutes, followed by drug washout. Parasites then were placed back into
435
culture for 72h, and growth assessed by the SybrGreen method. Growth characteristics were
436
plotted as the growth of each line relative to itself, employing 0 M as maximal growth and
437
1000 M MMS as no growth. Asterisks indicate a difference from the CE11 sample at a
438
significance of p 0.05. The presence of BbACc3_Bbrad51wt complemented ko1H5
439
sensitivity to 250 M MMS to a level that was not significantly different from wild-type
440
parasites. The presence of BbACc3 sequences, however, appears to impact somewhat the
441
overall robustness of parasites. 443
Discussion. With increasing human population density and environmental encroachment,
444
as well as climate change, many zoonotic diseases are emerging and/or expanding in range,
445
including babesiosis [38-40]. It is thus important that we understand well the biology of these
446
parasites, and prepare to defend against their expansion. 417
Complementation of Bbrad51 knockout phenotype. Because we were 418
unable to complement the loss of Bbrad51 by knock-in of the gene via double crossover HR, we
419
attempted complementation with the entire Bbrad51 locus provided via the BbACc3 platform. . CC-BY 4.0 International license
a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
this version posted April 11, 2019. ;
https://doi.org/10.1101/606590
doi:
bioRxiv preprint . CC-BY 4.0 International license
a
ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
this version posted April 11, 2019. ;
https://doi.org/10.1101/606590
doi:
bioRxiv preprint 420
CE11 wild type and ko1H5 knockout parasites were transformed by transfection with the artificial
421
chromosome constructs, BbACc3 (as negative control) and BbACc3_Bbrad51wt. 422
pBbACc3_Bbrad51wt is built upon pBbACc3 but also contains the entire uninterrupted Bbrad51
423
locus, including promoter and 3’-termination sequences to facilitate proper regulation (Fig. 5B). 424
ko1H5/ BbAC_Bbrad51wt parasites displayed reduced sensitivity to MMS compared with ko1H5
425
or ko1H5/ BbACc3 parasites, that was not significantly different from wild-type. While not quite
426
reaching wild type levels, this result indicates successful complementation by providing the
427
Bbrad51 locus on the BbACc3 platform. The contribution of ko1H5/ BbBACc3_Bbrad51wt is
428
especially clear when compared with ko1H5/ BbACc3 parasites, which appear to be somewhat
429
less robust overall than ko1H5 (Fig. 8). 430
431
Fig. 8. Complementation of the MMS-sensitivity phenotype by Bbrad51 expressed from
432
the BbACc3 platform. B. bovis parasites of the CE11 (wild type), ko1H5 (Bbrad51 knockout),
433
ko1H5/BbACc3_Bbrad51wt, and ko1Ht/BbACc3 lines were exposed to varying concentrations
434
of MMS for 90 minutes, followed by drug washout. Parasites then were placed back into
435
culture for 72h, and growth assessed by the SybrGreen method. Growth characteristics were
436
plotted as the growth of each line relative to itself, employing 0 M as maximal growth and
437
1000 M MMS as no growth. Asterisks indicate a difference from the CE11 sample at a
438
significance of p 0.05. The presence of BbACc3_Bbrad51wt complemented ko1H5
439
sensitivity to 250 M MMS to a level that was not significantly different from wild-type
440
parasites. 417
Complementation of Bbrad51 knockout phenotype. Because we were The presence of BbACc3 sequences, however, appears to impact somewhat the
441
overall robustness of parasites. 442
443
Discussion. With increasing human population density and environmental encroachment,
444
as well as climate change, many zoonotic diseases are emerging and/or expanding in range,
445
including babesiosis [38-40]. It is thus important that we understand well the biology of these
446
parasites, and prepare to defend against their expansion. Key to understanding the biology of
447
parasites at the molecular level is the ability to manipulate them genetically, something which is 420
CE11 wild type and ko1H5 knockout parasites were transformed by transfection with the artificial
421
chromosome constructs, BbACc3 (as negative control) and BbACc3_Bbrad51wt. 422
pBbACc3_Bbrad51wt is built upon pBbACc3 but also contains the entire uninterrupted Bbrad51
423
locus, including promoter and 3’-termination sequences to facilitate proper regulation (Fig. 5B). 424
ko1H5/ BbAC_Bbrad51wt parasites displayed reduced sensitivity to MMS compared with ko1H5
425
or ko1H5/ BbACc3 parasites, that was not significantly different from wild-type. While not quite
426
reaching wild type levels, this result indicates successful complementation by providing the
427
Bbrad51 locus on the BbACc3 platform. The contribution of ko1H5/ BbBACc3_Bbrad51wt is
428
especially clear when compared with ko1H5/ BbACc3 parasites, which appear to be somewhat
429
less robust overall than ko1H5 (Fig. 8). 430
431
Fig. 8. Complementation of the MMS-sensitivity phenotype by Bbrad51 expressed from
432
the BbACc3 platform. B. bovis parasites of the CE11 (wild type), ko1H5 (Bbrad51 knockout),
433
ko1H5/BbACc3_Bbrad51wt, and ko1Ht/BbACc3 lines were exposed to varying concentrations
434
of MMS for 90 minutes, followed by drug washout. Parasites then were placed back into
435
culture for 72h, and growth assessed by the SybrGreen method. Growth characteristics were
436
plotted as the growth of each line relative to itself, employing 0 M as maximal growth and
437
1000 M MMS as no growth. Asterisks indicate a difference from the CE11 sample at a
438
significance of p 0.05. The presence of BbACc3_Bbrad51wt complemented ko1H5
439
sensitivity to 250 M MMS to a level that was not significantly different from wild-type
440
parasites. The presence of BbACc3 sequences, however, appears to impact somewhat the
441
overall robustness of parasites. 442
443
Discussion. With increasing human population density and environmental encroachment,
444
as well as climate change, many zoonotic diseases are emerging and/or expanding in range,
445
including babesiosis [38-40]. 417
Complementation of Bbrad51 knockout phenotype. Because we were This was initially attempted
467
with pBbrad51wt_comp, which was designed to replace sequences associated with pBbrad51ko
468
and reinsert the Bbrad51 gene back into the genome at its native locus, where it would be under
469
control of its own promoter and terminator elements. However, in 11 attempts, eight performed
470
with linearized vector and three with circular plasmid, no successful transformations were
471
achieved, whereas three out of three attempts were successful in wild-type B. bovis CE11
472
parasites, using linearized vector. We interpret this difference as support for the pivotal
473
importance of Rad51 proteins to HR, including in B. bovis. However, while supportive, these
474
results also left the Bbrad51 gene without complementation to provide formal proof of its
475
involvement. Therefore, we sought to achieve complementation by another means. 476
477
In recent years, progress has been made in the ability to genetically modify B. bovis, with the
478
development of methods for transient and stable transfection with episomally-maintained
479
plasmids [9, 11, 14, 44-46], and by targeted integration via single- [12, 47] or double-crossover 450
parasite which, if compromised, could lead to its control during infection [41]. At least two
451
mechanisms are used to evade an ongoing immune response: cytoadhesion in the deep
452
vasculature and rapid antigenic variation of the cytoadhesion ligand [6]. Previously, we have
453
shown that antigenic variation relies in large part upon segmental gene conversion (SGC) for
454
modification of the expressed member of the ves gene family [42]. With SGC assumed to be a
455
form of homologous recombination, a process thought to be dependent upon Rad51 proteins, we
456
knocked out the Bbrad51 gene to begin an assessment of its contribution to this phenomenon. 457
Here, we demonstrate that a lack of BbRad51 led to hypersensitivity to MMS, an alkylating
458
agent that methylates adenosine bases [43]. The methyl adducts can result in stalled replication
459
forks and/or single-stranded DNA breaks, both of which can advance to double-stranded DNA
460
breaks, and lead to MMS-hypersensitivity in organisms lacking Rad51 [29-31]. 461 462
We confirmed in two ways that the MMS-hypersensitive phenotype observed in the Bbrad51
463
knockout lines was due to the loss of BbRad51 and not some artifactual secondary effect caused
464
by modification of the genome. 417
Complementation of Bbrad51 knockout phenotype. Because we were Key to understanding the biology of
447
parasites at the molecular level is the ability to manipulate them genetically, something which is
448
not yet well developed for babesial parasites. We have been especially interested in the
449
mechanisms of immune evasion employed by B. bovis as a potential “Achilles heel” of this . CC-BY 4.0 International license
a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
this version posted April 11, 2019. ;
https://doi.org/10.1101/606590
doi:
bioRxiv preprint . CC-BY 4.0 International license
a
ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
this version posted April 11, 2019. ;
https://doi.org/10.1101/606590
doi:
bioRxiv preprint 450
parasite which, if compromised, could lead to its control during infection [41]. At least two
451
mechanisms are used to evade an ongoing immune response: cytoadhesion in the deep
452
vasculature and rapid antigenic variation of the cytoadhesion ligand [6]. Previously, we have
453
shown that antigenic variation relies in large part upon segmental gene conversion (SGC) for
454
modification of the expressed member of the ves gene family [42]. With SGC assumed to be a
455
form of homologous recombination, a process thought to be dependent upon Rad51 proteins, we
456
knocked out the Bbrad51 gene to begin an assessment of its contribution to this phenomenon. 457
Here, we demonstrate that a lack of BbRad51 led to hypersensitivity to MMS, an alkylating
458
agent that methylates adenosine bases [43]. The methyl adducts can result in stalled replication
459
forks and/or single-stranded DNA breaks, both of which can advance to double-stranded DNA
460
breaks, and lead to MMS-hypersensitivity in organisms lacking Rad51 [29-31]. 461
462
We confirmed in two ways that the MMS-hypersensitive phenotype observed in the Bbrad51
463
knockout lines was due to the loss of BbRad51 and not some artifactual secondary effect caused
464
by modification of the genome. First, we integrated pBbrad51wt_comp into the Bbrad51 locus
465
and demonstrated that the phenotype was identical to wt upon exposure to MMS. Secondly, we
466
complemented the phenotype in parasites lacking the Bbrad51 gene. 417
Complementation of Bbrad51 knockout phenotype. Because we were First, we integrated pBbrad51wt_comp into the Bbrad51 locus
465
and demonstrated that the phenotype was identical to wt upon exposure to MMS. Secondly, we
466
complemented the phenotype in parasites lacking the Bbrad51 gene. This was initially attempted
467
with pBbrad51wt_comp, which was designed to replace sequences associated with pBbrad51ko
468
and reinsert the Bbrad51 gene back into the genome at its native locus, where it would be under
469
control of its own promoter and terminator elements. However, in 11 attempts, eight performed
470
with linearized vector and three with circular plasmid, no successful transformations were
471
achieved, whereas three out of three attempts were successful in wild-type B. bovis CE11
472
parasites, using linearized vector. We interpret this difference as support for the pivotal
473
importance of Rad51 proteins to HR, including in B. bovis. However, while supportive, these
474
results also left the Bbrad51 gene without complementation to provide formal proof of its
475
involvement. Therefore, we sought to achieve complementation by another means. 476 477
In recent years, progress has been made in the ability to genetically modify B. bovis, with the
478
development of methods for transient and stable transfection with episomally-maintained
479
plasmids [9, 11, 14, 44-46], and by targeted integration via single- [12, 47] or double-crossover
480
recombination [15, 45]. Although each approach is very useful, each also has its own limitations. . CC-BY 4.0 International license
a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
this version posted April 11, 2019. ;
https://doi.org/10.1101/606590
doi:
bioRxiv preprint . CC-BY 4.0 International license
a
ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
this version posted April 11, 2019. ;
https://doi.org/10.1101/606590
doi:
bioRxiv preprint . CC-BY 4.0 International license
a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
this version posted April 11, 2019. 417
Complementation of Bbrad51 knockout phenotype. Because we were ;
https://doi.org/10.1101/606590
doi:
bioRxiv preprint 481
Transient transfection provides a short-term phenotype only, does not affect the entire parasite
482
population, and may not accurately reflect gene regulation. Segregation of episomes during
483
mitosis is inaccurate, resulting in a heterogeneous parasite population and plasmid loss from the
484
population over time. Such heterogeneity could compromise the ability to interpret
485
complementation of a phenotype that is not dramatic, such as the MMS-sensitivity phenotype of
486
Bbrad51 knockout parasites. This problem was partially solved by the inclusion of centromeric
487
sequences into the plasmid which dramatically improved segregation and resulted in sufficient
488
stability for a plasmid to be maintained in the absence of drug selection for two months [12]. 489
However, a circular construct retains the potential to integrate by single-crossover through
490
sequence elements within the plasmid, potentially compromising ongoing studies. We sought to
491
address this issue through the construction of a linear mini-chromosome, BbACc3, containing
492
centromeric and subtelomeric sequences, and telomeric repeats. BbACc3 is manipulated as a
493
plasmid (pBACc3) in E. coli for modification and production. It is then linearized for
494
transfection into B. bovis, where it is maintained as a linear mini-chromosome with legitimate
495
telomeric ends. Once established, BbACc3_Bbrad51 was found to assemble with nucleosomes
496
into chromatin (Fig. 7A), and to undergo expansion of the telomeric repeat regions (Fig. 7B). 497
These behaviors suggest that, once established, the BbACc3 platform behaves in a manner
498
consistent with a native chromosome. Here, BbACc3_Bbrad51, carrying the Bbrad51 gene and
499
its native flanking 5’ and 3’ regulatory sequences, was demonstrated to complement the lack of
500
BbRad51 in knockout parasites with regard to MMS-sensitivity. Moreover, complementation
501
occurred at levels not significantly different from wild-type, strongly supporting the notion that
502
BbRad51 contributes to DNA repair and consistent with a possible role in segmental gene
503
conversion. The ability to express sequences in a regulated fashion opens the possibility of using
504
BbACc3 as a platform to complement knockout of essential genes, or to perform studies of
505
promoter structure and functional interactions. Moreover, constructs carrying exogenous genes
506
could be used to engender transmission-blocking immunity by including vector proteins, to
507
immunize against other co-transmitted parasites by inclusion of appropriate vaccine genes, or
508
other similar applications. 417
Complementation of Bbrad51 knockout phenotype. Because we were 509
510
In the course of these studies we found it useful to construct and employ an artificial 481
Transient transfection provides a short-term phenotype only, does not affect the entire parasite
482
population, and may not accurately reflect gene regulation. Segregation of episomes during
483
mitosis is inaccurate, resulting in a heterogeneous parasite population and plasmid loss from the
484
population over time. Such heterogeneity could compromise the ability to interpret
485
complementation of a phenotype that is not dramatic, such as the MMS-sensitivity phenotype of
486
Bbrad51 knockout parasites. This problem was partially solved by the inclusion of centromeric
487
sequences into the plasmid which dramatically improved segregation and resulted in sufficient
488
stability for a plasmid to be maintained in the absence of drug selection for two months [12]. 489
However, a circular construct retains the potential to integrate by single-crossover through
490
sequence elements within the plasmid, potentially compromising ongoing studies. We sought to
491
address this issue through the construction of a linear mini-chromosome, BbACc3, containing
492
centromeric and subtelomeric sequences, and telomeric repeats. BbACc3 is manipulated as a
493
plasmid (pBACc3) in E. coli for modification and production. It is then linearized for
494
transfection into B. bovis, where it is maintained as a linear mini-chromosome with legitimate
495
telomeric ends. Once established, BbACc3_Bbrad51 was found to assemble with nucleosomes
496
into chromatin (Fig. 7A), and to undergo expansion of the telomeric repeat regions (Fig. 7B). 497
These behaviors suggest that, once established, the BbACc3 platform behaves in a manner
498
consistent with a native chromosome. Here, BbACc3_Bbrad51, carrying the Bbrad51 gene and
499
its native flanking 5’ and 3’ regulatory sequences, was demonstrated to complement the lack of
500
BbRad51 in knockout parasites with regard to MMS-sensitivity. Moreover, complementation
501
occurred at levels not significantly different from wild-type, strongly supporting the notion that
502
BbRad51 contributes to DNA repair and consistent with a possible role in segmental gene
503
conversion. The ability to express sequences in a regulated fashion opens the possibility of using
504
BbACc3 as a platform to complement knockout of essential genes, or to perform studies of
505
promoter structure and functional interactions. Moreover, constructs carrying exogenous genes
506
could be used to engender transmission-blocking immunity by including vector proteins, to
507
immunize against other co-transmitted parasites by inclusion of appropriate vaccine genes, or
508
other similar applications. 417
Complementation of Bbrad51 knockout phenotype. Because we were 509
510
In the course of these studies we found it useful to construct and employ an artificial 481
Transient transfection provides a short-term phenotype only, does not affect the entire parasite
482
population, and may not accurately reflect gene regulation. Segregation of episomes during
483
mitosis is inaccurate, resulting in a heterogeneous parasite population and plasmid loss from the
484
population over time. Such heterogeneity could compromise the ability to interpret 510
In the course of these studies we found it useful to construct and employ an artificial
511
chromosome, BbACc3, as a platform for the reintroduction of the Bbrad51 gene into B. bovis. . CC-BY 4.0 International license
a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
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https://doi.org/10.1101/606590
doi:
bioRxiv preprint . CC-BY 4.0 International license
a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
this version posted April 11, 2019. ;
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doi:
bioRxiv preprint 512
This allowed us to circumvent the need for homologous recombination to reintegrate Bbrad51
513
sequences into the genome in order to acquire a uniform population. Significantly,
514
BbACc3_Bbrad51wt enabled the complementation of the Bbrad51 knockout phenotype at near-
515
normal levels in response to MMS challenge. Once established, the BbACc3 platform behaved
516
like a native chromosome, forming chromatin and lengthening its telomeres. Despite these
517
positive attributes, a cautionary note must also be provided. Southern blot results suggest that
518
within CE11 wild-type parasites, the major parasite population to grow up during selection
519
appears to have undergone a crossover event between BbACc3 and one of the native
520
chromosomes (Fig. 7B). This appears to have occurred through subtelomeric or telomeric repeat
521
sequences at the “left” end near the -lactamase coding sequences, resulting in significant
522
expansion of that end of BbACc3, and an overall much longer artificial chromosome than in
523
ko1H5/BbACc3_Bbrad51wt, even after expansion of its telomeric repeats. An event of this nature
524
would always be a potential hazard in any HR-competent parasite line. 542
Parasites and culture conditions. B. bovis parasites of the CE11 clonal line were 542
Parasites and culture conditions. B. bovis parasites of the CE11 clonal line were
543
used in experiments [7]. Parasites were grown under microaerophilous stationary phase culture
544
conditions, with an atmosphere of 90:5:5 nitrogen: oxygen: carbon dioxide [49, 50]. Defibrinated
545
bovine blood products were obtained locally from Holstein cows (approved by University of
546
Florida Institutional Animal Care and Use Committee, protocol #201102216) or commercially
547
(Hemostat Laboratories; Dixon, CA). Parasite sensitivity to pyrimethamine was determined as
548
described [51]. Transformed parasites were maintained in medium containing 2.0 M
549
pyrimethamine. 542
Parasites and culture conditions. B. bovis parasites of the CE11 clonal line were
543
used in experiments [7]. Parasites were grown under microaerophilous stationary phase culture
544
conditions, with an atmosphere of 90:5:5 nitrogen: oxygen: carbon dioxide [49, 50]. Defibrinat
545
bovine blood products were obtained locally from Holstein cows (approved by University of
546
Florida Institutional Animal Care and Use Committee, protocol #201102216) or commercially
547
(Hemostat Laboratories; Dixon, CA). Parasite sensitivity to pyrimethamine was determined as
548
described [51]. Transformed parasites were maintained in medium containing 2.0 M
549
pyrimethamine. 550
551
Validation of Bbrad51 gene and BbRad51 identity. Identification of
552
BbRad51 (accession no. XP_001609877) was achieved by reciprocal Psi-BLAST searches of th
553
B. bovis T2Bo genome [25], using S. cerevisiae Rad51 (accession no. CAA45563) to initiate th
554
query. Alignments used sequences publicly available through Genbank, EuPathDB database
555
(http://eupathdb.org/eupathdb/), and/or Wellcome- Sanger Institute pathogens ftp site
556
(ftp://ftp.sanger.ac.uk/pub/pathogens/). Annotation of Bbrad51 gene structure was confirmed a
557
both the genome (GeneID: 5478106) and transcript (XM_001609827.1) levels as described
558
below. Virtual translation and routine manipulations performed with CLC-Bio Main Workbenc
559
(CLC-Bio, Arrhenius, Denmark). Conserved Domains search was performed through the
560
National Center for Biotechnology CDD webserver
561
(http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml) [52, 53]. Three-dimensional structural
562
modeling was performed through the Robetta webserver (http://robetta.bakerlab.org/) [22, 54]. 563
Structural superimpositions and graphics were created with the Chimera software package of th
564
Resource for Biocomputing, Visualization, and Informatics at the University of California, San
565
Francisco (www.cgl.ucsf.edu/chimera/), using the Matchmaker algorithm [24]. 566 417
Complementation of Bbrad51 knockout phenotype. Because we were A useful improvement of
525
BbACc3 could be made through the inclusion of replication origin sequences that might improve
526
the ease of initial establishment of the chromosome, which in these experiments was no faster
527
than selection of double crossover integration mutants. Thus, while artificial chromosome
528
technology holds considerable promise for certain applications and was invaluable in this study,
529
it will require optimization to meet its full potential. Regardless, BbACc3 provides a new option
530
in the toolkit for genetic manipulation of B. bovis and perhaps other babesial parasites where its
531
centromeric and regulatory sequences may function, and will be made freely available to the
532
scientific community. . CC-BY 4.0 International license
a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
this version posted April 11, 2019. ;
https://doi.org/10.1101/606590
doi:
bioRxiv preprint . CC-BY 4.0 International license
a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
this version posted April 11, 2019. ;
https://doi.org/10.1101/606590
doi:
bioRxiv preprint 551
Validation of Bbrad51 gene and BbRad51 identity. Identification of 552
BbRad51 (accession no. XP_001609877) was achieved by reciprocal Psi-BLAST searches of the
553
B. bovis T2Bo genome [25], using S. cerevisiae Rad51 (accession no. CAA45563) to initiate the
554
query. Alignments used sequences publicly available through Genbank, EuPathDB database
555
(http://eupathdb.org/eupathdb/), and/or Wellcome- Sanger Institute pathogens ftp site
556
(ftp://ftp.sanger.ac.uk/pub/pathogens/). Annotation of Bbrad51 gene structure was confirmed at
557
both the genome (GeneID: 5478106) and transcript (XM_001609827.1) levels as described
558
below. Virtual translation and routine manipulations performed with CLC-Bio Main Workbench
559
(CLC-Bio, Arrhenius, Denmark). Conserved Domains search was performed through the
560
National Center for Biotechnology CDD webserver 561
(http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml) [52, 53]. Three-dimensional structural
562
modeling was performed through the Robetta webserver (http://robetta.bakerlab.org/) [22, 54]. 563
Structural superimpositions and graphics were created with the Chimera software package of the
564
Resource for Biocomputing, Visualization, and Informatics at the University of California, San
565
Francisco (www.cgl.ucsf.edu/chimera/), using the Matchmaker algorithm [24]. 567
Validation of Bbrad51 gene structure. B. bovis gDNAs were isolated as described Tobacco acid pyrophosphatase was used to remove the 5’-cap structure for adapter
590
addition, and reverse transcription using M-MuLV and random decamers. Nested PCR
591
amplification of 5’-untranslated sequences was performed using primers EAM2 and 5’-RACE
592
outer, and EAM4 and 5’-RACE inner primers. Products were cloned into pCR-TOPO-Blunt and
593
sequenced. The 3’-untranslated sequences were obtained by addition of the 3’-RACE adapter,
594
and nested PCR amplification with primers EAM1 and 3’-RACE outer, and EAM3 and 3’-
595
RACE inner. Products were directly cloned into pCR-TOPO-Blunt and sequenced. 596 572
[58]. When needed, cultures were grown to elevated levels of percent parasitized erythrocytes by
573
“dilution enrichment” [59]. Packed erythrocytes were emulsified with either TRIzol reagent
574
(Invitrogen; Waltham, MA) or RiboZol reagent (Amresco; Solon, OH), extracted twice with
575
chloroform and precipitated from 2-propanol. A 2244 bp segment containing the 1134 bp
576
Bbrad51 gene and its 5´ and 3´ intergenic regions was amplified from B. bovis C9.1 gDNA by
577
polymerase chain reaction (PCR) with Phusion High-Fidelity DNA Polymerase (New England
578
BioLabs; Beverley, MA) using primers EAM6 and EAM18. This segment was cloned into
579
pCR2.1-TOPO-TA (Invitrogen) to create plasmid pBbRad51. Bbrad51 structure was confirmed
580
by PCR amplification of the 1134 bp gene within the 2244 bp amplicon, using flanking primers
581
EAM6 and EAM18. The amplicon was cloned into pCR2.1-TOPO-TA (Invitrogen) and
582
sequenced through the University of Florida Sanger Sequencing Core by primer-walking. To
583
characterize Bbrad51 transcripts, cDNA was made from B. bovis C9.1 line total RNA, using M-
584
MuLV Reverse Transcriptase (New England Biolabs; Beverley, MA) and oligo-d(T) primer. 585
Full-length coding sequences were obtained by PCR amplification of cDNA with primers
586
EAM50 and EAM51. The amplicon was cloned into pCR2.1-TOPO-TA and sequenced. To
587
obtain non-coding transcript sequences, RNA Ligase-mediated rapid amplification of cDNA
588
ends (RLM-RACE) was performed, using the FirstChoice RLM-RACE kit (Ambion; Waltham,
589
MA). Tobacco acid pyrophosphatase was used to remove the 5’-cap structure for adapter
590
addition, and reverse transcription using M-MuLV and random decamers. Nested PCR
591
amplification of 5’-untranslated sequences was performed using primers EAM2 and 5’-RACE
592
outer, and EAM4 and 5’-RACE inner primers. Products were cloned into pCR-TOPO-Blunt and
593
sequenced. 567
Validation of Bbrad51 gene structure. B. bovis gDNAs were isolated as described 567
Validation of Bbrad51 gene structure. B. bovis gDNAs were isolated as described
568
[55, 56], but with prior ammonium chloride lysis of the erythrocytes [57]. Alternatively, QIAmp
569
Mini Spin Columns (Qiagen) were sometimes used, following manufacturer’s instructions. For
570
RNA extractions, cultures were grown in erythrocytes from which host leukocytes were removed
571
by filtration through Whatman CF-11 cellulose (GE Healthcare Life Sciences; Pittsburgh, PA) . CC-BY 4.0 International license
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certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
this version posted April 11, 2019. ;
https://doi.org/10.1101/606590
doi:
bioRxiv preprint . CC-BY 4.0 International license
a
ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
this version posted April 11, 2019. ;
https://doi.org/10.1101/606590
doi:
bioRxiv preprint 572
[58]. When needed, cultures were grown to elevated levels of percent parasitized erythrocytes by
573
“dilution enrichment” [59]. Packed erythrocytes were emulsified with either TRIzol reagent
574
(Invitrogen; Waltham, MA) or RiboZol reagent (Amresco; Solon, OH), extracted twice with
575
chloroform and precipitated from 2-propanol. A 2244 bp segment containing the 1134 bp
576
Bbrad51 gene and its 5´ and 3´ intergenic regions was amplified from B. bovis C9.1 gDNA by
577
polymerase chain reaction (PCR) with Phusion High-Fidelity DNA Polymerase (New England
578
BioLabs; Beverley, MA) using primers EAM6 and EAM18. This segment was cloned into
579
pCR2.1-TOPO-TA (Invitrogen) to create plasmid pBbRad51. Bbrad51 structure was confirmed
580
by PCR amplification of the 1134 bp gene within the 2244 bp amplicon, using flanking primers
581
EAM6 and EAM18. The amplicon was cloned into pCR2.1-TOPO-TA (Invitrogen) and
582
sequenced through the University of Florida Sanger Sequencing Core by primer-walking. To
583
characterize Bbrad51 transcripts, cDNA was made from B. bovis C9.1 line total RNA, using M-
584
MuLV Reverse Transcriptase (New England Biolabs; Beverley, MA) and oligo-d(T) primer. 585
Full-length coding sequences were obtained by PCR amplification of cDNA with primers
586
EAM50 and EAM51. The amplicon was cloned into pCR2.1-TOPO-TA and sequenced. To
587
obtain non-coding transcript sequences, RNA Ligase-mediated rapid amplification of cDNA
588
ends (RLM-RACE) was performed, using the FirstChoice RLM-RACE kit (Ambion; Waltham,
589
MA). 567
Validation of Bbrad51 gene structure. B. bovis gDNAs were isolated as described The 3’-untranslated sequences were obtained by addition of the 3’-RACE adapter,
594
and nested PCR amplification with primers EAM1 and 3’-RACE outer, and EAM3 and 3’-
595
RACE inner. Products were directly cloned into pCR-TOPO-Blunt and sequenced. 596 597
Bbrad51 knockout plasmid assembly. The Bbrad51 gene was knocked out by a
598
double crossover homologous recombination strategy [60], with plasmid pRad51KO (Fig. 2A). 599
Plasmid pRad51KO was designed to replace all but the 3’-terminal 388 bp of Bbrad51 coding
600
sequences with a selectable gfp-bsd marker (a gift from C.E. Suarez). This region was retained
601
because of the close proximity (320 bp) of Bbrad51 to the gene downstream encoding a
602
conserved mechanosensitive channel-like protein, XP_001609876. To assemble pRad51KO, the 597
Bbrad51 knockout plasmid assembly. The Bbrad51 gene was knocked out by a
598
double crossover homologous recombination strategy [60], with plasmid pRad51KO (Fig. 2A). 599
Plasmid pRad51KO was designed to replace all but the 3’-terminal 388 bp of Bbrad51 coding
600
sequences with a selectable gfp-bsd marker (a gift from C.E. Suarez). This region was retained
601
because of the close proximity (320 bp) of Bbrad51 to the gene downstream encoding a
602
conserved mechanosensitive channel-like protein, XP_001609876. To assemble pRad51KO, the . CC-BY 4.0 International license
a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
this version posted April 11, 2019. ;
https://doi.org/10.1101/606590
doi:
bioRxiv preprint . CC-BY 4.0 International license
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ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
this version posted April 11, 2019. ;
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doi:
bioRxiv preprint 603
EF1 gene “B” hemi-promoter for driving the bsd selectable marker was PCR-amplified from B. 604
bovis CE11 line gDNA with primers EAM42 and EAM43. Bbrad51 3’-end targeting sequences
605
were amplified from plasmid pBbRAD51, using primers EAM49 and EAM56. The EF1-B
606
segment and Bbrad51 3’ targeting sequences were combined by crossover PCR [61] with
607
EAM43 and EAM56 to form fragment 3’-rad51-EF1-B. The 92 bp B. bovis -tubulin
608
terminator region (T3) was amplified from plasmid pDS-bsd [11], using primers EAM45 and
609
EAM46. 567
Validation of Bbrad51 gene structure. B. bovis gDNAs were isolated as described The gfp-bsd fusion gene from plasmid pGFP-Bsd [14], was combined with the T3
610
fragment by crossover PCR, using primers EAM44 and EAM46 and including the T3 fragment
611
in the reaction. The Bbrad51 upstream targeting sequence was added to the gfp-bsd-T3 fragment
612
by crossover PCR with primers EAM44 and EAM47, and including pBbRad51 in the reaction, to
613
form fragment gfp-bsd-T3-5’-rad51. The two fragments were combined by crossover PCR, using
614
primers EAM56 and EAM47 and inserted into pBluescript II-KS(+). A single missense mutation
615
was identified within the bsd sequence. This was corrected by PCR-amplification of the two
616
halves of the insert with EAM61 and EAM47, and EAM62 and EAM56. The two fragments
617
were fused by crossover PCR, using only EAM47 and EAM56, and reinserted into pBluescript
618
II-KS(+). The corrected plasmid, pRAD51KO, was transformed into Escherichia coli DH5. 619
Proper construction was confirmed by full sequencing, and the corrected plasmid used for
620
subsequent plasmid production. 622
Validation of Bbrad51 knock-out. Parasite populations recovered initially were
623
screened directly with the Phusion Blood Direct PCR Kit (Thermo Scientific), using primers
624
EAM75 and EAM77 within the coding sequences of the immediately flanking genes. Non-
625
mutated parasites generated a band of 2.4 Kbp, whereas a proper knockout yielded a band of 3.4
626
Kbp. RT-PCR with primers EAM50 and EAM51 confirmed the lack of Bbrad51 transcripts. 627
Southern blots were performed as described [42]. DNA fragments, alkaline-transferred to nylon
628
membranes (Amersham Hybond-N+, GE Healthcare), were cross-linked with 50 mJ ultraviolet in
629
a GS Gene Linker UV chamber (BioRad; Hercules, CA). Oligonucleotide probes were end-
630
labeled with -[32P]ATP, using polynucleotide kinase. 631 632
Parasite growth assays. Parasite growth was assayed by counting Giemsa-stained . CC-BY 4.0 International license
a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
this version posted April 11, 2019. ;
https://doi.org/10.1101/606590
doi:
bioRxiv preprint . CC-BY 4.0 International license
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ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
this version posted April 11, 2019. 567
Validation of Bbrad51 gene structure. B. bovis gDNAs were isolated as described ;
https://doi.org/10.1101/606590
doi:
bioRxiv preprint 633
smears, with samples collected at 0, 24, and 48h growth (approximately 0, 3 and 6 cell cycles
634
[62]). Alternatively, in some experiments a DNA-based SYBR Green I method was performed,
635
essentially as described [63, 64], on parasites grown in bovine erythrocytes depleted of
636
leukocytes [58]. For measuring sensitivity to pyrimethamine, parasites were grown in complete
637
medium containing serial 10-fold dilutions of pyrimethamine for 72 hours, at concentrations
638
from 0 to 100 M. For experiments involving MMS, parasites were exposed at 2% ppe to
639
various concentrations of MMS (diluted in complete medium) for 90 minutes at room
640
temperature, then were washed two times at 6000 x g.min at room temperature with 1x VYM
641
buffer [65] to remove MMS. Parasites then were resuspended in complete medium, and placed
642
under normal culture conditions. In initial experiments, cells were diluted 1/10 into a 10%
643
packed cell volume suspension of untreated uninfected erythrocytes, and were placed back into
644
culture. Smears were made at 0, 24, 48, and 72 hours post-treatment, and Giemsa-stained for
645
microscopic counting of percent parasitized erythrocytes. After validation of effect, subsequent
646
experiments were performed using the SybrGreen method, with starting values of 0.2%
647
parasitized erythrocytes, and terminal collection at 72 hours only. An initial titration experiment
648
with B. bovis CE11 established 125 M to 1000 M as the informative range. 649 647
parasitized erythrocytes, and terminal collection at 72 hours only. An initial titration experiment
648
with B. bovis CE11 established 125 M to 1000 M as the informative range. 650
Assembly of complementation constructs. Two different complementation 650
Assembly of complementation constructs. Two different complementation
651
strategies were used in this project. (i) In the first strategy, plasmid pBbrad51wt_comp (S5 Fig.)
652
was engineered to replace all sequences associated with the knockout plasmid by double
653
crossover homologous recombination. The goal was to replace them with the wild-type Bbrad51
654
locus, concomitant with integration of a human dihydrofolate reductase (hDHFR) expression
655
cassette, to enable selection of transformants with pyrimethamine [51]. pBbrad51wt_comp was
656
made by PCR-amplifying Bbrad51 3’ terminator sequences from B. bovis C9.1 line gDNA with
657
primers DA255 + DA256. The hDHFR selection cassette was moved from pBACc3 by
658
amplification with primers DA254 + DA253. pBluescript-SK(+) was linearized with XhoI +
659
HindIII restriction endonucleases, and the three sequences were assembled simultaneously, using
660
InFusion reagents (Clontech). The Bbrad51 locus, including complete promoter through
661
immediate 3’ sequences, was amplified from B. bovis C9.1 line gDNA with primers DA248 +
662
DA252. The Bbrad51 locus was inserted, using InFusion reagents, into the intermediate
663
construct that had been opened with XbaI + HindIII. (ii) The second strategy was to reintroduce . CC-BY 4.0 International license
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ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
this version posted April 11, 2019. ;
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doi:
bioRxiv preprint . CC-BY 4.0 International license
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certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
this version posted April 11, 2019. ;
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doi:
bioRxiv preprint 664
the Bbrad51 locus in the context of the BbACc3 artificial chromosome, which did not require
665
reintegration into the genome but allowed expression controlled by autologous regulatory
666
elements. Construction of pBACc3 (circular plasmid form) was begun with destruction of the
667
NotI site (nt 669-676) within the multiple cloning site (mcs) of pBluescript-KS(+), by a fill-in
668
reaction with T4 DNA polymerase and blunt ligation [66]. This product was partially digested
669
with RcaI, and amplified by inverse-PCR with Phusion Hotstart II polymerase (New England
670
Biolabs; Beverley, MA), using primers DA162 and DA163 [67]. 650
Assembly of complementation constructs. Two different complementation PCR-amplification of the hDHFR coding sequences
700
with DA164 + DA165, the B. bovis actin promoter with DA166 + DA167, and the Rap1 gene 3'
701
terminator sequences with DA168 + DA169 was used to recover these segments. A two-step
702
crossover PCR was used to combine hDHFR coding sequence with promoter and terminator
703
[61], simultaneously removing all HindIII, EcoRV, and PstI sites. The full-length cassette first
704
was inserted into the SalI site of pBS2. The complete cassette then was removed with KpnI and
705
PstI, and integrated into the artificial chromosome construct, using Gibson Assembly reagent
706
mix (New England Biolabs; Beverley, MA) [69], yielding pBACc3. The final pBACc3 basic
707
vector construct is 11,030 bp (Fig. 5A). To make BbACc3_Bbrad51wt, the entire Bbrad51 locus
708
was amplified from pBbrad51wt_comp with primers DA248 + DA281, and inserted with
709
NEBuilder reagents (New England Biolabs) into pBACc3 opened with BamHI and PstI. All
710
added sequences and modified regions of the plasmid were fully Sanger-sequenced to confirm
711
proper construction. Constructs were transfected into E. coli, using strains DH5 (Invitrogen),
712
NEB10 (New England Biolabs), or Stellar (Clontech), by electroporation. For transfection,
713
pBACc3_Bbrad51wt was linearized with PmeI to release the stuffer sequence and expose the
714
telomeric ends (Fig. 5B). pBACc3 will be made available upon request. 650
Assembly of complementation constructs. Two different complementation CC-BY 4.0 International license
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ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
this version posted April 11, 2019. ;
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doi:
bioRxiv preprint 695
single candidate was identified from chromosome 3, corresponding to position 2922284-
696
2925108. Centromeric sequences were amplified with primers DA174 and DA175, and inserted
697
into the NotI site by ligation with T4 DNA ligase. The hDHFR selection cassette was moved
698
from the plasmid pDHFR-gfp-Bbcent2 (a gift from Shin-Ichiro Kawazu [12]) into the
699
intermediate construct, in multiple steps. PCR-amplification of the hDHFR coding sequences
700
with DA164 + DA165, the B. bovis actin promoter with DA166 + DA167, and the Rap1 gene 3'
701
terminator sequences with DA168 + DA169 was used to recover these segments. A two-step
702
crossover PCR was used to combine hDHFR coding sequence with promoter and terminator
703
[61], simultaneously removing all HindIII, EcoRV, and PstI sites. The full-length cassette first
704
was inserted into the SalI site of pBS2. The complete cassette then was removed with KpnI and
705
PstI, and integrated into the artificial chromosome construct, using Gibson Assembly reagent
706
mix (New England Biolabs; Beverley, MA) [69], yielding pBACc3. The final pBACc3 basic
707
vector construct is 11,030 bp (Fig. 5A). To make BbACc3_Bbrad51wt, the entire Bbrad51 locus
708
was amplified from pBbrad51wt_comp with primers DA248 + DA281, and inserted with
709
NEBuilder reagents (New England Biolabs) into pBACc3 opened with BamHI and PstI. All
710
added sequences and modified regions of the plasmid were fully Sanger-sequenced to confirm
711
proper construction. Constructs were transfected into E. coli, using strains DH5 (Invitrogen),
712
NEB10 (New England Biolabs), or Stellar (Clontech), by electroporation. For transfection,
713
pBACc3_Bbrad51wt was linearized with PmeI to release the stuffer sequence and expose the
714
telomeric ends (Fig. 5B). pBACc3 will be made available upon request. 715 695
single candidate was identified from chromosome 3, corresponding to position 2922284-
696
2925108. Centromeric sequences were amplified with primers DA174 and DA175, and inserted
697
into the NotI site by ligation with T4 DNA ligase. The hDHFR selection cassette was moved
698
from the plasmid pDHFR-gfp-Bbcent2 (a gift from Shin-Ichiro Kawazu [12]) into the
699
intermediate construct, in multiple steps. 650
Assembly of complementation constructs. Two different complementation Upon circularization of the
671
amplicon nt 2888-2940 were deleted, replacing the RcaI site at position 2881-2886 with a new
672
NotI site, and creating the 2920 bp intermediate plasmid, pBS2. B. bovis C9.1 line telomeric
673
sequences were inserted into the remaining RcaI site at nt 1873-1878 (original numbering), as
674
follows: B. bovis C9.1 line genomic DNA (gDNA) was PCR-amplified with Phusion polymerase
675
(New England Biolabs; Beverley, MA), using primers specific to unique sequences at nt
676
2,592,052-2,592,072 (DA170) and 948-923 (DA171) of the B. bovis T2Bo chromosome 3,
677
coupled with primers DA172 or DA173, respectively. The identification of centromeric
678
sequences is provided in S6 Fig.. The 5’ halves of DA170 and DA171 were complementary to
679
vector sequences flanking the remaining RcaI site. The 3’ halves of primers DA172 and DA173
680
each represented three telomeric repeats. The 3'-most repeat of each contained an extra T, an
681
infrequent imperfection in the telomeric repeats of B. bovis (T2Bo isolate reference genome,
682
accession no. AAXT01000001.1) [25], whereas the 5’ halves overlapped with Anaplasma
683
marginale genomic sequences. A 483 bp stuffer fragment, corresponding to nucleotides 145014-
684
145482 of the A. marginale Florida isolate genome (accession no. NC_012026), then was
685
amplified with primers DA152 and DA153, and a two-step crossover PCR approach was used to
686
assemble all three amplicons into one fragment containing PmeI restriction sites flanking the
687
stuffer [61]. The fused amplicon was inserted into the RcaI site, using InFusion reagents
688
(Clontech Laboratories, Inc.; Mountain View, CA). Putative centromeres were identified in the
689
B. bovis C9.1 line genome (ftp://ftp.sanger.ac.uk/pub/pathogens/Babesia/). Artemis v.16.0.0
690
(http://www.sanger.ac.uk/science/tools/artemis) was used to observe sequence %GC content,
691
identifying stretches > 2.5 standard deviations below the mean in a running window of 1000 nt
692
[68]. Sequences were considered candidates when the AT-rich segment was 2 Kbp or longer and
693
possessed no annotated open reading frames. Candidate sequences were further analyzed by dot-
694
plot to identify those containing significant internal repetitive sequence structure (S6 Fig.). A . CC-BY 4.0 International license
a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
this version posted April 11, 2019. ;
https://doi.org/10.1101/606590
doi:
bioRxiv preprint . 716
Parasite transformation, and maintenance of BbACc3. Parasites were 717
washed into cytomix buffer [70], then transfected with knockout or complementation vectors
718
dissolved in cytomix. Transfection was achieved by electroporation with 10 pmol of DNA, under
719
conditions of 1.25 kV, 25 F, and 200 resistance, as described [11]. Prior to transfection,
720
pRad51ko and pBbrad51wt_comp were linearized with NotI. pBACc3_Bbrad51wt linearized
721
with PmeI, releasing the A. marginale "stuffer" DNA and generating linear BbACc3_Bbrad51wt
722
with telomeric ends. It was found not to be necessary to purify BbACc3 away from cleaved
723
stuffer DNA prior to transfection. In some experiments parasites were transfected with pBACc3
724
as supercoiled plasmid. Following transfection, parasites were placed into culture for 24 h in the
725
absence of drug selection. To effect selection, pyrimethamine then was added and maintained at . CC-BY 4.0 International license
a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
this version posted April 11, 2019. ;
https://doi.org/10.1101/606590
doi:
bioRxiv preprint . CC-BY 4.0 International license
a
ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
this version posted April 11, 2019. ;
https://doi.org/10.1101/606590
doi:
bioRxiv preprint 726
a concentration of 2 M (the approximate IC90 concentration), beginning 24h post-transfection. 727
In some experiments, drug pressure was removed for 21 days (approximately ≥ 60 cell cycles)
728
from already-transformed parasites, then was reapplied at varying concentrations to assess for
729
loss of drug-resistance. 730
731
Observation of chromosome maintenance and chromatin assembly. 732
Maintenance of BbACc3_Bbrad51wt as a linear chromosomal element was confirmed by
733
isolation of genomic DNA [55], with resolution of uncut and restriction-digested DNAs on a
734
0.7% agarose gel. DNAs were alkaline-transferred to nylon membranes as described [42], and
735
hybridized with probes to Bbrad51, -lactamase, centromeric, and hDHFR sequences. Probes
736
were generated by PCR amplification of pBACc3_Bbrad51wt sequences with primers EAM8 +
737
EAM11, DA290 + DA291, DA189 + DA190, or DA164 + DA165, etc., respectively. One g of
738
each amplicon was labeled with digoxigenin, and detected with anti-digoxigenin-HRP antibodies
739
and CSPD substrate, according to supplier’s instructions (Sigma Chemical; St. Louis, MO). 716
Parasite transformation, and maintenance of BbACc3. Parasites were Blots
740
were imaged with a FluorChemR instrument (Protein Simple; San Jose, CA) by detection of
741
luminescence. The stability of telomeric end lengths was tested by assessing the lengths of
742
BamHI restriction fragments of PmeI-linearized BbACc3 constructs used to transfect, and
743
BbACc3 constructs recovered after establishment of transformed parasites in culture. gDNAs
744
were cleaved with BamHI, and probed with the above-mentioned probes. pBACc3 digested with
745
PmeI to release stuffer sequences was used as control. Probes were labeled with 11-dUTP-
746
digoxigenin, following manufacturer’s instructions, and detected as described above. To observe
747
assembly of BbACc3 and BbACc3_Bbrad51wt into chromatin, nuclei were isolated from
748
transformed parasites, the chromatin partially digested with micrococcal nuclease (MNase), and
749
the fragments isolated as described [37]. To observe overall nucleosomal assembly and spacing,
750
digestion products were stained in-gel with SybrGold. Similar gels were also alkaline-blotted to
751
nylon membranes and probed by Southern blotting, as described above. Alkali-labile probes
752
were used to facilitate stripping of blots and re-probing 726
a concentration of 2 M (the approximate IC90 concentration), beginning 24h post-transfection. 727
In some experiments, drug pressure was removed for 21 days (approximately ≥ 60 cell cycles)
728
from already-transformed parasites, then was reapplied at varying concentrations to assess for
729
loss of drug-resistance. 731
Observation of chromosome maintenance and chromatin assembly. 732
Maintenance of BbACc3_Bbrad51wt as a linear chromosomal element was confirmed by
733
isolation of genomic DNA [55], with resolution of uncut and restriction-digested DNAs on a
734
0.7% agarose gel. DNAs were alkaline-transferred to nylon membranes as described [42], and
735
hybridized with probes to Bbrad51, -lactamase, centromeric, and hDHFR sequences. Probes
736
were generated by PCR amplification of pBACc3_Bbrad51wt sequences with primers EAM8 +
737
EAM11, DA290 + DA291, DA189 + DA190, or DA164 + DA165, etc., respectively. One g of
738
each amplicon was labeled with digoxigenin, and detected with anti-digoxigenin-HRP antibodies
739
and CSPD substrate, according to supplier’s instructions (Sigma Chemical; St. Louis, MO). Blots
740
were imaged with a FluorChemR instrument (Protein Simple; San Jose, CA) by detection of
741
luminescence. The stability of telomeric end lengths was tested by assessing the lengths of
742
BamHI restriction fragments of PmeI-linearized BbACc3 constructs used to transfect, and
743
BbACc3 constructs recovered after establishment of transformed parasites in culture. gDNAs
744
were cleaved with BamHI, and probed with the above-mentioned probes. pBACc3 digested with
745
PmeI to release stuffer sequences was used as control. Probes were labeled with 11-dUTP-
746
digoxigenin, following manufacturer’s instructions, and detected as described above. To observe
747
assembly of BbACc3 and BbACc3_Bbrad51wt into chromatin, nuclei were isolated from
748
transformed parasites, the chromatin partially digested with micrococcal nuclease (MNase), and
749
the fragments isolated as described [37]. To observe overall nucleosomal assembly and spacing,
750
digestion products were stained in-gel with SybrGold. Similar gels were also alkaline-blotted to 754
Acknowledgments. The authors thank Carlos Suarez and Shen-ichi Kawazu for
755
providing plasmids pGFP-Bsd and pDHFR-gfp-Bbcent2, respectively, and Kevin Brown and . CC-BY 4.0 International license
a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
this version posted April 11, 2019. ;
https://doi.org/10.1101/606590
doi:
bioRxiv preprint . CC-BY 4.0 International license
a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
this version posted April 11, 2019. ;
https://doi.org/10.1101/606590
doi:
bioRxiv preprint 756
Linda Bloom for providing critical early comments. 731
Observation of chromosome maintenance and chromatin assembly. We are indebted to Allison Vansickle for
757
assistance with bovine blood collection and maintenance of in vitro cultures. . CC-BY 4.0 International license
a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
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doi:
bioRxiv preprint 758
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65. Vega CA, Buening GM, Rodriguez SD, Carson CA. Concentration and enzyme content
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66. Maniatis T, Fritsch EF, Sambrook J. Molecular Cloning: A Laboratory Manual. Cold
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67. Triglia T, Peterson MG, Kemp DJ. A procedure for in vitro amplification of DNA
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segments that lie outside the boundaries of known sequences. Nucleic Acids Res. 1988;16:8186. 934
segments that lie outside the boundaries of known sequences. Nucleic Acids Res. 1988;16:8186. 935
68. Rutherford K, Parkhill J, Crook J, Horsnell T, Rice P, Rajandream M-A, et al. Artemis:
936
sequence visualization and annotation. Bioinformatics (Oxford, England). 2000;16(10):944-5. 937
69
Gibson DG Young L Chuang R Y Venter JC Hutchison CA III Smith HO 934
segments that lie outside the boundaries of known sequences. Nucleic Acids Res. 1988;16:8186. 935
68. Rutherford K, Parkhill J, Crook J, Horsnell T, Rice P, Rajandream M-A, et al. Artemis:
936
sequence visualization and annotation. Bioinformatics (Oxford, England). 2000;16(10):944-5. 937
69. Gibson DG, Young L, Chuang R-Y, Venter JC, Hutchison CA, III., Smith HO. 938
Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods. 939
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936
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69. Gibson DG, Young L, Chuang R-Y, Venter JC, Hutchison CA, III., Smith HO. 938
Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods. 935
68. Rutherford K, Parkhill J, Crook J, Horsnell T, Rice P, Rajandream M-A, et al. Artemis:
936
sequence visualization and annotation. Bioinformatics (Oxford, England). 2000;16(10):944-5. 938
Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods. 939
2009;6:343-5. 940
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certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
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bioRxiv preprint 945
Supporting Information Captions 946
S1 Fig.. Similarity of B. bovis RecA/RadA/Rad51 superfamily-related proteins to known or
947
putatively annotated Rad51 proteins from other species. Those proteins for which there is
948
experimental support for catalysis of canonical Rad51 functions are indicated in blue in Fig. 1. 949
The Walker A and Walker B motifs are indicated here by red and blue box overlays,
950
respectively. This alignment provided the basis for the phylogenetic tree shown in Fig. 1A. 946
S1 Fig.. Similarity of B. bovis RecA/RadA/Rad51 superfamily-related proteins to known or
947
putatively annotated Rad51 proteins from other species. Those proteins for which there is
948
experimental support for catalysis of canonical Rad51 functions are indicated in blue in Fig. 1. 949
The Walker A and Walker B motifs are indicated here by red and blue box overlays,
950
respectively. This alignment provided the basis for the phylogenetic tree shown in Fig. 1A. 0
respectively. This alignment provided the basis for the phylogenetic tree show 951
S2 Fig.. RT-PCR amplification of Bbrad51 and gfp-bsd transcripts. Total RNAs were
952
isolated from B. bovis CE11 subclones B8, C2, and C5, the initial uncloned CE11Bbrad51
953
knockout and three clonal lines derived from it (ko1C3, ko1H5, and ko1H6), and lines
954
CE11rad51ko2 and CE11rad51ko3. A. cDNAs made with oligo[dT] primer were amplified
955
with primers EAM8 and EAM11 [11, 51] for detection of Bbrad51 transcripts. B. cDNAs were
956
amplified with XW119 and XW121 [11, 51] for detection of gfp-bsd transcripts. Results
957
demonstrate Bbrad51 transcription by wild type lines but not by knockouts. Conversely, gfp-bsd
958
transcripts are present in knockout but not wild type lines. 959
S3 Fig.. Southern blot analysis of Bbrad51 locus. A. Schematic diagram of the Bbrad51 locus
960
in B. bovis CE11 wild type (bottom) and knockout (top) parasites, and the locations of restriction
961
endonuclease and primer binding sites. The tables provide the anticipated sizes (in bp) of specific
962
fragments detected with the indicated probes, based upon the genome sequence. B. Southern
963
blots of B. bovis CE11 wild type, and the initial CE11Bbrad51 knockout parasite population
964
both early and late (12 months later) in selection prior to cloning (left panel), after probing with
965
Bbrad51-specific oligonucleotide probe, EAM10. Initial B. bovis CE11Bbrad51 clonal lines
966
ko1C3, ko1H5, and ko1H6 are shown in the right panel. C. 945
Supporting Information Captions The same blots shown in panel B are
967
shown after being stripped and re-probed with oligonucleotide DA101R, specific for gfp
968
sequences. The numbers above the bands indicate the sizes of the bands in bp. 969
S4 Fig.. Titration of B. bovis CE11 wild type parasite sensitivity to MMS. In initial 970
experiments to determine useful concentrations of MMS to observe phenotype, parasites were
971
exposed to varying concentrations of MMS for 90 minutes at room temperature, then washed and
972
placed back into culture for 72h. Smears were made at 0, 48, and 72h post-treatment, fixed and
973
stained with Giemsa stain, and manually counted by microscopy in a blinded fashion. Growth is . CC-BY 4.0 International license
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certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
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ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
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indicated as percent parasitized erythrocytes (PPE). This experiment indicated that a range from
975
250 M to 1000 M MMS provided useful results. 974
indicated as percent parasitized erythrocytes (PPE). This experiment indicated that a range from
975
250 M to 1000 M MMS provided useful results. ructure of the pBbrad51wt_comp double crossover complementation plasmid. 977
This plasmid was designed to integrate into the genome by double-crossover homologous
978
recombination via sequences upstream and downstream to the Bbrad51 coding sequences. In
979
doing so, the strategy was to recreate the Bbrad51 locus, but with a short 3’-untranslated region
980
for regulation, followed by the hDHFR selection cassette. The plasmid was introduced into
981
parasites after NotI linearization, and selected for growth in the presence of pyrimethamine. This
982
occurred as intended in three of three attempts with wild type parasites, but failed in 11 attempts
983
with B. bovis ko1H5 parasites. 984
S6 Fig.. Identification of the B. bovis C9.1 chromosome 2 centromere. A. B. bovis T2Bo
985
isolate genomic sequences [25] were scanned with the “GC Content (%) with 2.5 SD Cutoff”
986
subroutine of Artemis v. 16.0.0 [68], using a sliding window of 1000 bp. Stretches of sequence >
987
2.5 standard deviations below the mean G+C content, and ≥ 2 Kbp in length, were identified. 988
Shown is the region of chromosome 3 identified by this strategy. Sequence comprising
989
nucleotides 2922285-2925108 of the B. bovis C9.1 line genome [36], corresponding to
990
nucleotides 29912-32736 of the B. bovis T2Bo isolate chromosome 3 [25], were recovered by
991
PCR and used in the construction of pBbACc3. B. Dot-plots of the putative B. bovis C9.1 line
992
chromosome 3 (left plot) and T2Bo isolate chromosome 2 (right plot) centromeres against
993
themselves. The internal repeat structure of each is apparent from the plots, with the
994
chromosome 2 centromere having a larger major repeat domain. The darkness of spots indicates
995
the degree of similarity, with the dark blue diagonal lines indicating identity. Values within the
996
range from 40-100% identity is shown. When plotted against one another there is no evidence for
997
any specific sequence relationship (not shown). 998 . CC-BY 4.0 International license
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certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. ructure of the pBbrad51wt_comp double crossover complementation plasmid. It is made available under
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Directional properties of polar paramagnetic molecules subject to congruent electric, magnetic and optical fields
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Directional properties of polar paramagnetic molecules subject to congruent electric
and optical fields
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2015 New J. Phys. 17 045017
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My IOPscience Abstract Weshowthat congruent electric, magneticand non-resonant opticalfieldsactingconcurrentlyon a
polar paramagnetic(and polarizable) moleculeoffer possibilitiesto both amplifyand controlthe
directionalityoftheensuingmolecular statesthat surpassthoseavailablein double-field combinations
or in singlefieldsalone.At thecoreofthesetriple-field effectsistheliftingofthedegeneracyofthe
projection quantum number M bythemagneticfield superimposed on theopticalfield and a
subsequent couplingofthemembersofthe‘doubled’ (for stateswith
≠
M
0) tunnelingdoubletsdue
to theopticalfield byeven aweakelectrostaticfield. Directionalpropertiesofpolarparamagneticmoleculessubjectto
congruentelectric,magneticandopticalfields Ketan Sharma and BretislavFriedrich1
Fritz-Haber-Institut der Max-Planck-Gesellschaft Faradayweg4-6,D-14195Berlin, Germany
1 Author to whom anycorrespondenceshould beaddressed. E-mail:ketan@fhi-berlin.mpg.deand bretislav.friedrich@fhi-berlin.mpg.de Keywords:Starkeffect, Zeeman effect,induced-dipoleinteraction, polarizability, nonresonant laser field,combined fields, alignment
orientation Content from thiswork
maybeused under the
termsoftheCreative
CommonsAttribution 3.0
licence. Directional properties of polar paramagnetic molecules subject to congruent electric, magnetic
and optical fields This content has been downloaded from IOPscience. Please scroll down to see the full text. 2015 New J. Phys. 17 045017
(http://iopscience.iop.org/1367-2630/17/4/045017) This content has been downloaded from IOPscience. Please scroll down to see the full text. View the table of contents for this issue, or go to the journal homepage for more Download details: Please note that terms and conditions apply. NewJ.Phys.17(2015) 045017 doi:10.1088/1367-2630/17/4/045017 PAPER
Directionalpropertiesofpolarparamagneticmoleculessubjectto
congruentelectric,magneticandopticalfields
Ketan Sharma and BretislavFriedrich1
Fritz-Haber-Institut der Max-Planck-Gesellschaft Faradayweg4-6,D-14195Berlin, Germany
1 Author to whom anycorrespondenceshould beaddressed.
E-mail:ketan@fhi-berlin.mpg.deand bretislav.friedrich@fhi-berlin.mpg.de
Keywords:Starkeffect, Zeeman effect,induced-dipoleinteraction, polarizability, nonresonant laser field,combined fields, alignment and OPENACCESS
RECEIVED
16January2015
REVISED
12March 2015
ACCEPTEDFORPUBLICATION
20March 2015
PUBLISHED
24April2015
Content from thiswork
maybeused under the
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CommonsAttribution 3.0
licence. Anyfurther distribution of
thiswork must maintain
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author(s) and thetitleof
thework, journalcitation
and DOI. Anyfurther distribution of
thiswork must maintain
attribution to the
author(s) and thetitleof
thework, journalcitation
and DOI. ©2015IOP PublishingLtd and DeutschePhysikalischeGesellschaft 1.Introduction Herein weshowthat amagneticand opticaldouble field interaction with apolar paramagneticmolecule
maycreatenear-degeneraciesofadditionallevelsthat can beeasilycoupled byeven aweak electricfield
(magneticand opticaland electrictriple-field combination). Thereby,thetriple-field combination could, for
instance,enablefast switchingofdipolar orientation and other dynamicaleffectsthat arenot availablein a
doublemagneticand electricor opticaland electricfield combinationsalone(not to speak about thesingle
fields).At thecoreofthesenoveltriple-field effectsistheliftingofthedegeneracyoftheprojection quantum
number M bythemagneticfield superimposed on theopticalfield and asubsequent couplingofthemembersof
the‘doubled’ (for stateswith
≠
M
0) tunnelingdoubletsdueto theopticalfield byaweakelectrostaticfield. Thispaper isorganized asfollows:In section 2weintroducetherotationalHamiltonian ofa Σ
2
polar
moleculeaswellasitsmatrixrepresentation in theHund’scase(b) basisset. In sections2.1–2.3wepresent,in
turn, thesingle-field Hamiltoniansfor theelectric, magnetic, and opticalpotentials. In section 2.4,wepresent
thefullHamiltonian for theelectricand magneticand opticaltriple-field interaction. In section 3wepresent and
discusstheresultsofour calculationsoftheeigenpropertiesofthepartialHamiltoniansaswellasofthefull
triple-field combined Hamiltonian. Section 4surveysand summarizesour results. Theappendixliststhekey
matrixelementsused in thecalculations, describestheproceduredeveloped to assign thestatesobtained bythe
diagonalization oftheHamiltonian matrix, and liststheconversion factorsneeded to evaluatethedimensionless
parametersused throughout thepaper in termsofcustomaryunits. Thispaper isorganized asfollows:In section 2weintroducetherotationalHamiltonian ofa Σ
2
polar
moleculeaswellasitsmatrixrepresentation in theHund’scase(b) basisset. In sections2.1–2.3wepresent,in
turn, thesingle-field Hamiltoniansfor theelectric, magnetic, and opticalpotentials. In section 2.4,wepresent
thefullHamiltonian for theelectricand magneticand opticaltriple-field interaction. In section 3wepresent and
discusstheresultsofour calculationsoftheeigenpropertiesofthepartialHamiltoniansaswellasofthefull
triple-field combined Hamiltonian. Section 4surveysand summarizesour results. Theappendixliststhekey
matrixelementsused in thecalculations, describestheproceduredeveloped to assign thestatesobtained bythe
diagonalization oftheHamiltonian matrix, and liststheconversion factorsneeded to evaluatethedimensionless
parametersused throughout thepaper in termsofcustomaryunits. 1.Introduction Interactionswith externalelectric, magneticor opticalfieldsprovidethechiefmeansto manipulatethe
rotationaland translationalmotion ofneutralgas-phasemolecules[1]. Theseinteractionscreatedirectional
statesin which themolecular multipolemomentsbecomenon-vanishingin thelaboratoryframeso that space-
fixed fieldscan act upon them. Directionalstatesareat thecoreofnumerousapplicationsin molecular physics,
such asorientation/alignment ofmolecules[2–30],deflection and focusingofmolecular translation [31–34],
molecular trapping[35], attainingtime-resolved photoelectron angular distributions[36–38],diffraction-
from-within [39],separation ofphotodissociation products[40–42],deracemization [43],high-order
harmonicgeneration and orbitalimaging[44–50],quantum simulation [51,52] or quantum computing
[53–59]. Herein, weexaminedirectionalstatescreated byatriple-combination ofcongruent (parallelor antiparallel)
electric, magneticand non-resonant opticalfieldsactingconcurrentlyon linear polar paramagnetic(and
polarizable) molecules. Whiletheelectricand magneticfieldsinteract, respectively, with thebody-fixed electric
and magneticdipolemomentsofthemolecule,thenon-resonant opticalfield couplesto themolecular
anisotropicpolarizabilitytensor. Themolecular effectsgenerated bythedouble-field combinations(electricand
magnetic, electricand optical,magneticand optical) areallsui generisand amount to morethan thesum oftheir
parts. And so doesthetriple-field combination (electricand magneticand optical) which not onlyoffersahigh
efficiencyand flexibilityin amplifyingthedirectionalityofmolecular statesbut isalso offundamentalinterest
per se,assupersymmetry[60–64] aswellasmonodromyand quantum chaos[65, 66] lurk behind the
combined-field effects. Polar paramagneticmoleculesareofpotentialimportancefor many-bodyphysicssimulations, studiesof
crossingsofStark and Zeeman molecular energylevels, and quantum computing. Amongthemost prominent
examplesoflinear polar paramagneticmoleculesaretheubiquitous Σ
2 , Σ
3 , and Π
2
linear species, such asSrF,
SO,and OH. Heteronuclear diatomicsor larger polar moleculesthat contain arare-Earth atom often exhibit
much higher orbitaland spin electronicangular momenta (e.g., CeO isa Φ
3
2 moleculein itselectronicground
state) and, therefore, correspondinglylarger magneticdipolemoments. Therecentlydiscovered LiHevan der
Waalsmolecule[67, 68], apolar and paramagnetichalo species,would also benefit from thestudyofits ©2015IOP PublishingLtd and DeutschePhysikalischeGesellschaft NewJ.Phys.17(2015) 045017 NewJ.Phys.17(2015) 045017 KSharmaand BFriedrich propertiesin combined fields, asthiswould likelyrevealadditionalparticularsabout itsstructureand the
dynamicsofitsformation.Asurveyoflinear polar paramagneticmoleculesalongwith their keypropertiesis
availablein table3of[9].However,our treatment hereisgeneric, makinguseofreduced molecular interaction
parameters,and thereforeapplicableto anypolar paramagneticand polarizablemoleculein agiven electronic
state.For thepurposesofthepresent study, wechosemoleculesin a Σ
2
stateasaprototype. Directionalstatesofmoleculesmayexhibit either orientation (visualized asasingle-headed arrowlibrating
about aspace-fixed axis) and/or alignment (visualized asadouble-headed arrowlibratingabout aspace-fixed
axis).Themoredirectionalthestate,thetighter thelibrationalamplitudeofthearrowand themorecomplete
theprojection ofthecorrespondingdipole(whether permanent or induced) on thespace-fixed axis. Sinceoriented statesmayonlybeofindefiniteparity—otherwisetheywould violatetheparityselection rule
[69]—arecipefor creatingoriented statesisto mixstatesofoppositeparity.Thecoupling—or hybridization—
ofoppositeparitystatescan begenerallyachieved bytheelectricdipoleinteraction, which isthemoreeffectivein
couplingtheopposite-paritylevelsthecloser theylieto oneanother. Close-lyingoppositeparitystatescan be
prepared for largeclassesoflinear moleculesbyeither opticalor magneticfields. In our previouswork aswellas
that ofothers, it hasbeen shown that, for linear molecules, theopposite-paritystatesamenableto facileelectric-
dipolecouplingareeither thequasi-degeneratemembersofthetunnelingdoubletscreated bytheinduced-
dipoleinteraction with anon resonant opticalfield (combination ofelectricand opticalfields)
[4,5, 16,21,22, 30] or theintersectingopposite-parityZeeman levelsthat becomeexactlydegenerateat their
intersection points(combination ofelectricand magneticfields) [9,10,70,71]. Herein weshowthat amagneticand opticaldouble-field interaction with apolar paramagneticmolecule
maycreatenear-degeneraciesofadditionallevelsthat can beeasilycoupled byeven aweak electricfield
(magneticand opticaland electrictriple-field combination). Thereby,thetriple-field combination could, for
instance,enablefast switchingofdipolar orientation and other dynamicaleffectsthat arenot availablein a
doublemagneticand electricor opticaland electricfield combinationsalone(not to speak about thesingle
fields).At thecoreofthesenoveltriple-field effectsistheliftingofthedegeneracyoftheprojection quantum
number M bythemagneticfield superimposed on theopticalfield and asubsequent couplingofthemembersof
the‘doubled’ (for stateswith
≠
M
0) tunnelingdoubletsdueto theopticalfield byaweakelectrostaticfield. 2.Rotational structureofa polar Σ
2
moleculein combined electric, magneticand optical
fields Thephenomenologicalfield-freerotationalHamiltonian ofa Σ
2
moleculeisgiven by[72] (1) γ
=
+
H
BN
N
S
· ,
(1)
0
2 γ
=
+
H
BN
N
S
· ,
0
2 where N and Sare, respectively, therotationaland electronicspin angular momenta,
≡
B
I
2 istherotational
constant,with I themolecule’smoment ofinertiain agiven vibrationalstatehosted bythe Σ
2
electronicstate,
and γisthespin-rotation couplingconstant. Hamiltonian (1) neglectsnuclear spin aswellasthe(small)
magneticmoment arisingdueto therotation ofthemolecule. 1 where N and Sare, respectively, therotationaland electronicspin angular momenta,
≡
B
I
2 istherotational
constant,with I themolecule’smoment ofinertiain agiven vibrationalstatehosted bythe Σ
2
electronicstate,
and γisthespin-rotation couplingconstant. Hamiltonian (1) neglectsnuclear spin aswellasthe(small)
magneticmoment arisingdueto therotation ofthemolecule. Whilefor a Σ
2
statetheelectronicspin angular momentum
=
S
1
2,theorbitalelectronicangular
momentum isidenticallyzero and so isthespin–orbit coupling. A Σ
2
statethusexhibitsaHund’scase(b)
couplingbetween therotationaland electronicangular momenta[72],with theprojectionsofthetotaland spin
electronicangular momenta on themolecular axis(an axisofcylindricalsymmetry) Ω
Σ
=
=
1
2, seefigure1. electronicangular momenta on themolecular axis(an axisofcylindricalsymmetry) Ω
Σ
=
=
1
2, seefigure1. TheHund’scase(b) basisfunctionsarean equallyweighted linear combination ofHund’scase(a) basis
functions, each aproduct ofasymmetrictop wavefunction 2
TheHund’scase(b) basisfunctionsarean equallyweighted linear combination ofHund’scase(a) basis
functions, each aproduct ofasymmetrictop wavefunction D
Ω
π
θ ϕ χ
=
−
+
Ω
Ω
−
−
−
J
M
J
,
,
( 1)
(2
1)
8
( ,
,
)
(2)
M
M
J
, (2) 2 NewJ.Phys.17(2015) 045017 KSharmaand BFriedrich Figure1. Definition ofEuler angles θ ϕ χ
,
,
describingtherotation ofthemolecule-fixed coordinatesx, y, zwith respect to thespace-
fixed coordinates X Y Z
,
,
for adiatomicmoleculedepicted asabar-bell. Thegreen axisisthelineofnodes,perpendicular to both z
and Z. Also shown aretherotational, N,electron spin, S, and total, J,angular momentaaswellastheprojectionsM and Ω
Σ
=
of J
on thespace- and body-fixed axis. Figure1. Definition ofEuler angles θ ϕ χ
,
,
describingtherotation ofthemolecule-fixed coordinatesx, y, zwith respect to thespace-
fixed coordinates X Y Z
,
,
for adiatomicmoleculedepicted asabar-bell. Thegreen axisisthelineofnodes,perpendicular to both z
and Z. Also shown aretherotational, N,electron spin, S, and total, J,angular momentaaswellastheprojectionsM and Ω
Σ
=
of J
on thespace- and body-fixed axis. Figure1. Definition ofEuler angles θ ϕ χ
,
,
describingtherotation ofthemolecule-fixed coordinatesx, y, zwith respect to thespace-
fixed coordinates X Y Z
,
,
for adiatomicmoleculedepicted asabar-bell. Thegreen axisisthelineofnodes,perpendicular to both z
and Z. Also shown aretherotational, N,electron spin, S, and total, J,angular momentaaswellastheprojectionsM and Ω
Σ
=
of J
on thespace- and body-fixed axis. 2.Rotational structureofa polar Σ
2
moleculein combined electric, magneticand optical
fields and aspin function and aspin function Σ
α
β
Σ
Σ
=
+
−
Σ
Σ
+
−
S
S
S
,
(
) !(
)! (3)
S
S (3) with
=
±
J
N
Sthetotal(rotation and electron spin) angular momentum quantum number, M and Ωthe
projectionsofthetotalangular momentum on, respectively, thespace-fixed Z-axisand thebody-fixed z-axis,
D
θ ϕ χ
Ω( ,
,
)
M
J
,
theWigner matrix, with θ ϕ χ
,
,
theEuler angles, and α β
,
thespin functions. Thusfor a Σ
2
state
(
=
S
1
2),therearetwo typesofHund’scase(b) basisfunctions ψ
Ω
Ω
±
=
±
−
−
± N
M
S
J
M
S
J
M
1
2 ,
1
2
, 1
2
,
,
,
1
2
,
,
(4)
⎜
⎟
⎛
⎝
⎞
⎠
⎡
⎣⎢
⎤
⎦⎥ (4) pertainingto
=
±
J
N
1
2, with parity −
( 1)N.Thecorrespondingeigenenergiesare γ
+
=
+
+
+
E
N
M
BN N
N
1
2 ,
(
1)
2
,
(5)
⎜
⎟
⎛
⎝
⎞
⎠
γ
−
=
+
−
+
−
E
N
M
BN N
N
1
2 ,
(
1)
2 (
1). (6)
⎜
⎟
⎛
⎝
⎞
⎠ (5) (6) The± statesofa Σ
2
moleculeareconventionallyreferred to asF1(when
=
+
J
N
1
2) and F2(when
=
−
J
N
1
2). Both Jand N but not Ωaregood quantum numbersfor afield-free Σ
2
molecule. The± statesofa Σ
2
moleculeareconventionallyreferred to asF1(when
=
+
J
N
1
2) and F2(when
=
−
J
N
1
2). Both Jand N but not Ωaregood quantum numbersfor afield-free Σ
2
molecule. 2.1.Interaction with an electricfield 2.1.Interaction with an electricfield 2.1.Interaction with an electricfield Theinteraction potentialfor alinear moleculewith an electricdipolemoment μel alongthemolecule-fixed z-
axissubject to an electrostaticfield εS (aStark field) definingaspace-fixed Z-axis, seefigure2,isgiven by η
θ
= −
V
B
cos ,
(7)
el
el (7) where η
μ ε
≡
B
(8)
S
el
el (8) μ
≡
B
el B isadimensionlessparameter characterizingthestrength oftheStark interaction. Wenotethat theattainable
externalelectricfield εS ismuch weaker than theinternalelectricfield produced bythemolecule’sconstituent
electronsand nucleiand thusitseffect on theelectronicstructureofthemoleculeisnegligible. In what follows
wewilldealsolelywith theeffect oftheexternalfieldson themolecular rotationalstructure. isadimensionlessparameter characterizingthestrength oftheStark interaction. Wenotethat theattainable
externalelectricfield εS ismuch weaker than theinternalelectricfield produced bythemolecule’sconstituent
electronsand nucleiand thusitseffect on theelectronicstructureofthemoleculeisnegligible. In what follows
wewilldealsolelywith theeffect oftheexternalfieldson themolecular rotationalstructure. The
θ
cos
operator (arisingfrom thedirection cosinematrix, seeappendix) mixesHund’scase(b) basis
functionswith thesameM but with
′
N s that differ by±1and thushaveoppositeparities.Asaresult, thestates
created bytheStark interaction areofindefiniteparityand N (and J) ceaseto begood quantum numbers. The 3 NewJ.Phys.17(2015) 045017 KSharmaand BFriedrich Figure2. An electrostaticfield εS directed alongthespace-fixed Z-axisactingon apolar
Σ
+
S
2
1
moleculewith an electricdipole
moment μel alongthemolecule-fixed z-axis. Also shown aretherotational,spin,and totalangular momenta N, S,and J aswellasthe
polar angleθbetween thespace- and molecule-fixed axesand theazimuthalangleϕ uniformlydistributed about theZ-axis.Seetext. Figure2. An electrostaticfield εS directed alongthespace-fixed Z-axisactingon apolar
Σ
+
S
2
1
moleculewith an electricdipole
moment μel alongthemolecule-fixed z-axis. Also shown aretherotational,spin,and totalangular momenta N, S,and J aswellasthe
polar angleθbetween thespace- and molecule-fixed axesand theazimuthalangleϕ uniformlydistributed about theZ-axis.Seetext. Figure3. Amagneticfield directed alongthespace-fixed Z-axisactingon a
Σ
+
S
2
1
moleculewith amagneticdipolemoment μm
alongtheelectronicspin vector S.Also shown aretherotationaland totalangular momenta N and J aswellasthepolar angleθ
between thespace- and molecule-fixed axesand theazimuthalangleϕ uniformlydistributed about theZ-axis.Seetext. Figure3. Amagneticfield directed alongthespace-fixed Z-axisactingon a
Σ
+
S
2
1
moleculewith amagneticdipolemoment μm
alongtheelectronicspin vector S.Also shown aretherotationaland totalangular momenta N and J aswellasthepolar angleθ
between thespace- and molecule-fixed axesand theazimuthalangleϕ uniformlydistributed about theZ-axis.Seetext. onlygood quantum number istheprojection M of J on theZ-axis. N and Jcan, nevertheless, beused asadiabatic
labelsofthestatesin thefield,in which casetheyarefurnished with atilde,
η
∣
→〉→∣
〉
N J M
N J M
˜ , ˜,
;
0
, ,
el
. onlygood quantum number istheprojection M of J on theZ-axis. N and Jcan, nevertheless, beused asadiabatic
labelsofthestatesin thefield,in which casetheyarefurnished with atilde,
η
∣
→〉→∣
〉
N J M
N J M
˜ , ˜,
;
0
, ,
el
. 2.1.Interaction with an electricfield 2.2.Interaction with a magneticfield 2.2.Interaction with a magneticfield
Theinteraction potentialfor a Σ
2
moleculesubject to amagneticfield (aZeeman field) definingaspace-fixed
Z-axis, seefigure3, isgiven by μ
η
= −
=
V
B
S ,
(9)
m
Z
m
m
Z (9) μ
η
= −
=
V
B
S ,
m
Z
m
m
Z μ
η
= −
=
V
B
S ,
(9)
m
Z
m
m
Z
where
η
μ
≡
B
(10)
m
m where η
μ
≡
B
(10)
m
m (10) B NewJ.Phys.17(2015) 045017 KSharmaand BFriedrich Figure4. Anonresonant opticalfield εL polarized alongthespace-fixed Z-axisactingon apolar
Σ
+
S
2
1
moleculewith paralleland
perpendicular componentsofthemolecular polarizability α∥and α⊥with respect to themolecular z-axis. Also shown arethe
rotational, spin,and totalangular momenta N, S,and J aswellasthepolar angleθbetween thespace- and molecule-fixed axesand
theazimuthalangleϕ uniformlydistributed about theZ-axis. Seetext. Figure4. Anonresonant opticalfield εL polarized alongthespace-fixed Z-axisactingon apolar
Σ
+
S
2
1
moleculewith paralleland
perpendicular componentsofthemolecular polarizability α∥and α⊥with respect to themolecular z-axis. Also shown arethe
rotational, spin,and totalangular momenta N, S,and J aswellasthepolar angleθbetween thespace- and molecule-fixed axesand
theazimuthalangleϕ uniformlydistributed about theZ-axis. Seetext. with μ
μ
= g
m
S
Btheelectronicmagneticdipolemoment ofthe Σ
2
molecule,
≅
g
2.0023
S
theelectronic
gyromagneticratio and μBtheBohr magneton. TheSZoperator couplesHund’scase(b) basisfunctionswith thesameM but with
′
N s that areeither the
sameor differ by±2and hencehavethesameparity.Theselection ruleon N moreover ensuresthat the
Hamiltonian matrixin theHund’scase(b) basisfor theZeeman interaction ofa Σ
2
moleculefactorsinto blocks
that areno greater than 2× 2,renderingthecorrespondingZeeman energyat most quadraticin .Apart from
M, also parity −
( 1)N˜ isagood quantum number. 2.3.Interaction with an optical field 2.3.Interaction with an optical field Asfor anylinear species, thepolarizabilitytensor ofa Σ
2
moleculeisanisotropic, with theprincipalcomponent
alongthemolecular axisexceedingthat perpendicular to theaxis, α
α
>
∥
⊥.When subject to an electricfield εL of
an electromagneticwaveofintensity linearlypolarized alongthespace-fixed Z- axis, figure4,whose
oscillation frequencyisfar removed from anymolecular resonance,themoleculeundergoesan interaction given
bythepotential η
θ
η
= −
−
⊥
V
B
B
cos
,
(11)
opt
opt
2 (11) where η
η
η
≡
−
∥
⊥
(12)
opt η
η
η
≡
−
∥
⊥
(12)
opt
and
η
πα
≡
∥⊥
∥⊥
Bc
2
(13)
,
,
with
π ε
=
c
4
. (14)
L
2 (12) η
η
η
≡
−
∥
⊥
opt and and η
πα
≡
∥⊥
∥⊥
Bc
2
(13)
,
, (13) with π ε
=
c
4
. (14)
L
2 (14) TheVopt potentialisadouble-wellpotentialwith two equivalent minimaat θ = 0 and 180°,separated byan
equatorialbarrier at θ = π
2. Asaresult, allstatesbound byVopt occur asdoublets, split bytunnelingthrough the
equatorialbarrier. The
θ
cos2
operator ofVopt hybridizesfree-rotor statesofsameparityand so thestatescreated
byVopt areofdefiniteparity, given by −
( 1)J˜.ThemembersofanyofthetunnelingdoubletshavesameM but ′J s
˜
that differ by±1and thusareofoppositeparity. Thetunnelingsplitting Δ
η
η
∝
−
E (
)
exp(
)
t
opt
opt
1
2
,see[73]. 5 NewJ.Phys.17(2015) 045017 KSharmaand BFriedrich Figure5. Congruent electrostatic, magneticand opticalfields εS, ,and εL directed alongthespace-fixed Z-axisactingon a polar
Σ
+
S
2
1
moleculewith body-fixed electricand magneticdipolemoments μel and μm and polarizabilitycomponents α∥and α⊥.Also
shown aretherotational, spin, and totalangular momenta N, S,and J aswellasthepolar angleθbetween thespace- and molecule-
fixed axesand theazimuthalangleϕ uniformlydistributed about theZ-axis. Seetext. Figure5. Congruent electrostatic, magneticand opticalfields εS, ,and εL directed alongthespace-fixed Z-axisactingon a polar
Σ
+
S
2
1
moleculewith body-fixed electricand magneticdipolemoments μel and μm and polarizabilitycomponents α∥and α⊥.Also
shown aretherotational, spin, and totalangular momenta N, S,and J aswellasthepolar angleθbetween thespace- and molecule-
fixed axesand theazimuthalangleϕ uniformlydistributed about theZ-axis. Seetext. Figure6. Dependence oftheeigenenergiesofapolar Σ
2
moleculeon thepermanent electricdipoleinteraction parameter ηel.F1and
F2levelsareshown,respectively, bydashed and fulllines.Red and bluecurvespertain, respectively, to stateswith
=
J˜
1
2 and
=
J˜
3
2. Notethat here η
η
=
= 0
m
opt
. Figure6. Dependence oftheeigenenergiesofapolar Σ
2
moleculeon thepermanent electricdipoleinteraction parameter ηel.F1and
F2levelsareshown,respectively, bydashed and fulllines.Red and bluecurvespertain, respectively, to stateswith
=
J˜
1
2 and
=
J˜
3
2. Notethat here η
η
=
= 0
m
opt
. 2.3.Interaction with an optical field 2.4.Interaction with congruent electric, magnetic, and optical fields
In thecongruent electric, magnetic, and opticalfields,thepotentialisgiven by 2.4.Interaction with congruent electric, magnetic, and optical fields
In thecongruent electric, magnetic, and opticalfields,thepotentialisgiven by
=
+
+
V
V
V
V
(15)
m
m
el,
,opt
el
opt (15) NewJ.Phys.17(2015) 045017 KSharmaand BFriedrich Figure7. Probabilitydensities, orientation and alignment cosinesofa Σ
2
moleculeasfunctionsofthepermanent electricdipole
interaction parameter ηel.Valuespertainingto theF1and F2statesareshown, respectively, bydashed and fulllines.Blueand red
curvespertain,respectively, to theleft (orientation) and right (alignment) ordinate. Notethat here η
η
=
= 0
m
opt
. Figure7. Probabilitydensities, orientation and alignment cosinesofa Σ
2
moleculeasfunctionsofthepermanent electricdipole
interaction parameter ηel.Valuespertainingto theF1and F2statesareshown, respectively, bydashed and fulllines.Blueand red
curvespertain,respectively, to theleft (orientation) and right (alignment) ordinate. Notethat here η
η
=
= 0
m
opt
. and thecorrespondingHamiltonian becomes and thecorrespondingHamiltonian becomes Theintegraloftheeigenfunction’ssquareover thespin variables Theintegraloftheeigenfunction’ssquareover thespin variables ∑
θ ϕ χ
ψ ψ
=
f ( ,
,
)
*
(20)
S ∑
θ ϕ χ
ψ ψ
=
f ( ,
,
)
*
(20)
S
i
lifi
t (20) D
D
∑
θ ϕ χ
π
θ ϕ χ
θ ϕ χ δ
=
−
×
+
′ +
×
Σ
Σ
Σ
Σ
Σ Σ
′
′
′
′
′
′
−
′
′
′
−
−
′
(
)(
)
f
c
c
J
J
( ,
, )
( 1)
2
1
2
1
64
( ,
,
)
( ,
,
)
. (21)
J M
J M
J M
J M
M K
M K
J
M
K
J
,
,
,
,
,
,
*
,
,
2
,
,
, D
D
θ ϕ χ
θ ϕ χ δ
×
Σ Σ
′
′
′
−
−
′
( ,
,
)
( ,
,
)
. (21)
M K
J
M
K
J
,
,
, (21) In order to visualizethedirectionalpropertiesofthemolecular statescreated,wepresent probability
distributionsofthespatialvariables, θ ϕ
,
,aspolar plotsof
θ ϕ χ =
f ( ,
,
0). In order to visualizethedirectionalpropertiesofthemolecular statescreated,wepresent probability
distributionsofthespatialvariables, θ ϕ
,
,aspolar plotsof
θ ϕ χ =
f ( ,
,
0). and thecorrespondingHamiltonian becomes (16) =
+
H
H
V
. (16)
m
m
el,
,opt
0
el,
,opt Themolecular axis,z,angular momenta, J, N, Sand thedipolemoments, μel, μm,and polarizability
components, α∥, α⊥aswellasthespace-fixed Z-axisareshown in figure5. Themolecular axis,z,angular momenta, J, N, Sand thedipolemoments, μel, μm,and polarizability
components, α∥, α⊥aswellasthespace-fixed Z-axisareshown in figure5. Themolecular axis,z,angular momenta, J, N, Sand thedipolemoments, μel, μm,and polarizability
components, α∥, α⊥aswellasthespace-fixed Z-axisareshown in figure5. BydividingHamiltonian (16) through therotationalconstant Band makinguseofequations(1),(7),(9),
and (11),weobtain thereduced Hamiltonian BydividingHamiltonian (16) through therotationalconstant Band makinguseofequations(1),(7),(9),
and (11),weobtain thereduced Hamiltonian γ
η
θ
η
η
θ
η
≡
=
+
′
−
+
−
−
⊥
H
B
H
S
N
N
S
·
cos
cos
(17)
m
m
Z
el,
,opt
2
el
opt
2 ≡
H
B
H
m
el,
,opt γ
η
θ
η
η
θ
η
=
+
′
−
+
−
−
⊥
S
N
N
S
·
cos
cos
(17)
m
Z
2
el
opt
2 (17) with γ′ ≡
γ
B. with γ′ ≡
γ
B. Theeigenfunction ofthetriple-field Hamiltonian (17) can bewritten as γ
B. Theeigenfunction ofthetriple-field Hamiltonian (17) can bewritten as B
Theeigenfunction ofthetriple-field Hamiltonian (17) can bewritten as ∑
ψ
η
η
η
Σ
≡
=
Σ
Σ
J N M
c
J K M
S
˜, ˜ ,
;
,
,
,
,
,
(18)
m
J
J
J N M
el
opt
,
,
˜, ˜ , (18) with anormalization with anormalization with anormalization
=
Σ
c
1. (19)
J
J N M
,
˜, ˜ ,
2 =
Σ
c
1. (19)
J
J N M
,
˜, ˜ ,
2 =
Σ
c
1. J
J N M
,
˜, ˜ ,
2 (19) NewJ.Phys.17(2015) 045017 KSharmaand BFriedrich Figure8. Dependence oftheeigenenergiesofapolar Σ
2
moleculeon themagneticdipoleinteraction parameter ηm.F1and F2levels
areshown,respectively,bydashed and fulllinesin panelspertainingto signed valuesofthegood quantum number M.Red and blue
curvespertain,respectively, to stateswith
=
J˜
1
2 and
=
J˜
3
2.Notethat here η
η
=
= 0
el
opt
. Figure8. Dependence oftheeigenenergiesofapolar Σ
2
moleculeon themagneticdipoleinteraction parameter ηm.F1and F2levels
areshown,respectively,bydashed and fulllinesin panelspertainingto signed valuesofthegood quantum number M.Red and blue
curvespertain,respectively, to stateswith
=
J˜
1
2 and
=
J˜
3
2.Notethat here η
η
=
= 0
el
opt
. 3.1.Single-field effects 3.1.Single-field effects 3.Results and discussion Theeigenenergiesand eigenvectorsofHamiltonian (17) wereobtained bynumericaldiagonalization ofthe
matrixrepresentation oftheHamiltonian in Hund’scase(a) basis. For collinear fields,considered here,M isa
good quantum number and so theHamiltonian matrixtakesablock-diagonalform for different valuesofM. For each M,theblock wastruncated at
=
J
15
2 to ensuretheconvergenceofeigenvaluesand eigenvectors. This
leadsto formation ofblock matricesofrank 30. Each oftheseblockswasdiagonalized separately.The
diagonalization wascarried out usingtheArmadillo C++ linear algebralibrary[74]. In order to track which stateiswhich astheinteraction parameters ηel, ηm,and ηopt werevaried, aprocedure
termed adiabaticfollowingwasdeveloped. Instead oflookingat thedependenceon theinteraction parametersof
thecomponentsoftheeigenvectors, wemonitored thescalar product ofthestatesbeforeand after a(small) 8 NewJ.Phys.17(2015) 045017 KSharmaand BFriedrich Figure9. Probabilitydensities, orientation and alignment cosinesofa Σ
2
moleculeasfunctionsofthemagneticdipoleinteraction
parameter ηm. Valuespertainingto theF1and F2statesareshown,respectively,bydashed and fulllines. Blueand red curvespertain,
respectively, to theleft (orientation) and right (alignment) ordinate.Notethat here η
η
=
= 0
el
opt
. Figure9. Probabilitydensities, orientation and alignment cosinesofa Σ
2
moleculeasfunctionsofthemagneticdipoleinteraction
parameter ηm. Valuespertainingto theF1and F2statesareshown,respectively,bydashed and fulllines. Blueand red curvespertain,
respectively, to theleft (orientation) and right (alignment) ordinate.Notethat here η
η
=
= 0
el
opt
. changeoftheinteraction parameter. Thescalar product wascalculated between agiven stateat theinitialvalueof
theinteraction parameter(s) and alltheother statesat thealtered valueoftheinteraction parameter(s). The
maximum ofthescalar product wasthen found and used to identifythestatethat makesthesmallest anglewith
thegiven state. Allthecalculationsbelowwerecarried out for ageneric Σ
2
moleculewith avalueofthereduced spin- changeoftheinteraction parameter. Thescalar product wascalculated between agiven stateat theinitialvalueof
theinteraction parameter(s) and alltheother statesat thealtered valueoftheinteraction parameter(s). The
maximum ofthescalar product wasthen found and used to identifythestatethat makesthesmallest anglewith
thegiven state. g
Allthecalculationsbelowwerecarried out for ageneric Σ
2
moleculewith avalueofthereduced spin-
rotation constant γ
γ
′ ≡
=
B
0.41(which pertains, e.g., to theNaO moleculein itsA Σ
2
state[75]). 3.1.1.PureStarkinteraction TheStarkpotential, equation (7),splitseach J˜ levelinto
+
J˜
1
2 stateswith different valuesof∣
∣
M but doesnot
undo the ± M degeneracy. Figure6showsthedependenceoftheeigenenergiesofthelowest sixstateson the
permanent electricdipoleinteraction parameter ηel,which isproportionalto theelectricfield strength. Note
that at zero field, theenergylevelsaregiven byequations(5) and (6).AllStarkstatesbecomehigh-field seeking
(i.e.,their eigenenergydecreaseswith increasingfield strength) at sufficientlyhigh field strengths. However, at a
lowfield,wheretheStark potentialmerelyhindersmolecular rotation, Stark stateswith
<
+
M
J J
˜(˜
1)
1
3
2
arefirst
high-field seeking(i.e., their eigenenergyincreaseswith increasingfield strength), asexemplified bythe
η
∣
=
=
∣
∣=
〉
J
N
M
˜
, ˜
1,
;
3
2
1
2
el and
η
∣=
=
∣
∣=
〉
J
N
M
˜
, ˜
2,
;
3
2
1
2
el states.Thisbehavior resultsfrom thetilt
angleoftheangular momentum (approximatelyconserved at lowfield strengths) with respect to thefield vector
(space-fixed Z-axis). When theangular momentum isnearlyperpendicular to thefield vector, themoleculeacts
likeaplanar rotor and spendsmost ofitstimeoriented oppositelyto thedirection oftheStark field, wherethe
rotor-fixed electricdipolemoment interactswith thefield repulsively. Oncethefield strength becomessufficient
for theStarkpotentialto confinethemolecular rotation and convert it into libration about thefield vector, the
body-fixed dipolegetsoriented alongthefield vector wherebytheStark interaction becomesattractive. 9 NewJ.Phys.17(2015) 045017 KSharmaand BFriedrich Figure10. Dependenceoftheeigenenergiesofapolar Σ
2
moleculeon theanisotropicpolarizabilityinteraction parameter ηopt.F1 and
F2levelsareshown,respectively, bydashed and fulllinesin panelspertainingto different valuesofthegood quantum number ∣
∣
M . Red and bluecurvespertain, respectively,to stateswith
=
J˜
1
2 and
=
J˜
3
2.Notethat here η
η
=
= 0
m
el
. Figure10. Dependenceoftheeigenenergiesofapolar Σ
2
moleculeon theanisotropicpolarizabilityinteraction parameter ηopt.F1 and
F2levelsareshown,respectively, bydashed and fulllinesin panelspertainingto different valuesofthegood quantum number ∣
∣
M . Red and bluecurvespertain, respectively,to stateswith
=
J˜
1
2 and
=
J˜
3
2.Notethat here η
η
=
= 0
m
el
. Figure7showstheorientation and alignment ofthelowest sixstatesasafunction of ηel.Theorientation and
alignment ofthemolecular axisischaracterized, respectively, bytheexpectation values
θ
〈
〉
cos
and
θ
〈
〉
cos2
.In
addition, thedirectionalityofthestatesand itsvariation with field strength isvisualized bythepolar diagrams
displaying, at intervals, theprobabilitydensity, equation (22).In thesepolar diagramsand in thosethat areto
followbelow, thefullrangeoftherainbowalwayscorrespondsto thefullrangeoftheprobability. Theabsolute
directionalityofthestatesisreflected in theshapeofthepolar plotsand theplotted valuesofthealignment and
orientation cosines. Asthemoleculebecomesoriented in the + Z direction, thelower lobeoftheprobabilitydistribution
becomessmaller and theupper lobelarger. At high electricfield strengthsthelower lobeishardlyvisible. For a
given J˜ and N˜ ,stateswith ∣
∣=
M
J˜ havethelowest energyand exhibit thehighest orientation. Wenotethat, by
theHellmann–Feynman theorem,
θ
〈
〉= −
η
∂
∂
cos
E B
(
)
el ,and so onecan glean thiskeymeasureofdirectionality
from theslopesoftheStark energies. 3.1.1.PureStarkinteraction Figure7also illustratesthevariation ofthedirectionalityofthe
η
∣=
=
∣
∣=
〉
J
N
M
˜
, ˜
1,
;
3
2
1
2
el and
η
∣
=
=
∣
∣=
〉
J
N
M
˜
, ˜
2,
;
3
2
1
2
el states, i.e.,the‘wrong-way’ orientation at lowfield strengthsand itsconversion to
the‘right-way’ orientation at high field strengths, asdescribed above. Alessintuitiveeffect oftheelectricfield on thepolar Σ
2
moleculeisatransfer oftheprobabilitydensityfrom
otationalto spin angular momentum, asreflected bytheincreaseofthesizeofthepolar plots. 3.1.2.PureZeemaninteraction TheZeeman potential, equation (9),undoesthe ± M degeneracyand splitseach J˜ levelinto
+
J
2˜
1stateswith
different signed valuesofM. Figure8showsthedependenceoftheeigenenergiesofa Σ
2
moleculefor thelowest twelvestateson the
magneticdipoleinteraction parameter ηm,which isproportionalto themagneticfield strength. The
eigenenergiesarelinear in ηm for stateswith
= ±
M
J˜ (so called stretched states) and at most quadraticfor other
states. In thestrong-field limit,F1statesarelow- or high-field seekingdependingon whether M ispositiveor
negative,whereasF2statesareallhigh-field seeking. In thestrong-field (Paschen–Back) limit, theelectron spin
and therotationalangular momentum uncoupleand thedependenceoftheZeeman levelson themagneticfield
strength becomes
η
≈M
E
B
S m, where
= ±
MS
1
2 istheprojection ofthespin angular momentum Son theZ-axis. 10 NewJ.Phys.17(2015) 045017 KSharmaand BFriedrich Figure11. Probabilitydensities, orientation and alignment cosinesofa Σ
2
moleculeasfunctionsoftheanisotropicpolarizability
interaction parameter ηopt.Valuespertainingto theF1and F2statesareshown,respectively, bydashed and fulllines.Blueand red
curvespertain,respectively, to theleft (orientation) and right (alignment) ordinate. Notethat here η
η
=
= 0
m
el
. Figure11. Probabilitydensities, orientation and alignment cosinesofa Σ
2
moleculeasfunctionsoftheanisotropicpolarizability
interaction parameter ηopt.Valuespertainingto theF1and F2statesareshown,respectively, bydashed and fulllines.Blueand red
curvespertain,respectively, to theleft (orientation) and right (alignment) ordinate. Notethat here η
η
=
= 0
m
el
. ThePaschen–Back uncouplingsetson at η
γ
≫
=
′
+
−
+
−
N
( ˜
)
m
E
E
B
1
2 ,i.e.,at η ⪆1
m
for themolecular example
considered. Sinceparity, −
( 1)N˜ ,and M aregood quantum numbers, thenumerouscrossingsoftheZeeman levelsthat
comeabout for a Σ
2
moleculearegenuine. Ofparticular interest arecrossingsoflevelswith sameM but opposite
parity,seesection 3.2.2.Wenotethat it isthePaschen–Back effect that precludestheoccurrenceofavoided
crossingsofthe Σ
2
Zeeman levels[9,10]. Figure9displaysthedirectionalpropertiesofa Σ
2
moleculesubject to amagneticfield. Sinceamagnetic
field cannot orient themolecular axis, theorientation cosinevanishesidentically. However, theaxiscan be
aligned.Thealignment cosine, concurrent for agiven statewith theexpectation valueofthemagneticdipole
moment [10],increases/decreasesmonotonouslywith ηm onlyfor thestretched stateswith
<
>
M
M
0
0,
whilefor therest it variesbetween ‘wrong-way’ (lessthan field-freevalue) and ‘right-way’ (morethan field-free
value) alignment. In thePaschen–Back limit,thealignment tendsto aconstant value. 3.2.Double-field effects In thissection wewillprovidea summaryofhowtwo collinear fieldsaffect apolar and polarizable Σ
2
molecule. 3.1.3.Purepolarizabilityinteractionwithanoptical field LiketheStark potential, equation (7),theanisotropicpolarizabilityinteraction with anonresonant opticalfield,
equation (11), splitseach J˜ levelinto
+
J˜
1
2 stateswith different valuesof∣
∣
M but doesnot undo the ± M
degeneracy. Figure10showsthedependenceoftheeigenenergiesofthelowest sixstatesofa Σ
2
moleculeon the
interaction parameter ηopt,which isproportionalto theopticalfield intensity .Onecan seetheformation of
theopposite-paritytunnelingdoubletswith increasing ηopt,which becomequasi-degenerateat high fields.Note
that themembersofagiven tunnelingdoublet havesame J˜.In contrast to theStark interaction, wherefor a 11 NewJ.Phys.17(2015) 045017 KSharmaand BFriedrich Figure12. Dependenceoftheeigenenergiesofa Σ
2
moleculeon theopticalfield strength parameter ηopt in thepresenceofan electric
field. F1 and F2levelsareshown,respectively,bydashed and fulllinesin panelspertainingto different valuesofthegood quantum
number ∣
∣
M . Red and bluecurvespertain, respectively,to stateswith
=
J˜
1
2 and
=
J˜
3
2. Notethat here η = 5
el
and η = 0
m
. Figure12. Dependenceoftheeigenenergiesofa Σ
2
moleculeon theopticalfield strength parameter ηopt in thepresenceofan electric
field. F1 and F2levelsareshown,respectively,bydashed and fulllinesin panelspertainingto different valuesofthegood quantum
number ∣
∣
M . Red and bluecurvespertain, respectively,to stateswith
=
J˜
1
2 and
=
J˜
3
2. Notethat here η = 5
el
and η = 0
m
. given J˜, stateswith lower ∣
∣
M haveahigher eigenenergy, theeigenenergyofstatescreated bytheanisotropic
polarizabilityinteraction increaseswith increasing∣
∣
M . Figure11displaysthedirectionalpropertiesofa Σ
2
moleculesubject to an opticalfield.Theopticalfield
doesnot orient themoleculebut greatlyenhancesitsalignment. Notethat thealignment ofthemembersofa
given tunnelingdoublet becomesthesameastheir eigenenergiesbecomeexponentiallyquasi-degenerateas
η
∝
−
exp(
)
opt
1
2
.Thisbehavior followsfrom theHellmann–Feynman theorem, accordingto which θ
〈
〉= −
η
∂
∂
cos2
( )
E
B
opt . Wenotethat thealignment ofthestatethat becomesthehigher member ofatunneling
˜
˜ doublet (and so hasahigher valueof N˜ ) alwaysexceedsthat ofthelower member (with alower valueof N˜ ). Interestingly, for apair ofStark stateswith same J˜, it istheonewith lower N˜ that hasthelarger alignment ofthe
two.Theopticalfield leadsto aconsiderabletransfer oftheprobabilitydensityfrom therotationalto thespin
angular momentum, asreflected bytheincreasein thesizeofthepolar plotswith increasinginteraction
parameter ηopt. 3.2.1.Congruent electricandoptical fields Figure12showsthedependenceoftheeigenenergiesofthelowest sixstatesofa Σ
2
moleculeon theinteraction
parameter ηopt in thepresenceofan electricfield such that thecorrespondinginteraction parameter η = 5
el
. Compared with figure10,weseethat theopposite-paritytunnelingdoubletsthat werequasi-degeneratein the
absenceoftheelectricfield havebeen readilysplit dueto thecouplingbytheelectricdipoleinteraction. The
tunnelingsplittingin thecombined fieldsat agiven ηopt isproportionalto ηel, Δ
η
η
η
=
∝
E (
const .,
)
t
opt
el
el
[5,62]. Figure12showsthedependenceoftheeigenenergiesofthelowest sixstatesofa Σ
2
moleculeon theinteraction
parameter ηopt in thepresenceofan electricfield such that thecorrespondinginteraction parameter η = 5
el
. Compared with figure10,weseethat theopposite-paritytunnelingdoubletsthat werequasi-degeneratein the
absenceoftheelectricfield havebeen readilysplit dueto thecouplingbytheelectricdipoleinteraction. The
tunnelingsplittingin thecombined fieldsat agiven ηopt isproportionalto ηel, Δ
η
η
η
=
∝
E (
const .,
)
t
opt
el
el
[5,62]. [ ,
]
Figure13displaysthedirectionalpropertiesofa Σ
2
moleculesubject to an opticalfield in thepresenceofan
electricfield.Theseexhibit quiteafewdistinct features, such asthesudden back-and-forth variationsofthe [ ,
]
Figure13displaysthedirectionalpropertiesofa Σ
2
moleculesubject to an opticalfield in thepresenceofan
electricfield.Theseexhibit quiteafewdistinct features, such asthesudden back-and-forth variationsofthe 12 NewJ.Phys.17(2015) 045017 KSharmaand BFriedrich Figure13. Probabilitydensities, orientation and alignment cosinesofa Σ
2
moleculeasfunctionsoftheanisotropicpolarizability
interaction parameter ηopt in thepresenceofan electricfield.Valuespertainingto theF1and F2 statesareshown, respectively, by
dashed and fulllines.Blueand red curvespertain,respectively, to theleft (orientation) and right (alignment) ordinate.Notethat here
η = 5
el
and η = 0
m
. Figure13. Probabilitydensities, orientation and alignment cosinesofa Σ
2
moleculeasfunctionsoftheanisotropicpolarizability
interaction parameter ηopt in thepresenceofan electricfield.Valuespertainingto theF1and F2 statesareshown, respectively, by
dashed and fulllines.Blueand red curvespertain,respectively, to theleft (orientation) and right (alignment) ordinate.Notethat here
η = 5
el
and η = 0
m
. orientation and alignment cosineswith ηopt.Most ofthesefeaturesareconnected with themutual‘repelling’ of
thelevelswithin agiven tunnelingdoublet—which lendsthecorrespondingstatesopposite-wayorientation—
and with intersectionsofthoselevelswith levelsofsame∣
∣
M but pertainingto different tunnelingdoublets. 1
1 and with intersectionsofthoselevelswith levelsofsame∣
∣
M but pertainingto different tunnelingdoublets. So, for instance,likeitstunneling-doublet partner, the∣=
=
∣
∣=
〉
J
N
M
˜
, ˜
1,
1
2
1
2 stateisinitiallyright-way
oriented but flipsitsorientation, at η
≈23
opt
,dueto itsinteraction with the∣=
=
∣
∣=
〉
J
N
M
˜
, ˜
1,
3
2
1
2 state. Thisisreflected in thepolar plotsoftheprobabilitydensitiesaswellin that theupper lobevanishesand thelower
lobebecomeshuge, portendingthewrongwayorientation ofthemolecular state. Likewise, the
∣=
=
∣
∣=
〉
J
N
M
˜
, ˜
1,
3
2
1
2 state, which isinitiallywrong-wayoriented, flipsitsorientation at η
≈23
opt
dueto its
˜
˜
1
1 Thisisreflected in thepolar plotsoftheprobabilitydensitiesaswellin that theupper lobevanishesand thelower
lobebecomeshuge, portendingthewrongwayorientation ofthemolecular state. 3.2.1.Congruent electricandoptical fields Likewise, the
∣=
=
∣
∣=
〉
J
N
M
˜
, ˜
1,
3
2
1
2 state, which isinitiallywrong-wayoriented, flipsitsorientation at η
≈23
opt
dueto its
interaction with the∣=
=
∣
∣=
〉
J
N
M
˜
, ˜
1,
1
2
1
2 stateand acquiresaright-wayorientation. The ∣=
=
∣
∣=
〉
J
N
M
˜
, ˜
1,
3
2
1
2 state, which isinitiallywrong-wayoriented, flipsitsorientation at η
≈23
opt
dueto its
interaction with the∣=
=
∣
∣=
〉
J
N
M
˜
, ˜
1,
1
2
1
2 stateand acquiresaright-wayorientation. The ∣=
=
∣
∣=
〉
J
N
M
˜
, ˜
2,
3
2
1
2 stateundergoestheflip twice, wherebythefirst flip isdueto theinteraction with the
∣=
=
∣
∣=
〉
J
N
M
˜
, ˜
1,
3
2
1
2 stateand thesecond flip comesabout becauseofthestate’sinteraction with the
∣=
=
∣=
〉
J
N
M
˜
, ˜
2,
5
2
1
2 stateat η
≈28
opt
(ahigher-lyingstatenot shown here).Thestateisright-wayoriented
between thesetwo flipsand iswrong-wayoriented in thehigh field region. Apart from that, thereis,asexpected,
aprobabilitydensitytransfer from therotationalangular momentum to thespin angular momentum. =
=
∣
∣=
〉
J
N
M
˜
, ˜
2,
3
2
1
2 stateundergoestheflip twice, wherebythefirst flip isdueto theinteraction with the
=
=
∣
∣=
〉
J
N
M
˜
, ˜
1,
3
2
1
2 stateand thesecond flip comesabout becauseofthestate’sinteraction with the
˜
˜
5
1 Theseflipsin theorientation ofthemoleculeareofparticular importancesincenot onlydo theseprovidethe
meansfor switchingtheorientation ofthemolecule, but, aswewillseein section 3.3, thevaluesoftheinteraction
parameter wheretheflipstakeplacecan becontrolled byintroducingathird field. Theseflipsin theorientation ofthemoleculeareofparticular importancesincenot onlydo theseprovidethe
meansfor switchingtheorientation ofthemolecule, but, aswewillseein section 3.3, thevaluesoftheinteraction
parameter wheretheflipstakeplacecan becontrolled byintroducingathird field. 13 NewJ.Phys.17(2015) 045017 KSharmaand BFriedrich Figure14. Dependenceoftheeigenenergiesofapolar Σ
2
moleculeon themagneticdipoleinteraction parameter ηm in thepresenceof
an electricfield.F1and F2levelsareshown, respectively, bydashed and fulllinesin panelspertainingto signed valuesofthegood
quantum number M.Red and bluecurvespertain, respectively, to stateswith
=
J˜
1
2 and
=
J˜
3
2.Notethat here η = 5
el
and η
= 0
opt
. Figure14. Dependenceoftheeigenenergiesofapolar Σ
2
moleculeon themagneticdipoleinteraction parameter ηm in thepresenceof
an electricfield.F1and F2levelsareshown, respectively, bydashed and fulllinesin panelspertainingto signed valuesofthegood
quantum number M.Red and bluecurvespertain, respectively, to stateswith
=
J˜
1
2 and
=
J˜
3
2.Notethat here η = 5
el
and η
= 0
opt
. 3.2.2.Congruent electricandmagneticfields Figure14showsthedependenceoftheeigenenergiesofthelowest twelvestatesofa Σ
2
moleculeon the
interaction parameter ηm in thepresenceofan electricfield such that thecorrespondinginteraction parameter
η = 5
el
.Compared with figure8, weseethat thegenuineintersection in theabsenceoftheelectricfield ofthe
opposite-paritylevelshavebecomeavoided crossingsdueto thecouplingbytheelectricdipoleinteraction. This
transformsthelow-field seekingstatesinto high-field seekersand viceversa. Theconcomitant directionalpropertiesareexemplified in figure15.For instance, the
=
=
= −
J
N
M
˜
, ˜
1,
1
2
1
2 statechangesitsshapefrom an oriented torusto an oriented double-lobed form
whilethecrossing
=
=
= −
J
N
M
˜
, ˜
1,
3
2
1
2 statechangesfrom an oriented double-lobeto an oriented torus. Wenotethat sincetheintersectinglevelsareexactlydegenerateat thecrossingpoint,even asmallelectricfield
can mixthem and thusgenerateorientation. For η
η
⩾
m
el,themaximum valueoftheorientation cosineis
determined just bytheintersectingpurelyZeeman statesand isindependent of ηel,see[9, 10]. Wenotethat sincetheintersectinglevelsareexactlydegenerateat thecrossingpoint,even asmallelectricfield
can mixthem and thusgenerateorientation. For η
η
⩾
m
el,themaximum valueoftheorientation cosineis
determined just bytheintersectingpurelyZeeman statesand isindependent of ηel,see[9, 10]. Wenotethat sincetheintersectinglevelsareexactlydegenerateat thecrossingpoint,even asmallelectricfield
can mixthem and thusgenerateorientation. For η
η
⩾
m
el,themaximum valueoftheorientation cosineis
determined just bytheintersectingpurelyZeeman statesand isindependent of ηel,see[9, 10]. 3.2.3.Congruent magneticandoptical fields In light ofthefact that themagneticdipoleinteraction onlycouplesstateswith sameparity, theopposite-parity
membersofthetunnelingdoubletscreated bythepolarizabilityinteraction with theopticalfield remain
uncoupled in thepresenceofthemagneticfield. However, themagneticfield liftsthe ± M degeneracyofthe
good quantum number ∣
∣
M that characterizeseach tunnelingdoublet in theopticalfield aloneand thus,for
∣
∣>
M
0,doublesthenumber ofthetunnelingdoublets. Thisisillustrated in figure16, which showsthedependenceoftheeigenenergiesofa Σ
2
moleculeon the
opticalfield in thepresenceofamagneticfield such that η = 2.5
m
.Thekeyfeatureofthe‘doubled’ tunneling
doubletsisthat theyallremain quasi-degenerateat high ηopt.However, thestatescreated bytheanisotropic
polarizabilityinteraction with theopticalfield arealso affected bythepresenceofthemagneticfield in other
waysthan removingthe ± M degeneracy. In particular, sincethemagneticfield movesthelevelsofa
paramagneticmoleculearound,seesection 3.1.2,someofthetunnelingdoubletsundergo a flip ofthepartner
levels:what wasalower member ofadoublet becomesahigher member and viceversa. 14 NewJ.Phys.17(2015) 045017 KSharmaand BFriedrich Figure15. Probabilitydensities, orientation and alignment cosinesofa Σ
2
moleculeasfunctionsofthemagneticdipoleinteraction
parameter ηm in thepresenceofan electricfield. Valuespertainingto theF1and F2statesareshown, respectively, bydashed and full
lines. Blueand red curvespertain,respectively, to theleft (orientation) and right (alignment) ordinate. Notethat here η = 5
el
and
η
= 0
opt
. Figure15. Probabilitydensities, orientation and alignment cosinesofa Σ
2
moleculeasfunctionsofthemagneticdipoleinteraction
parameter ηm in thepresenceofan electricfield. Valuespertainingto theF1and F2statesareshown, respectively, bydashed and full
lines. Blueand red curvespertain,respectively, to theleft (orientation) and right (alignment) ordinate. Notethat here η = 5
el
and
η
= 0
opt
. Figure17showsthedirectionalpropertiesofa Σ
2
moleculeasafunction ofan opticalfield in thepresenceof
amagneticfield.Aswehaveseen in figure9, themagneticfield doesnot alter thedirectionalpropertiesofa
molecular stateascreated bytheopticalfield unlessthestateencountersanother statethat couplesto it. Since
neither amagneticnor an opticalfield can orient a molecule,
θ
〈
〉
cos
vanishesidenticallyfor allstatescreated by
thesefields. Finally, weobservethat theopticalfield leadsto asignificant transfer ofprobabilitydensityfrom rotationalto
spin angular momentum in thecombined magneticand opticalfields. p
In thissection westudytheeffectsofallthreefieldsactingon apolar and polarizable Σ
2
molecule
simultaneously. p
In thissection westudytheeffectsofallthreefieldsactingon apolar and polarizable Σ
2
molecule
simultaneously. Variationoftheelectricfield.Figure18showsthedependenceoftheeigenenergiesofthelowest sixstateson
theelectricdipoleinteraction parameter ηel in thepresenceofconstant magnetic (η = 2.5
m
) and opticalfields
(η
= 15
opt
).Sincethepresenceofthemagneticfield liftsthe ± M degeneracy, thefigureissplit into four panels,
each pertainingto agiven valueofM, asstateswith
>
M
0 behavedifferentlyfrom stateswith
<
M
0. Wesee
that thestatesarepaired up at η →0
el
dueto theformation ofthequasi-degeneratetunnelingdoubletsbythe
opticalfield. For η > 0
el
thedoubletsareincreasinglycoupled bytheelectricdipoleinteraction and split up asa
result.Themagneticfield bringsabout arelativeshift ofthedoublet levelswhich leadsto avoided crossings. Figure19showsthedirectionalpropertiesofa Σ
2
moleculeasafunction oftheelectricinteraction
parameter at constant magneticand opticalfields.Weagain observeabrupt changesin thesenseofthemolecular
axisorientation. However, thefield strengthsat which theseabrupt changestakeplacecan becontrolled by 15 NewJ.Phys.17(2015) 045017 KSharmaand BFriedrich Figure16. Dependenceoftheeigenenergiesofapolar Σ
2
moleculeon theanisotropicpolarizabilityinteraction parameter ηopt in the
presenceofamagneticfield.F1and F2levelsareshown,respectively, bydashed and fulllinesin panelspertainingto signed valuesof
thegood quantum number M. Red and bluecurvespertain, respectively,to stateswith
=
J˜
1
2 and
=
J˜
3
2. Notethat here η = 2.5
m
and
η = 0
el
. Figure16. Dependenceoftheeigenenergiesofapolar Σ
2
moleculeon theanisotropicpolarizabilityinteraction parameter ηopt in the
presenceofamagneticfield.F1and F2levelsareshown,respectively, bydashed and fulllinesin panelspertainingto signed valuesof
thegood quantum number M. Red and bluecurvespertain, respectively,to stateswith
=
J˜
1
2 and
=
J˜
3
2. Notethat here η = 2.5
m
and
η = 0
el
. tuningthevalueofthesuperimposed magneticfield.For instance, the∣=
=
∣
∣=
〉
J
N
M
˜
, ˜
1,
1
2
1
2 statein the
absenceofthemagneticfield changesitsorientation at η ≈4
el
;herethechangetakesplaceat ahigher valueof
theelectricfield for the∣
=
=
= −〉
J
N
M
˜
, ˜
1,
1
2
1
2 state (η ≈6
el
at η = 2.5
m
) and for the
∣=
=
=
〉
J
N
M
˜
, ˜
1,
1
2
1
2 stateat alower valueofelectricfield (η ≈2
el
at η = 2.5
m
). ∣=
=
=
〉
J
N
M
˜
, ˜
1,
1
2
1
2 stateat alower valueofelectricfield (η ≈2
el
at η = 2.5
m
). 2
2
In general, for stateswith
<
M
0,thehigher thevalueofthemagneticfield,thegreater istheelectricfield
strength required to flip theorientation ofthestate–and viceversa for stateswith
>
M
0:thehigher thevalueof
magneticfield, thelower istheelectricfield strength required to flip theorientation. Thishappensbecausethe
avoided crossingsformed that lead to aflip in orientation ariseat alower electricfield for stateswith
<
M
0 and
ahigher electricfield for stateswith
>
M
0 asthemagneticfield strength isincreased.For
<
M
0, thelower of
thetwo statesformingtheavoided crossingishigh-field seekingand thehigher stateislow-field seekingunder
themagneticfield alone. p
In thissection westudytheeffectsofallthreefieldsactingon apolar and polarizable Σ
2
molecule
simultaneously. Thisresultsin an increasein theenergysplittingbetween thesetwo statesasthe
magneticfield isincreased. This,in turn, leadsto theformation ofavoided crossings, and henceto theflippingof
theorientation ofthestateat ahigher electricfield. On theother hand,for
>
M
0 states,thehigher ofthetwo
statesformingtheavoided crossingishigh-field seekingand thelower stateislow-field seekingunder the
magneticfield alone.Thisresultsin adecreaseoftheenergysplittingbetween thesetwo statesasthemagnetic
field isincreased and theformation ofavoided crossings, and henceto theflippingoftheorientation ofthestates
at alower electricfield. Theabovefeatureofthetriple-field interaction lendsitselfasameansto controlthesense
ofthemolecular axisorientation with thesuperimposed magneticfield asacontrolparameter. Variationofthemagneticfield.Figure20displaysthedependenceoftheeigenenergiesofa Σ
2
moleculeon
themagneticfield in thepresenceofan electricfield (η = 5
el
) and opticalfield (η
= 15
opt
). Aswecan see, the
tunnelingdoubletsareno longer quasi-degenerateastheyaresplit bytheelectricfield. Figure20bearsa
similaritywith figure14;however, dueto thelevelshiftsbrought about bytheopticalfield, theenergysplittingat
theavoided crossingof, e.g.,the∣=
=
= −〉
J
N
M
˜
, ˜
1,
1
2
1
2 and ∣=
=
= −〉
J
N
M
˜
, ˜
1,
3
2
1
2 statesislessthan in
theabsenceoftheopticalfield. Thisleadsto a much moreabrupt variation oftheorientation cosinein the
vicinityofthecrossing, ascan beseen in figure21.In addition,bycomparingfigures21and 15, weseethat the 16 NewJ.Phys.17(2015) 045017 KSharmaand BFriedrich Figure17. Probabilitydensities, orientation and alignment cosinesofa Σ
2
moleculeasfunctionsoftheanisotropicpolariuzability
interaction parameter ηopt in thepresenceofamagneticfield.Valuespertainingto theF1and F2statesareshown, respectively, by
dashed and fulllines.Blueand red curvespertain,respectively, to theleft (orientation) and right (alignment) ordinate.Notethat here
η = 2.5
m
and η = 0
el
. Figure17. Probabilitydensities, orientation and alignment cosinesofa Σ
2
moleculeasfunctionsoftheanisotropicpolariuzability
interaction parameter ηopt in thepresenceofamagneticfield.Valuespertainingto theF1and F2statesareshown, respectively, by
dashed and fulllines.Blueand red curvespertain,respectively, to theleft (orientation) and right (alignment) ordinate.Notethat here
η = 2.5
m
and η = 0
el
. Figure17. Probabilitydensities, orientation and alignment cosinesofa Σ
2
moleculeasfunctionsoftheanisotropicpolariuzability
interaction parameter ηopt in thepresenceofamagneticfield.Valuespertainingto theF1and F2statesareshown, respectively, by
dashed and fulllines.Blueand red curvespertain,respectively, to theleft (orientation) and right (alignment) ordinate.Notethat here
η = 2.5
m
and η = 0
el
. presenceoftheopticalfield can lead to ahigher orientation ofthestates(i.e.,greater valuesof
θ
∣〈
〉∣
cos
.The
flippingoftheorientation can beconvenientlycontrolled bymakinguseoftheopticalfield asacontrol
parameter. For stateswith
<
M
0,thehigher theopticalfield,thelower isthemagneticfield required to flip the
orientation and viceversafor stateswith
>
M
0.Thisisbecausetheelectricfield couplesthetunnelingdoublets
formed bytheopticalfield. For
<
M
0 states,thelowest statefor everyM isahigh-field seekingstatewhich,
therefore, doesnot haveanypointsofinflection.Theavoided crossings, wheretheflippingoftheorientation
takesplace, areformed between statesofdifferent J˜. Theenergysplittingbetween thesestatesdecreaseswith
increasingopticalfield asthetunnelingdoubletsformed bytheopticalfield arecoupled bytheelectricfield.This
leadsto adecreasein themagneticfield strength required to flip theorientation ofthestatewith increasing
opticalfield. On theother hand,for stateswith
>
M
0,thelowest statefor each M isalow-field seekingstate
under themagneticfield interaction. So theavoided crossingswheretheflip in orientation takesplacearewithin
thesametunnelingdoublet. Theenergysplittingbetween thetwo statesincreaseswith increasingopticalfield
becausethetunnelingdoubletsarecoupled bytheelectricfield, therebyrequiringagreater magneticfield to flip
theorientation. Variationof theoptical field. p
In thissection westudytheeffectsofallthreefieldsactingon apolar and polarizable Σ
2
molecule
simultaneously. Figure22showsthedependenceoftheeigenenergieson theopticalfield strength
parameter ηopt in thepresenceofelectric (η = 5
el
) and magnetic (η = 2.5
m
) fields.Likein figure16,weseethat
thetunnelingdoubletssplit bytheelectricdipoleinteraction. However, dueto theZeeman shifts, someofthe
levelshaveinterchanged their order. So thelower member ofthe
=
=
=
J
M
N
˜
,
, ˜
0, 1
1
2
1
2
tunnelingdoublet
hasbecomethehigher member and thehigher member hasbecomethelower member. Such an interchangein
theorder ofthestatesoccursbecausethetwo statesgenuinelycrosseach other under theeffect ofmagneticfield,
seefigure8. 17 NewJ.Phys.17(2015) 045017 KSharmaand BFriedrich Figure18. Dependenceoftheeigenenergiesofapolar Σ
2
moleculeon theelectricdipoleinteraction parameter ηel in thepresenceofa
magneticand an opticalfield.F1and F2 levelsareshown,respectively, bydashed and fulllinesin panelspertainingto signed valuesof
thegood quantum number M. Red and bluecurvespertain, respectively,to stateswith
=
J˜
1
2 and
=
J˜
3
2. Notethat here η = 2.5
m
and
η
= 15
opt
. Figure18. Dependenceoftheeigenenergiesofapolar Σ
2
moleculeon theelectricdipoleinteraction parameter ηel in thepresenceofa
magneticand an opticalfield.F1and F2 levelsareshown,respectively, bydashed and fulllinesin panelspertainingto signed valuesof
thegood quantum number M. Red and bluecurvespertain, respectively,to stateswith
=
J˜
1
2 and
=
J˜
3
2. Notethat here η = 2.5
m
and
η
= 15
opt
. Thisisdetailed in figure23which showsthedependenceon theopticalfield ηopt ofthetunnelingsplitting,
ΔE
B
t,between the∣
=
=
∣
∣=
〉
J
N
M
˜
, ˜
0,
1
2
1
2 and ∣
=
=
∣
∣=
〉
J
N
M
˜
, ˜
1,
1
2
1
2 states(thelowest tunnelingdoublet)
in theabsence(red curve) and presence(bluecurves) ofthemagneticfield.Themagneticfield separatesthe
tunnelingdoublet into two, with each pertainingeither to
=
M
1
2 or
= −
M
1
2.Anegativetunnelingsplitting
correspondsto a reversaloftheorder ofthemembersofthetunnelingdoublets. Wenotethat thetunneling
doubletsdepicted becomequasi-degeneratein thehigh field region, followingthe
η
∝
−
Δ
exp(
)
E
B
opt
t
1
2
asymptotic
dependence. Figure24detailswhat happenswhen an electricfield (correspondingto η = 5
el
) issuperimposed. Thered
curveshowsthetunnelingsplitting ΔE B
t
in theabsenceofthemagneticfield.Sincetheelectricfield couplesthe
∣=
=
∣
∣=
〉
J
N
M
˜
, ˜
0,
1
2
1
2 and ∣=
=
∣
∣=
〉
J
N
M
˜
, ˜
1,
1
2
1
2 statesin question, they‘repel’ each other asaresult. Initially,thetunnelingsplittingrapidlyincreaseswith theopticalfield onlyto taper offin thehigh field region. When themagneticfield isswitched on,thistunnelingdoublet dividesinto two separatetunnelingdoublets, one
with
= −
M
1
2 and another with
=
M
1
2.Thetunnelingsplittingofthetwo tunnelingdoubletsformed isshown
bythedashed blueline. Whilethedependenceon ηopt ofthetunnelingsplittingofthedoublet pertainingto
= −
M
1
2 resemblesthat ofthetunnelingdoublet in theabsenceofthemagneticfield, thetunnelingsplitting
keepson increasingwith theopticalfield strength for thedoublet pertainingto
=
M
1
2. exchanged their labels;in thislatter caseit isthestatewith lower N˜ that exhibitsawrongwayorientation at high
opticalfields. exchanged their labels;in thislatter caseit isthestatewith lower N˜ that exhibitsawrongwayorientation at high
opticalfields. p
In thissection westudytheeffectsofallthreefieldsactingon apolar and polarizable Σ
2
molecule
simultaneously. Notethat in thelatter
case, themembersofthetunnelingdoublet interchanged their order, asreflected in thechangeofsign of ΔE B
t
from positiveto negative. Figure25showsthedirectionalpropertiesofa Σ
2
moleculeasafunction oftheopticalfield in thepresence
ofboth electric (η = 5
el
) and magnetic (η = 2.5
m
) fields. Thepresenceofthemagneticfield can beused to
controltheopticalfield strength at which theorientation cosinechangessign.Theopticalfield strength required
to flip theorientation decreaseswith increasingmagneticfield for stateswith
<
M
0 and viceversafor states
with
>
M
0.Finally,wenotethat thetunnelingdoublet member with greater N˜ exhibitsawrongway
orientation in thehigh field region, except for thecasewhen thetwo membersofthetunnelingdoublet have 18 NewJ.Phys.17(2015) 045017 KSharmaand BFriedrich Figure19. Probabilitydensities, orientation and alignment cosinesofa Σ
2
moleculeasfunctionsoftheelectricdipoleinteraction
parameter ηel in thepresenceofamagneticand an opticalfield.Valuespertainingto theF1and F2statesareshown, respectively, by
dashed and fulllines.Blueand red curvespertain,respectively, to theleft (orientation) and right (alignment) ordinate.Notethat here
η = 2.5
m
and η
= 15
opt
. Figure19. Probabilitydensities, orientation and alignment cosinesofa Σ
2
moleculeasfunctionsoftheelectricdipoleinteraction
parameter ηel in thepresenceofamagneticand an opticalfield.Valuespertainingto theF1and F2statesareshown, respectively, by
dashed and fulllines.Blueand red curvespertain,respectively, to theleft (orientation) and right (alignment) ordinate.Notethat here
η = 2.5
m
and η
= 15
opt
. 4.Conclusions Weexamined theeigenpropertiesofpolar, paramagnetic, and polarizablelinear moleculesin congruent electric,
magnetic, and nonresonant opticalfieldsbynumericaldiagonalization ofthecorrespondingHamiltonian
matrix.Wefound that thedirectionalityofthemolecular stateswhich can beachieved with thetriple-field
combination supersedes—in itsmagnitudeaswellascontrollability—that obtained bythedouble-field
combinationsor singlefields,asstudied previously.Thehighlydirectionalmolecular statescreated bythetriple-
field combination can bethen acted upon byspacefixed fields,permittingto manipulatereadilyand efficiently
both therotation and translation ofthemolecules. Possibleapplicationsabound, but herewewould liketo
emphasizethepotentialfor manipulatingcold molecules. Not onlyarecold moleculesgenerallymore
susceptibleto manipulation byexternalfieldsdueto their lowtranslationalenergy, but someoftheir
applications, such asquantum computing[76] or thesearch for theelectricdipolemoment oftheelectron [70],
havealreadyenvisioned theuseofcombined fieldsfor both trappingand probing. Thepresent paper expandson
what theuseofthetriple-field combination would entail. Thecombination ofan opticaland amagneticfield createsamultitudeofdegenerateor quasi-degenerate
statesofoppositeparitythat can undergo afacilecouplingbyasuperimposed (weak) electricfield. Thisisthe
essenceoftheeffectsofthethreecongruent fieldsand thebasisfor their synergy. That oneofthefields—the
opticalone—can bevaried or switched on and offat timescaleson theorder oftherotationalperiod would lend 19 NewJ.Phys.17(2015) 045017 KSharmaand BFriedrich Figure20. Dependenceoftheeigenenergiesofapolar Σ
2
moleculeon themagneticdipoleinteraction parameter ηm in thepresenceof
an electricand an opticalfield. F1and F2levelsareshown,respectively,bydashed and fulllinesin panelspertainingto signed valuesof
thegood quantum number M. Red and bluecurvespertain, respectively,to stateswith
=
J˜
1
2 and
=
J˜
3
2. Notethat here η = 5
el
and
η
= 15
opt
. Figure20. Dependenceoftheeigenenergiesofapolar Σ
2
moleculeon themagneticdipoleinteraction parameter ηm in thepresenceof
an electricand an opticalfield. F1and F2levelsareshown,respectively,bydashed and fulllinesin panelspertainingto signed valuesof
thegood quantum number M. Red and bluecurvespertain, respectively,to stateswith
=
J˜
1
2 and
=
J˜
3
2. Notethat here η = 5
el
and
η
= 15
opt
. amanipulation techniquebased on thetriple-field effectsadegreeofcontrollabilitythat isneeded for such
applicationsasstereo-dynamicalcollisionalstudiesor quantum computing. In our forthcomingwork wewillexaminethenon adiabaticeffectsthat areexpected [30] to arisewhen the
opticalfield isvaried at timescalesshorter than therotationalperiod ofthemolecule.Also worthyofexploringis
thedependenceofthetriple-field effectson thetilt anglesamongthethreefield vectors[21,71].Relevant to both
isthetopologyoftheeigenenergysurfacesspanned bythe ηel, ηm,and ηopt interaction parametersthat mayresult
in conicalintersections[63,64], another subject ofour forthcomingstudy. Acknowledgments Discussionswith Dr Burkhard Schmidt, Prof. John Doyleand Dr MikeTarbutt aswellassupport bytheDFG
through grant FR3319/3-1 aregratefullyacknowledged. AppendixA. Direction cosinematrixelements in thesymmetrictop basis Thenon-vanishingelementsofthedirection cosinematrix, ϕI
j, used in thiswork aregiven by Ω
ϕ
Ω
Ω
Ω
′
′
′
=
′
′
′
×
′
′
(
) (
)
J
M
J
M
f J
J g
J
J
h
J
M
J M
,
,
,
,
,
,
, ,
( ,
, ,
)
(22)
I
j
j
I (22) with
′
f J
J
( , ),
Ω
Ω
′
′
g J
J
( ,
, ,
)
j
,and
′
′
h
J
M
J M
( ,
, ,
)
I
listed in tablesA1 –A5,see[72]. with
′
f J
J
( , ),
Ω
Ω
′
′
g J
J
( ,
, ,
)
j
,and
′
′
h
J
M
J M
( ,
, ,
)
I
listed in tablesA1 –A5,see[72]. with
′
f J
J
( , ),
Ω
Ω
′
′
g J
J
( ,
, ,
)
j
,and
′
′
h
J
M
J M
( ,
, ,
)
I
listed in tablesA1 –A5,see[72]. A.1. Matrixelements in Hund’s case(a) basis A.1. Matrixelements in Hund’s case(a) basis
For theelectricfield interaction,weneed matrixelementsoftheoperator
θ
cos
which arelisted in tableA6 . For theopticalfield interaction, weneed matrixelementsoftheoperator
θ
cos2 ,which arelisted in tableA7 . ( )
For theelectricfield interaction,weneed matrixelementsoftheoperator
θ
cos
which arelisted in tableA6
For theopticalfield interaction, weneed matrixelementsoftheoperator
θ
cos2 ,which arelisted in tableA7 20 NewJ.Phys.17(2015) 045017 KSharmaand BFriedrich Figure21. Probabilitydensities, orientation and alignment cosinesofa Σ
2
moleculeasfunctionsofthemagneticdipoleinteraction
parameter ηm in thepresenceofamagneticand an opticalfield.Valuespertainingto theF1and F2statesareshown, respectively, by
dashed and fulllines.Blueand red curvespertain,respectively, to theleft (orientation) and right (alignment) ordinate.Notethat here
η = 5
el
and η
= 15
opt
. Figure21. Probabilitydensities, orientation and alignment cosinesofa Σ
2
moleculeasfunctionsofthemagneticdipoleinteraction
parameter ηm in thepresenceofamagneticand an opticalfield.Valuespertainingto theF1and F2statesareshown, respectively, by
dashed and fulllines.Blueand red curvespertain,respectively, to theleft (orientation) and right (alignment) ordinate.Notethat here
η = 5
el
and η
= 15
opt
. AppendixA. Direction cosinematrixelements in thesymmetrictop basis For themagneticfield interaction, weneed thematrixelementsoftheSZoperator For themagneticfield interaction, weneed thematrixelementsoftheSZoperator ϕ
ϕ
ϕ
=
+
+
+
−
−
+
(
)
S
S
S
S
1
2
,
(23)
Z
Z
Z
Z
z
z (23) wherethesuperscriptspertain to thebody-fixed and thesubscriptsto thespace-fixed frame.Theelectron spin
matrixelementsare wherethesuperscriptspertain to thebody-fixed and thesubscriptsto thespace-fixed frame.Theelectron spin
matrixelementsare wherethesuperscriptspertain to thebody-fixed and thesubscriptsto thespace-fixed frame.Theelectron spin
matrixelementsare Ω
Ω
±
±
=
±
S
J
M S
S
J
M
,
1
2
,
,
,
1
2
,
,
1,
(24)
Ω
Ω
±
∓
=
S
J
M S
S
J
M
,
1
2
,
,
,
1
2
,
,
1
2 . (25)
z (24) (25) AppendixB. Conversion factors Dependenceofthetunnelingsplitting Δ
=
−
=
=
∣
∣=
=
=
∣
∣=
E B
E
E
B
(
)
t
J
N
M
J
N
M
˜
, ˜
1,
˜
, ˜
0,
1
2
1
2
1
2
1
2
on theopticalfield interaction
parameter ηopt for η = 5
el
(red curve) and η = 2.5
m
(bluecurves). Figure25. Probabilitydensities, orientation and alignment cosinesofa Σ
2
moleculeasfunctionsoftheanisotropicpolarizability
interaction parameter ηopt in thepresenceofamagneticand an electricfield.Valuespertainingto theF1and F2statesareshown,
respectively, bydashed and fulllines.Blueand red curvespertain, respectively,to theleft (orientation) and right (alignment) ordinate. Notethat here η = 5
el
and η = 2.5
m
. Figure24. Dependenceofthetunnelingsplitting Δ
=
−
=
=
∣
∣=
=
=
∣
∣=
E B
E
E
B
(
)
t
J
N
M
J
N
M
˜
, ˜
1,
˜
, ˜
0,
1
2
1
2
1
2
1
2
on theopticalfield interaction
parameter ηopt for η = 5
el
(red curve) and η = 2.5
m
(bluecurves). Figure24. Dependenceofthetunnelingsplitting Δ
=
−
=
=
∣
∣=
=
=
∣
∣=
E B
E
E
B
(
)
t
J
N
M
J
N
M
˜
, ˜
1,
˜
, ˜
0,
1
2
1
2
1
2
1
2
on theopticalfield interaction
parameter ηopt for η = 5
el
(red curve) and η = 2.5
m
(bluecurves). Figure25. Probabilitydensities, orientation and alignment cosinesofa Σ
2
moleculeasfunctionsoftheanisotropicpolarizability
interaction parameter ηopt in thepresenceofamagneticand an electricfield.Valuespertainingto theF1and F2statesareshown,
respectively, bydashed and fulllines.Blueand red curvespertain, respectively,to theleft (orientation) and right (alignment) ordinate. Notethat here η = 5
el
and η = 2.5
m
. Figure25. Probabilitydensities, orientation and alignment cosinesofa Σ
2
moleculeasfunctionsoftheanisotropicpolarizability
interaction parameter ηopt in thepresenceofamagneticand an electricfield.Valuespertainingto theF1and F2statesareshown,
respectively bydashed and fulllines Blueand red curvespertain respectively to theleft (orientation) and right (alignment) ordinate Figure25. Probabilitydensities, orientation and alignment cosinesofa Σ
2
moleculeasfunctionsoftheanisotropicpolarizability
interaction parameter ηopt in thepresenceofamagneticand an electricfield.Valuespertainingto theF1and F2statesareshown,
respectively, bydashed and fulllines.Blueand red curvespertain, respectively,to theleft (orientation) and right (alignment) ordinate
Notethat here η = 5
el
and η = 2.5
m
. TableA1.The
′
f J
J
( , ) term ofthe
direction cosinematrix,
equation (22). AppendixB. Conversion factors With quantitiesexpressin customaryunits, thedimensionlessinteraction parametersaregiven by: η
μ
ε
=
−
−
B
0.0168
(Debye)
(kV cm )
(cm ),
S
el
el
1
1
η =
−
B
0.9347
(Tesla)
(cm ),
m
1
η
Δα
=
×
Å
−
−
−
B
1.05
10
[
]
(W cm )
(cm ). opt
11
3
2
1 η
μ
ε
=
−
−
B
0.0168
(Debye)
(kV cm )
(cm ),
S
el
el
1
1
η =
−
B
0.9347
(Tesla)
(cm ),
m
1
3 η
Δα
=
×
Å
−
−
−
B
1.05
10
[
]
(W cm )
(cm ). opt
11
3
2
1 η
Δα
=
×
Å
−
−
−
B
1.05
10
[
]
(W cm )
(cm ). opt
11
3
2
1 21 NewJ.Phys.17(2015) 045017 KSharmaand BFriedrich Figure22. Dependenceoftheeigenenergiesofapolar Σ
2
moleculeon theanisotropicpolarizabilityinteraction parameter ηopt in the
presenceofan electricand amagneticfield.F1and F2levelsareshown,respectively, bydashed and fulllinesin panelspertainingto
signed valuesofthegood quantum number M.Red and bluecurvespertain,respectively, to stateswith
=
J˜
1
2 and
=
J˜
3
2.Notethat
here η = 2.5
m
and η = 5
el
. Figure22. Dependenceoftheeigenenergiesofapolar Σ
2
moleculeon theanisotropicpolarizabilityinteraction parameter ηopt in the
presenceofan electricand amagneticfield.F1and F2levelsareshown,respectively, bydashed and fulllinesin panelspertainingto
signed valuesofthegood quantum number M.Red and bluecurvespertain,respectively, to stateswith
=
J˜
1
2 and
=
J˜
3
2.Notethat
here η = 2.5
m
and η = 5
el
. Figure23. Dependenceofthetunnelingsplitting Δ
=
=
∣
∣=
−
=
=
=
∣
∣=
E B
E
N
M
E
B
(
˜
1,
t
J
J
N
M
˜
,
1
2
˜
, ˜
0,
)
1
2
1
2
1
2
on theopticalfield
interaction parameter ηopt for η = 0
m
(red curve) and η = 2.5
m
(bluecurves). Figure23. Dependenceofthetunnelingsplitting Δ
=
=
∣
∣=
−
=
=
=
∣
∣=
E B
E
N
M
E
B
(
˜
1,
t
J
J
N
M
˜
,
1
2
˜
, ˜
0,
)
1
2
1
2
1
2
on theopticalfield
interaction parameter ηopt for η = 0
m
(red curve) and η = 2.5
m
(bluecurves). TableB1 showswhat valuestheinteraction parameters ηel, ηm, and ηopt takeat particular valuesofthe
respectivefield strengthsfor choice Σ
2
molecules. 22 NewJ.Phys.17(2015) 045017 KSharmaand BFriedrich Figure24. AppendixB. Conversion factors ′
′
h
J
M
J M
( ,
; ,
)
Z
′ =
+
J
J
1
+
+
−
+
J
M
J
M
2 (
1)(
1)
′ =
J
J
M
2
′ =
−
J
J
1
+
−
J
M
J
M
2 (
)(
) Table A5.The
′
′ ±
h
J
M
J M
( ,
1; ,
)
X
and ±
′
′ ±
h
J
M
J M
i
( ,
1; ,
)
Y
termsofthedirection cosinematrix,equation (22). ′
′ ±
h
J
M
J M
( ,
1; ,
)
X
or ±
′
′ ±
h
J
M
J M
i
( ,
1; ,
)
Y
′ =
+
J
J
1
∓
±
+
±
+
J
M
J
M
(
1)(
2)
′ =
J
J
∓
∓
+
J
M
J
M
(
)(
1)
′ =
−
J
J
1
∓
∓
∓
−
J
M
J
M
(
)(
1) Table A6. Non-vanishingmatrixelementsofthe
θ
cos
operator in thesymmetrictop basisset. Ω
θ
Ω
′
∣
∣
J
M
J
M
,
,
cos
,
,
′ =
+
J
J
1
Ω
Ω
+
+
−
+
+
+
−
+
+
+
+
J
J
J
M
J
M
J
J
J
(
1)(
1)(
1)(
1)
(
1) (2
1)(2
3)
′ =
J
J
Ω
+
M
J J(
1)
′ =
−
J
J
1
Ω
Ω
+
−
+
−
+
−
J
J
J
M
J
M
J
J
J
(
)(
)(
)(
)
(2
1)(2
1) Table A7. Nonvanishingelementsofthe
θ
cos2
operator in thesymmetrictop basisset. AppendixB. Conversion factors ′
f J
J
( ; )
′ =
+
J
J
1
+
+
+
J
J
J
1
4(
1) (2
1)(2
3)
′ =
J
J
+
J J
1
4 (
1)
′ =
−
J
J
1
+
+
−
J
J
J
1
4(
1) (2
1)(2
1) 23 NewJ.Phys.17(2015) 045017 KSharmaand BFriedrich NewJ.Phys.17(2015) 045017 TableA2. The
Ω
Ω
′
′
g J
J
( ,
; ,
)
z
term ofthe
direction cosinematrix, equation (22). Ω
Ω
′
′
g J
J
( ,
; ,
)
z
′ =
+
J
J
1
Ω
Ω
+
+
−
+
J
J
2 (
1)(
1)
′ =
J
J
Ω
2
′ =
−
J
J
1
Ω
Ω
+
−
J
J
2 (
)(
) TableA2. The
Ω
Ω
′
′
g J
J
( ,
; ,
)
z
term ofthe
direction cosinematrix, equation (22). Ω
Ω
′
′
g J
J
( ,
; ,
)
z
′ =
+
J
J
1
Ω
Ω
+
+
−
+
J
J
2 (
1)(
1)
′ =
J
J
Ω
2
′ =
−
J
J
1
Ω
Ω
+
−
J
J
2 (
)(
) TableA3.The
Ω
Ω
′
′ ±
g
J
J
( ,
1; ,
)
x
and
Ω
Ω
∓
′
′ ±
g
J
J
i
( ,
1; ,
)
y
termsofthedirection cosinematrix,equation (22). Ω
Ω
′
′ ±
g
J
J
( ,
1; ,
)
x
or
Ω
Ω
∓
′
′ ±
g
J
J
i
( ,
1; ,
)
y
′ =
+
J
J
1
Ω
Ω
∓
±
+
±
+
J
J
(
1)(
2)
′ =
J
J
Ω
Ω
∓
∓
+
J
J
(
)(
1)
′ =
−
J
J
1
Ω
Ω
∓
∓
∓
−
J
J
(
)(
1) TableA3.The
Ω
Ω
′
′ ±
g
J
J
( ,
1; ,
)
x
and
Ω
Ω
∓
′
′ ±
g
J
J
i
( ,
1; ,
)
y
termsofthedirection cosinematrix,equation (22). Table A4.The
′
′
h
J
M
J M
( ,
; ,
)
Z
term ofthe
direction cosinematrix, equation (22). AppendixB. Conversion factors Ω
θ
Ω
′J
M
J M
cos2
′ =
+
J
J
2
Ω
Ω
Ω
Ω
+
+
+
+
−
+
−
+
+
+
+
+
−
+
−
+
+
+
+
+
+
J
J
J
J
J
M
J
M
J
M
J
M
J
J
J
J
J
(
2)(
1)(
2)(
1)(
2)(
1)(
2)(
1)
(
1)(
2)(2
3) (2
1)(2
5)
′ =
+
J
J
1
+
Ω
Ω
Ω
+
+
−
+
+
+
−
+
+
+
+
+
M
J
J
J
M
J
M
J
J
J
J
J
(
1)(
1)(
1)(
1)
(
1)
(2
1)(2
3)
1
1
2
2
⎡
⎣
⎤
⎦
′ =
J
J
+
+
Ω
Ω
Ω
−
−
−
+
+
−
+
−
+
+
−
J
J
M
J
J
M
J
J
J
J
M
J
J
(
)(
)
(4
1)
(
1)
((
1)
)((
1)
)
(
1) (4(
1)
1)
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
′ =
−
J
J
1
+
Ω
Ω
Ω
+
−
+
−
+
−
−
+
M
J
J
J
M
J
M
J
J
J
J
J
(
)(
)(
)(
)
(2
1)(2
1)
1
1
1
1
2
⎡
⎣
⎤
⎦
′ =
−
J
J
2
Ω
Ω
Ω
Ω
+
+
−
−
−
−
+
+
−
−
−
−
−
−
+
−
J
J
J
J
J
M
J
M
J
M
J
M
J J
J
J
J
(
)(
1)(
)(
1)(
)(
1)(
)(
1)
(
1)(2
1) (2
1)(2
3) Table A7. Nonvanishingelementsofthe
θ
cos2
operator in thesymmetrictop basisset. 24 NewJ.Phys.17(2015) 045017 NewJ.Phys.17(2015) 045017 KSharmaand BFriedrich TableB1.Rotationalconstants, B,spin-rotation constants, γ,electricdipolemoments, μel,polarizabilityanisotropies, Δα,and valuesof
thedimensionlessinteraction parameters ηel, ηm,and ηopt at electric, magnetic, and opticalfieldsof100 kV cm−1,1Tesla, and 1012
W cm−2, respectively,for choice Σ
2
molecules, seetext.Dipolemomentsand polarizabilitiesgiven in parenthesesareestimates. Compi-
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]
p
[18] Friedrich B2003Manipulatingcold moleculeswith non resonant fieldsModernTrendsChemical ReactionDynami [18] Friedrich B2003Manipulatingcold moleculeswith no AppendixB. Conversion factors Molecule
B(cm−1)
γ(cm−1)
μ(D)
Δα (Å3)
ηel @100 kV cm−1
ηm @1 T
ηopt @1012 W cm−2
NaO
0.462
0.193
7.88a,c
2.88a,c
3.63
2.02
180.1
CaH
4.28
0.045
2.94
1.95a,b
1.15
0.22
4.81
CaF
0.34
0.0013
3.34
4.83a,b
16.4
2.75
150
MgH
5.83
0.020
1.27
2.29
0.37
0.16
4.15
RbO
0.24
−0.019
(8.5)
0.422a,c
60
3.9
18.57
BeH
10.32
0.005
0.1069
1.70
0.16
0.09
1.74
CN
1.99
0.0073
1.45
0.383
1.22
0.47
2.03
CaCl
0.15
0.0014
4.47
1.05a,b
50.06
6.23
73.92
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|
https://openalex.org/W2803448435
|
http://old.scielo.br/pdf/urbe/v10n3/2175-3369-urbe-2175-3369010003AO01.pdf
|
Portuguese
| null |
Financeirização da moradia e segregação socioespacial: Minha Casa, Minha Vida em São José dos Campos, Taubaté e Jacareí/SP
|
Urbe. Revista Brasileira de Gestão Urbana
| 2,018
|
cc-by
| 7,496
|
Financeirização da moradia e segregação
socioespacial: Minha Casa, Minha Vida em São
José dos Campos, Taubaté e Jacareí/SP Housing financing and socio-spatial segregation: “Minha Casa Minha Vida” in
São José dos Campos, Taubaté and Jacareí/SP Daniela das Neves Alvarenga[a], Paulo Romano Reschilian[a] [a] Universidade do Vale do Paraíba (Univap), Instituto de Pesquisa e Desenvolvimento (IP&D), São José dos Campos, SP, Brasil Resumo O presente artigo aborda o processo de internacionalização do capital financeiro no Brasil após a década de
1990, no período denominado neoliberal, e a política pública habitacional na fase neodesenvolvimentista,
entre 2002 e 2016, por intermédio da produção de moradia vinculada ao Programa Federal Minha Casa,
Minha Vida (MCMV). A discussão foi feita a partir do entendimento da forma pela qual a moradia de mercado
se tornou um ativo financeiro, uma nova fronteira de ganhos para o mercado de capitais no espaço urbano,
evidenciando a sofisticação do Sistema Financeiro Imobiliário (SFI) criado em 1997, e seu distanciamento
do conceito da universalização da habitação enquanto direito. O objetivo do trabalho é demonstrar que as
unidades habitacionais produzidas nas cidades de São José dos Campos, Taubaté e Jacareí, situadas na Região
Metropolitana do Vale do Paraíba e Litoral Norte (RMVPLN), no Estado de São Paulo, no período de 2009 a
2014, passaram a atender a esse novo mercado. Palavras-chave: Financeirização. Política habitacional. Espaço urbano. Segregação socioespacial. MCMV. DNA é mestre, e-mail: danielaneves.arq@gmail.com
PRR é doutor, e-mail: pauloromano@univap.br DOI: 10.1590/2175-3369.010.003.AO01 ISSN 2175-3369
Licenciado sob uma Licença Creative Commons DOI: 10.1590/2175-3369.010.003.AO01 ISSN 2175-3369
Licenciado sob uma Licença Creative Commons urbe. Revista Brasileira de Gestão Urbana (Brazilian Journal of Urban Management), 2018 set./dez., 10(3), 473-484 Introdução O Programa Minha Casa, Minha Vida (MCMV),
instituído em 2009 com base em um Sistema Nacional
de Habitação (SNH) focado no mercado, foi responsável
pela produção de moradia por meio do financiamento
ao setor privado. Segundo Maricato (2015), a retomada
da visão empresarial da política habitacional, com
a finalidade explícita de enfrentamento à crise
econômica de 2008, foi determinante não só para
construção de novas casas, mas também pelo boom
imobiliário ocorrido no Brasil. Contribuiu também para
o aumento do preço da terra, resultando na expulsão
dos pobres para a periferia, além do espraiamento
das cidades. Ferreira (2015) define o MCMV como
um programa de financiamento, e não uma política
urbana e habitacional, focado, portanto, no lucro do
setor imobiliário e das construtoras, mas distante do
interesse público. Segundo Santos (2004), a lógica do mercado global
se impõe ao mercado interno. A ciência, a tecnologia
e a informação, vetores fundamentais do processo
social, são a base da produção, da utilização e do
funcionamento do espaço, cada vez mais equipado
para facilitar a circulação da informação. Esse nexo
informacional, presente no processo de globalização,
é representado por capitais fixos (estradas, pontes
etc.), constantes (maquinário, veículos etc.) e pelos
fluxos, inclusive financeiro, que atuam na requalificação
dos espaços para atender aos interesses dos atores
hegemônicos da economia, da política e da cultura. Da mesma forma que a incorporação imobiliária, o
capital financeiro imobiliário busca moldar o espaço
urbano. Os lobbies imobiliários sobre o executivo e o
legislativo, no início do que se configurou como fase
neodesenvolvimentista, foram decisivos para o desenho
do Programa MCMV, discutido fora do Ministério das
Cidades, sem a participação do Conselho das Cidades
ou da Habitação, diretamente na Casa Civil. O entusiasmo neoliberal, principalmente a partir
da criação do Sistema Financeiro Imobiliário (SFI) em
1997, foi responsável por incorporar a racionalidade das
finanças na organização do financiamento da política
habitacional, por meio de instrumentos financeiros,
como os créditos imobiliários, distanciando-se do
conceito da universalização da moradia enquanto
direito (Royer, 2009). De acordo com Fix (2011),
a financeirização da política habitacional carrega
a lógica da habitação como frente da acumulação
capitalista, e não como um direito social, colaborando
para a construção de espaços urbanos cada vez mais
distanciados do direito à cidade. Introdução Estado e um mercado fortes, por meio de uma
política nacional focada no crescimento econômico,
atrelada ao aumento da renda da população e ao
fortalecimento do mercado de massas (inclusão pelo
consumo). A Política Nacional de Desenvolvimento
Regional (PNDR), criada em 2003 no Governo Lula,
foi responsável pela instituição de programas de
transferência de renda e de infraestrutura social,
especialmente, para as regiões Norte e Nordeste do
país, com destaque para o Programa Territórios da
Cidadania criado em 2008, que trabalhava com o
conceito de territórios rurais sustentáveis. O Programa
de Aceleração do Crescimento (PAC), criado em 2007,
responde aos princípios da Política Nacional de
Habitação (PNH) de 2004 e promoveu a integração
tecnológica e econômica, abrindo novos territórios
ao mercado (Senra, 2011). Estado e um mercado fortes, por meio de uma
política nacional focada no crescimento econômico,
atrelada ao aumento da renda da população e ao
fortalecimento do mercado de massas (inclusão pelo
consumo). A Política Nacional de Desenvolvimento
Regional (PNDR), criada em 2003 no Governo Lula,
foi responsável pela instituição de programas de
transferência de renda e de infraestrutura social,
especialmente, para as regiões Norte e Nordeste do
país, com destaque para o Programa Territórios da
Cidadania criado em 2008, que trabalhava com o
conceito de territórios rurais sustentáveis. O Programa
de Aceleração do Crescimento (PAC), criado em 2007,
responde aos princípios da Política Nacional de
Habitação (PNH) de 2004 e promoveu a integração
tecnológica e econômica, abrindo novos territórios
ao mercado (Senra, 2011). No Brasil, o período denominado neoliberal
(décadas de 1990-2000) ocorreu em um contexto de
redemocratização do país, dentro de um cenário global
de avanços tecnológicos nas áreas de computação e
de transportes, possibilitando a internacionalização
do capital financeiro por meio de uma conexão direta
e imediata do global com o local, baseado em um
modelo de acumulação flexível, ao mesmo tempo que
as desigualdades entre países centrais e periféricos
se acentuavam. Apoiado nos princípios do Consenso de Washington,
especialmente na liberalização financeira, na
liberalização do comércio exterior e na eliminação
de restrições ao capital externo, durante o governo
de Fernando Henrique Cardoso (1994-2002) houve
uma forte desestatização da economia, baseada em
uma política pública fundamentada na criação dos
Eixos Nacionais de Integração e Desenvolvimento em
1996, com o objetivo de conectar espaços produtivos
nacionais aos mercados internacionais (Senra, 2011). Abstract This article discusses the post 1990’s process of internationalization of financial capital in Brazil, in the
so-called neoliberal period, and public housing policy in the neodevelopmental phase, between 2002 and 2016,
through the production of housing linked to the federal program “Minha Casa Minha Vida” (MCMV - My House
My Life). The discussion was based on the understanding of how the market housing became a financial asset,
a new frontier of gains for the capital market in urban space. We evidence the sophistication of the Housing
Financial System created in 1997, and its distancing from the concept of the universalization of housing as
a right. The objective of this work is to demonstrate that the housing units produced in the cities of São José
dos Campos, Taubaté and Jacareí, located in the Metropolitan Region of the Vale do Paraíba e Litoral Norte
(RMVPLN) - SP, from 2009 to 2014, began to fulfill this market. Keywords: Financialization. Housing policy. Urban space. Socio-spatial segregation. MCMV. 474 Alvarenga, D. N., & Reschilian, P. R. urbe. Revista Brasileira de Gestão Urbana (Brazilian Journal of Urban Management), 2018 set./dez., 10(3), 473-484 Introdução Para este trabalho, foram obtidos dados primários
do MCMV, referentes às construtoras e à localização dos
empreendimentos contratados na Região Metropolitana
do Vale do Paraíba e Litoral Norte (RMVPLN), no
Estado de São Paulo, a partir da Caixa Econômica
Federal, bem como dados secundários fornecidos pelo
Ministério das Cidades, compilados no site deepAsk. A fase neodesenvolvimentista ocorrida no Brasil,
a partir de 2000, é marcada pela presença de um urbe. Revista Brasileira de Gestão Urbana (Brazilian Journal of Urban Management), 2018 set./dez., 10(3), 473-484 475 Financeirização da moradia e segregação socioespacial De acordo com Fix (2011), a política habitacional
estava pautada na criação de fluxos contínuos, por
meio do FGTS e do SBPE, bases financeiras para
constituição do circuito imobiliário. No entanto, a
única opção pós-1964 foi a oferta da casa própria, em
benefício da indústria da construção, principalmente
no subsetor de edificações. Mesmo com a construção
de quatro milhões de unidades habitacionais, o
cenário urbano nacional que se formava, sobretudo
com a crise de 1980, era de crescimento das favelas
nas médias e grandes cidades. com, relativos ao número de unidades contratadas e
concluídas e aos recursos aplicados nas três faixas do
programa, no período de 2009 a 2014. O objetivo desta
pesquisa é analisar a financeirização da habitação e
seu efeito no espaço urbano a partir dos municípios
de São José dos Campos, Taubaté e Jacareí, maiores
déficits habitacionais da RMVPLN, no ano de 2010. A hipótese é de que os empreendimentos produzidos
tenham atendido ao mercado de capitais, reafirmando a
lógica do MCMV enquanto programa de financiamento,
focado no lucro do setor imobiliário e das construtoras,
acentuando a segregação socioespacial. O processo de internacionalização do capital financeiro,
especialmente a partir da década de 1980, induziu
diversos países em desenvolvimento a adotar políticas
neoliberais, inclusive o Brasil, que reduziu despesas
do Estado, promovendo a liberação da economia e
ampliando as exportações para pagamento da dívida
externa. Após 1994, com Fernando Henrique Cardoso
na presidência, foram priorizadas políticas públicas
destinadas à promoção do aumento da competitividade
das empresas nacionais, para posterior inserção no
mercado internacional (Senra, 2011). Surgimento e configuração do
circuito imobiliário no Brasil Após a Segunda Guerra Mundial e, principalmente,
a partir da década de 1970, o mercado global vem
se consolidando com base no desenvolvimento da
ciência, da tecnologia e da informação, em que os
espaços atendem, sobretudo, aos interesses dos atores
hegemônicos da economia, da cultura e da política
(Santos, 2014). Segundo Senra (2011), a fase neoliberal foi
acompanhada também pela globalização, com os
avanços tecnológicos de comunicação e transportes,
conectando o global ao local. As políticas públicas de
desenvolvimento regional implementadas na época
tinham o objetivo de diminuir as desigualdades regionais
e promover a ocupação do território. O Programa dos
Eixos Nacionais de Integração e Desenvolvimento foi
uma das frentes adotadas pelo Governo FHC. A crescente penetração de práticas financeiras em
todas as relações econômicas e sociais alcança então
a produção do espaço urbano, em um processo em
que as instituições financeiras passam a buscar novas
fontes de lucratividade. O mercado imobiliário é então
absorvido pela dinâmica financeira, responsável pela
definição do capitalismo mais recente (Sanfelici, 2013). Foi nesse contexto que o Plano Plurianual 1996/1999
introduziu a proposta de sete Eixos Nacionais
de Integração e Desenvolvimento, considerada
a política de desenvolvimento regional mais
relevante do período, que teve como principal
objetivo conectar espaços produtivos nacionais
aos mercados internacionais (Senra, 2011, p. 181). Para entender como esse fluxo financeiro
transformou a moradia de mercado em uma nova
fronteira de ganhos para o mercado de capitais no
espaço urbano brasileiro, faz-se necessário retornar
ao período da ditadura militar, analisando a montagem
do sistema de financiamento habitacional, a partir
da criação do Banco Nacional de Habitação (BNH). O desenvolvimento do crédito imobiliário no Brasil
foi construído a partir de 1964, quando da criação
do BNH, do Sistema Financeiro de Habitação (SFH),
do Fundo de Garantia do Tempo de Serviço (FGTS),
em 1967, e do Sistema Brasileiro de Poupança e
Empréstimos (SBPE). O SFH, funding composto por
recursos públicos, privados e paraestatais, evidenciava
o papel do Estado na dinamização do setor imobiliário
e das políticas públicas de habitação durante o regime
militar (Royer, 2009). Segundo Rolnik (2015), o Governo FHC não se
preocupou em respeitar os direitos sociais estabelecidos
pela Constituição Federal de 1988, focando no retraimento
da intervenção estatal, no ajuste e na consolidação de
instituições financeiras internacionais, como o Banco
Mundial, o Fundo Monetário Internacional (FMI) e a
Organização Mundial do Comércio (OMC). urbe. Revista Brasileira de Gestão Urbana (Brazilian Journal of Urban Management), 2018 set./dez., 10(3), 473-484 Surgimento e configuração do
circuito imobiliário no Brasil A partir da carência de investimentos públicos, o
capital privado passou a apresentar suas propostas,
baseadas nos pontos estabelecidos no Consenso 476 Alvarenga, D. N., & Reschilian, P. R. de Washington, privatizando os serviços públicos,
desregulamentando os contratos de trabalho e
flexibilizando os direitos sociais (Maricato, 2013). do caráter de mercadoria da habitação, dentro de um
contexto mundial, que afetou de modo estrutural a
universalização do direito à moradia digna. A política habitacional no Governo FHC ficou
subordinada à Secretaria de Política Urbana do
Ministério do Planejamento e Orçamento. O Programa
Pró-Moradia e o Programa de Arrendamento Residencial
(PAR) foram seguidos pela instituição do SFI em
1997, com a finalidade de promover o financiamento
imobiliário em geral. Segundo Royer (2009), instituiu-se
também a racionalidade das finanças na organização
do financiamento da política de crédito imobiliário
e habitacional, por intermédio dos instrumentos
financeiros, uma vez que o SFI, diferentemente do
SFH, procurava captar fundos no mercado de capitais. A fase neodesenvolvimentista ocorrida a partir de
2000 foi responsável pela definição de políticas sociais,
que possibilitaram uma nova geração de políticas
públicas regionais, fomentando a competitividade
entre sistemas regionais de produção, com a
finalidade de promover o crescimento econômico e
o desenvolvimento social. Nesse cenário de competição em um sistema
global de produção, foram criadas, em 2003, a PNDR
e as mesorregiões diferenciadas, além da política dos
Territórios da Cidadania, a fim de evitar uma fragmentação
ainda maior no território brasileiro, promovendo a
adoção de políticas sociais com impacto territorial,
como os programas de transferência de renda e de
infraestrutura social, como o Bolsa Família e o Programa
Luz Para Todos, que privilegiaram as regiões Norte e
Nordeste, as mais vulneráveis do Brasil (Senra, 2011). Somando-se aos instrumentos financeiros já
existentes, como as Debêntures, as Letras Hipotecárias
e as Cédulas de Crédito Bancário, foram criados os
Certificados de Recebíveis Imobiliários (CRI), as
Cédulas de Crédito Imobiliário (CCI) e as Letras
de Crédito Imobiliário (LCI), fazendo com que o
SFI se configurasse com uma nova arquitetura do
financiamento imobiliário, fundada na crença de que
o mercado de capitais seria a solução para a provisão
de fundos para o sistema imobiliário. A construção da política habitacional do início
do Governo Lula foi inspirada no Projeto Moradia —
formulado no Instituto Cidadania, em 2000 —, sendo
já prevista a criação do Ministério das Cidades. urbe. Revista Brasileira de Gestão Urbana (Brazilian Journal of Urban Management), 2018 set./dez., 10(3), 473-484 O MCMV e a produção do espaço urbano
na Região Metropolitana do Vale do
Paraíba e Litoral Norte (RMVPLN) O ano de 2006 foi marcado pela influência de grandes
incorporadoras, como a Cyrela, a PDG e a MRV, as quais
levantavam capital na bolsa de valores, estabelecendo
vínculo com o mercado de capitais, configurando o
poder crescente das finanças (Sanfelici, 2013). O Programa MCMV, na sua fase 1, de 2009 a 2011,
previa um investimento total de R$ 28 bilhões a
serem destinados para faixa 1, modalidade FAR, FDS
(Entidades) e Rural, além das faixas 2 e 3, modalidade
FGTS, para construção de um milhão de unidades
habitacionais. Na fase 2, até 2014, o valor desse
investimento subiu para R$ 125 bilhões, a serem
direcionados, principalmente, para a construção
de moradias para as famílias com renda de zero a
três salários mínimos (60% de todo investimento),
prevendo a construção de dois milhões de unidades
habitacionais (Amore, 2015). Segundo Rolnik (2015), quando estourou a crise
hipotecária e financeira nos Estados Unidos no
final de 2008, os empresários atingidos passaram a
intensificar o lobby no Ministério da Fazenda para
a implementação de um pacote habitacional. Como
forma de salvar as empresas e, ao mesmo tempo,
funcionar como medida contracíclica para garantir
empregos e crescimento econômico, o presidente
Lula propôs a construção de um milhão de moradias. Rolnik (2015) ressalta que Em 2010, os maiores déficits habitacionais absolutos
da RMVPLN estavam presentes nos municípios de
São José dos Campos, Taubaté e Jacareí, com 21.910,
8.969 e 7.053 unidades, respectivamente (Vieira,
2015). Segundo dados da Fundação SEADE (2016)
São José dos Campos apresentava a maior população,
com 660.008 habitantes, seguida por Taubaté, com
296.449, e Jacareí, com 221.650 habitantes. [...] de pacote de salvamento de incorporadoras
financeirizadas, o MCMV transformou-se
na política habitacional do país, baseado no
modelo único de promoção de casa própria,
acessada via mercado e crédito hipotecário
(Rolnik, 2015, p. 309). Desde 2009, os três municípios localizados no
eixo da Rodovia Presidente Dutra, conforme Figura 1,
passaram a receber um grande aporte de recursos para
construção de unidades habitacionais contratadas
nas faixas 1, 2 e 3 do Programa MCMV. Até 2014,
foram contratadas 15.367 unidades em São José
dos Campos, 8.725 em Taubaté e 6.317 na cidade de
Jacareí (DeepAsk, 2016). O Programa MCMV, criado em 2009, foi elaborado
sob a liderança da Casa Civil e do Ministério da Fazenda,
promovendo “[...] um diálogo direto com representantes
da construção, e sem a participação popular [...]”
(Fix, 2011, p. 140). Surgimento e configuração do
circuito imobiliário no Brasil A
ampliação dos recursos e subsídios para a habitação
de interesse social teve como partida a Resolução nº
460 do Conselho Gestor do FGTS em 2005, seguida
da aprovação do Fundo Nacional de Habitação de
Interesse Social (FNHIS) no mesmo ano, resultado
de um projeto de lei de iniciativa popular. Como se pode depreender dos principais
instrumentos e da forma como foi estruturado,
o SFI constitui na verdade um marco regulatório
da participação e da operação de instituições
financeiras e correlatas no financiamento
imobiliário e no mercado de capitais, diferente
do SFH, que buscava constituir um aparato
estatal para o financiamento de habitação
(Royer, 2009, p. 102). Em síntese, o volume de recursos cresceu
significativamente combinando diversas
fontes, como o Orçamento Geral da União, o
Fundo de Amparo ao Trabalhador (FAT), o
Fundo de Arrendamento Residencial (FAR),
o Fundo de Desenvolvimento Social (FDS), o
Tesouro Nacional e, principalmente, o FGTS
(Fix, 2011, p. 134). Fix (2011) comenta que, embora o objetivo do
SFI fosse integrar o crédito imobiliário ao mercado
de capitais, a caderneta de poupança, o FGTS e o
binômio SFH/BNH permaneceram como base de
suas operações. A criação do Ministério das Cidades, inspirada
nas reivindicações trazidas pelo Fórum Nacional da
Reforma Urbana (FNRU), contribuiu para redução
das desigualdades sociais e urbanas, principalmente
a partir da Resolução nº 460, que permitiu a redução
do custo dos financiamentos com recursos do FGTS, e
por meio da criação do FNHIS, que passou a fornecer
subsídios diretos para habitação de interesse social. Como ressaltam Cardoso & Aragão (2013), essas duas
medidas demonstraram o compromisso do governo Shimbo (2016) ressalta a forma capitalista de
produção de habitação a partir dos anos 2000, por meio
do alinhamento entre Estado, finanças e construção
civil, responsável pela mobilização de diversos agentes
produtores — proprietários de terras, construtoras
subcontratadas, investidores internacionais e agentes
financeiros —, pela centralização do capital e pela
concentração de terras urbanas nos denominados
land banks. Segundo a autora, ocorreu a exacerbação urbe. Revista Brasileira de Gestão Urbana (Brazilian Journal of Urban Management), 2018 set./dez., 10(3), 473-484 477 Financeirização da moradia e segregação socioespacial federal em subsidiar a produção de moradia para as
camadas de mais baixa renda. urbe. Revista Brasileira de Gestão Urbana (Brazilian Journal of Urban Management), 2018 set./dez., 10(3), 473-484 O MCMV e a produção do espaço urbano
na Região Metropolitana do Vale do
Paraíba e Litoral Norte (RMVPLN) O pacote proposto direcionava os
recursos para o Fundo de Arrendamento Residencial
(FAR), em que o FGTS e o Orçamento Geral da União
(OGU) tornaram-se as fontes de financiamento. A Figura 2 traz o número de unidades contratadas pelo
programa na faixa 1 entre 2009 e 2014, evidenciando
a contratação de empreendimentos de interesse social
já em 2009 no município de Jacareí, a partir de 2012
em Taubaté e desde 2013 em São José dos Campos –
esta última cidade com o maior número de unidades
contratadas (4.914). Segundo Fix (2011), o Programa MCMV carrega
a lógica do BNH, em que a habitação é tratada como
mercadoria a ser produzida em modelos capitalistas,
favorecendo, especialmente, as grandes empresas que
passaram a atuar no “segmento econômico” (faixas
de quatro a dez salários mínimos). Em que pesem as dimensões não equacionadas pelo
Programa MCMV, relativas principalmente ao privilégio
concedido ao setor privado no combate ao déficit
habitacional, a proposta trazida pelo governo previa
a destinação de 40% de todo investimento às famílias
com renda de até três salários mínimos. Tratava-se,
portanto, de um subsídio jamais visto em programas
anteriores, seja por meio do Fundo de Arrendamento
Residencial (FAR), seja pelo Fundo de Desenvolvimento
Social (FDS), no caso da modalidade Entidades. De acordo com Suriano (2012), o processo de
expansão das periferias e a consequente segregação
socioespacial se consolidaram em São José dos Campos
no período de 1997 a 2011, por meio de planos e
programas habitacionais da Prefeitura Municipal
em parceria com a Caixa Econômica Federal ou com
a Companhia de Desenvolvimento Habitacional e
Urbano (CDHU). Conforme dados da Caixa Econômica
Federal (CEF, 2017), das 4.914 unidades contratadas
pelo Programa MCMV na faixa 1, 1.318 estavam urbe. Revista Brasileira de Gestão Urbana (Brazilian Journal of Urban Management), 2018 set./dez., 10(3), 473-484 478 Alvarenga, D. N., & Reschilian, P. R. Figura 1 - Mapa da RMVPLN, municípios e sub-regiões
Fonte: elaboração própria, a partir de EMPLASA (2012). Figura 2 - Unidades contratadas pelo Programa MCMV na faixa 1, de 2009 a 2014
Fonte: elaboração própria, a partir de DeepAsk (2016). localizadas na Região Leste da cidade, sob a forma de
condomínio, a 18 Km do centro. urbe. Revista Brasileira de Gestão Urbana (Brazilian Journal of Urban Management), 2018 set./dez., 10(3), 473-484 O MCMV e a produção do espaço urbano
na Região Metropolitana do Vale do
Paraíba e Litoral Norte (RMVPLN) O único loteamento
aprovado dentro do programa foi o “Pinheirinho
dos Palmares II”, destinado às famílias removidas
Sudeste, totalmente desarticulado da malha urban
próximo à Rodovia Carvalho Pinto, a quase 20 Km d
Região Central, e foi construído para 1.461 família
Da mesma forma que programas anteriores, as nov Figura 1 - Mapa da RMVPLN, municípios e sub-regiões
Fonte: elaboração própria, a partir de EMPLASA (2012). Figura 2 - Unidades contratadas pelo Programa MCMV na faixa 1, de 2009 a 2014
Fonte: elaboração própria, a partir de DeepAsk (2016). urbe. Revista Brasileira de Gestão Urbana (Brazilian Journal of Urban Management), 2018 set./dez., 10(3), 473-484
478/484
Figura 2 - Unidades contratadas pelo Programa MCMV na faixa 1, de 2009 a 2014
Fonte: elaboração própria, a partir de DeepAsk (2016). localizadas na Região Leste da cidade, sob a forma de
condomínio, a 18 Km do centro. O único loteamento
aprovado dentro do programa foi o “Pinheirinho
dos Palmares II”, destinado às famílias removidas
do antigo Pinheirinho. O loteamento fica na Região
Sudeste, totalmente desarticulado da malha urbana,
próximo à Rodovia Carvalho Pinto, a quase 20 Km da
Região Central, e foi construído para 1.461 famílias. Da mesma forma que programas anteriores, as novas
localidades da moradia de interesse social no município Figura 2 - Unidades contratadas pelo Programa MCMV na faixa 1, de 2009 a 2014
Fonte: elaboração própria, a partir de DeepAsk (2016). Figura 2 - Unidades contratadas pelo Programa MCMV na faixa 1, de 2009 a 2014
Fonte: elaboração própria, a partir de DeepAsk (2016). Sudeste, totalmente desarticulado da malha urbana,
próximo à Rodovia Carvalho Pinto, a quase 20 Km da
Região Central, e foi construído para 1.461 famílias. Da mesma forma que programas anteriores, as novas
localidades da moradia de interesse social no município localizadas na Região Leste da cidade, sob a forma de
condomínio, a 18 Km do centro. O único loteamento
aprovado dentro do programa foi o “Pinheirinho
dos Palmares II”, destinado às famílias removidas
do antigo Pinheirinho. O loteamento fica na Região urbe. Revista Brasileira de Gestão Urbana (Brazilian Journal of Urban Management), 2018 set./dez., 10(3), 473-484 479 Financeirização da moradia e segregação socioespacial A Figura 3 aponta o número de unidades habitacionais
contratadas nas faixas 2 e 3 (segmento econômico),
atingindo famílias com renda de quatro a dez salários
mínimos. O MCMV e a produção do espaço urbano
na Região Metropolitana do Vale do
Paraíba e Litoral Norte (RMVPLN) Considerando o gráfico relativo à faixa 2
do programa, destaca-se, novamente, o município
de São José dos Campos, com 7.353 unidades
contratadas. Taubaté e Jacareí aparecem juntas,
com aproximadamente 4 mil unidades contratadas
no MCMV. O gráfico relativo à faixa 3 reflete o maior
número de unidades contratadas em São José dos
Campos (3.100 unidades habitacionais), seguida por
Taubaté (2.231) e Jacareí (859). de São José dos Campos refletem a negação do direito
à cidade para 2.779 famílias com renda de até três
salários mínimos, em oposição aos princípios do Plano
Local de Habitação de Interesse Social (PLHIS), que
prevê a garantia do direito à moradia digna como
vetor de inclusão social. Como ressalta Rolnik (2015), os conjuntos
habitacionais voltados para as famílias enquadradas na
faixa 1 continuam ocorrendo nas periferias, próximos
às margens das cidades, reproduzindo a segregação
socioespacial de programas habitacionais anteriores. No caso de Jacareí, de acordo com Vieira (2015),
os sete condomínios financiados pelo Programa
MCMV, na faixa 1, foram construídos na Região Sul,
em uma área de franja urbana do município, apesar da
diretriz constante no PLHIS de 2010, de se construir
moradia para a população de mais baixa renda nos
vazios urbanos e em áreas centrais. A localização dos
empreendimentos construídos para 1.248 famílias sob
a forma de condomínio revela a dinâmica segregadora
e o espraiamento da cidade, visto também em São
José dos Campos. Segundo Sanfelici (2013), a lógica estabelecida
enormemente a partir de 2000, com a integração das
incorporadoras imobiliárias à dinâmica da Bolsa de
Valores, é a da maximização da apropriação de rendas: A oferta de grandes empreendimentos, somente
tornada possível pela capacidade de levantar somas
assombrosas no mercado de capitais, funciona
como uma espécie de economia de escala, por
intermédio da qual as incorporadoras podem
ampliar a diferença entre a renda do solo atual urbe. Revista Brasileira de Gestão Urbana (Brazilian Journal of Urban Management), 2018 set./dez., 10(3), 473-484
479/484
Figura 3 - Unidades contratadas pelo Programa MCMV nas faixas 2 e 3, de 2009 a 2014
Fonte: elaboração própria, a partir de DeepAsk (2016). a 3 Unidades contratadas pelo Programa MCMV nas faixas 2 e 3 de 2009 a 2014 Figura 3 - Unidades contratadas pelo Programa MCMV nas faixas 2 e 3, de 2009 a 2014
Fonte: elaboração própria, a partir de DeepAsk (2016). urbe. O MCMV e a produção do espaço urbano
na Região Metropolitana do Vale do
Paraíba e Litoral Norte (RMVPLN) Revista Brasileira de Gestão Urbana (Brazilian Journal of Urban Management), 2018 set./dez., 10(3), 473-484 480 Alvarenga, D. N., & Reschilian, P. R. (aquela paga ou permutada com o proprietário
do terreno) e a renda do solo potencial, colocada
pela localização do terreno na totalidade do
tecido urbano (Sanfelici, 2013, p. 41). Para Maricato (2013), o problema da moradia
persiste por estar vinculado a um pedaço de solo que
se valoriza de acordo com os atributos do entorno,
estando relacionado então à renda da terra. De acordo
com Ferreira (2015), a ordem fundiária e urbana
vigente não foi alterada pelo Programa MCMV. A lógica
de produzir segregação e desigualdade permanece. Segundo o autor, a negação do direito à cidade e a
responsabilidade pelo impacto urbano-ambiental
passam pelas construtoras, mas é, essencialmente,
culpa dos governos locais, responsáveis pelas políticas
territorial, urbana e habitacional, capazes de As formas assumidas pelos empreendimentos
são inúmeras, segundo o autor, passando por
empreendimentos multifuncionais de alto padrão,
mesclando o uso residencial com os usos comerciais
e de serviços, até condomínios de baixa renda nas
franjas dos tecidos metropolitanos e loteamentos
de unidades unifamiliares no âmbito do Programa
MCMV, contribuindo para construção de um espaço
urbano cada vez mais fragmentado. “[...] alterar o equilíbrio de forças políticas, de
enfrentar de fato a desigualdade sócio espacial
e mexer no poder dos proprietários fundiários
e das elites urbanas [...]” (Ferreira, 2015, p. 8). O espraiamento urbano, segregação socioespacial,
consolidação de grandes vazios urbanos, bairros
monofuncionais, formação de áreas isoladas ou
desarticuladas da malha urbana, sem diversidade
de usos e grupos sociais, caracterizam padrão
de produção das cidades que já se mostrou
insustentável do ponto de vista ambiental,
econômico e social (Ferreira, 2012, p. 64). Com relação aos recursos aplicados nos três
municípios, o valor total dos financiamentos e
subsídios contratados para a aquisição ou produção
das unidades habitacionais do Programa MCMV destaca
a cidade de São José dos Campos, com um valor total
de R$ 1.311.302.935,24, conforme a Figura 4, seguida
por Taubaté, com R$ 637.744.677,63, e Jacareí, com
R$ 473.476.409,94. A Figura 5 apresenta o valor dos
financiamentos contratados nos três municípios, sem
considerar os subsídios da União. São José dos Campos
também aparece com o maior valor de financiamento
contratado, R$ 783.360.975,69, seguida de Taubaté, com
R$ 402.484.050,16, e Jacareí, com R$ 304.246.655,67. O MCMV e a produção do espaço urbano
na Região Metropolitana do Vale do
Paraíba e Litoral Norte (RMVPLN) Os diferentes agentes da
construção civil têm por isso obrigação de assumir
corresponsabilidades quanto à sustentabilidade
e efeitos futuros da urbanização que promovem,
em relação à qualidade da arquitetura que bela 1 - São José dos Campos, Taubaté e Jacareí: unidades contratadas por faixa de renda – Programa MCMV (2009-2014) Tabela 1 - São José dos Campos, Taubaté e Jacareí: unidades contratadas por faixa de renda – Programa MCMV (2009-2014) Tabela 1 - São José dos Campos, Taubaté e Jacareí: unidades contratadas por faixa de renda – Programa MCMV (2009-2014)
Município
Faixa 1
Faixa 2
Faixa 3
Total nas 3 faixas
(0 a 3 SM)
(3 a 6 SM)
(6 a 10 SM)
São José dos Campos
4.914
7.353
3.100
15.367
Taubaté
2.124
4.370
2.231
8.725
Jacareí
1.248
4.210
859
6.317
Total
8.286
15.933
6.190
30.049
Fonte: elaboração própria, a partir de DeepAsk (2016). urbe. Revista Brasileira de Gestão Urbana (Brazilian Journal of Urban Management), 2018 set./dez., 10(3), 473-484
482/484
Tabela 1 - São José dos Campos, Taubaté e Jacareí: unidades contratadas por faixa de renda – Programa MCMV (2009-2014)
Município
Faixa 1
Faixa 2
Faixa 3
Total nas 3 faixas
(0 a 3 SM)
(3 a 6 SM)
(6 a 10 SM)
São José dos Campos
4.914
7.353
3.100
15.367
Taubaté
2.124
4.370
2.231
8.725
Jacareí
1.248
4.210
859
6.317
Total
8.286
15.933
6.190
30.049
Fonte: elaboração própria, a partir de DeepAsk (2016). Tabela 2 - São José dos Campos, Taubaté e Jacareí: relação entre o déficit habitacional e o número de unidades concluídas por meio do Programa MCMV
(2009-2014)
Município
Déficit absoluto 2010
Unidades concluídas
PMCMV
Déficit absoluto restante
Redução do déficit
relativo (%)
(Faixas 1, 2 e 3)
São José dos Campos
21.910
9.661
12.249
45
Taubaté
8.969
4.235
4.734
47
Jacareí
7.053
6.186
867
88
Fonte: elaboração própria, a partir de DeepAsk (2016). Fonte: elaboração própria, a partir de DeepAsk (2016). O MCMV e a produção do espaço urbano
na Região Metropolitana do Vale do
Paraíba e Litoral Norte (RMVPLN) Esse padrão de urbanização que tem marcado o
período neoliberal, a partir de 2000, reflete o poder
financeiro dos grandes investidores globais por
trás dos agentes imobiliários, contribuindo para a
continuidade das dinâmicas segregadoras no território
nacional. Mais uma vez os interesses particulares
prevalecem nas recentes transformações do espaço,
impedindo a realização da vida urbana e de uma
sociedade mais justa. A Figura 6 aponta o valor dos subsídios da União
para os empreendimentos MCMV oriundos do b R i
B
il i d G
ã U b
(B
ili
J
l f U b
M
) 2018
/d
10(3) 473 484
480/484
Figura 4 - Valor total dos financiamentos e subsídios do Programa MCMV, de 2009 a 2014
Fonte: elaboração própria, a partir de DeepAsk (2016). Figura 4 - Valor total dos financiamentos e subsídios do Programa MCMV, de 2009 a 2014
Fonte: elaboração própria, a partir de DeepAsk (2016). urbe. Revista Brasileira de Gestão Urbana (Brazilian Journal of Urban Management), 2018 set./dez., 10(3), 473-484 urbe. Revista Brasileira de Gestão Urbana (Brazilian Journal of Urban Management), 2018 set./dez., 10(3), 473-484 481 Financeirização da moradia e segregação socioespacial Figura 5 - Valor dos financiamentos contratados, de 2009 a 2014
Fonte: elaboração própria, a partir de DeepAsk (2016). Figura 5 - Valor dos financiamentos contratados, de 2009 a 2014
Fonte: elaboração própria, a partir de DeepAsk (2016). Figura 5 - Valor dos financiamentos contratados, de 2009 a 2014
Fonte: elaboração própria, a partir de DeepAsk (2016). Figura 5 - Valor dos financiamentos contratados, de 2009 a 2014
Fonte: elaboração própria, a partir de DeepAsk (2016). b R i t B
il i d G tã U b
(B
ili
J
l f U b
M
t) 2018
t /d
481/484
Figura 6 - Valor dos subsídios da União e do FGTS, de 2009 a 2014
Fonte: elaboração própria, a partir de DeepAsk (2016). urbe Revista Brasileira de Gestão Urbana (Brazilian Journal of Urban Management) 2018 set /dez
481/484
Figura 6 - Valor dos subsídios da União e do FGTS, de 2009 a 2014
Fonte: elaboração própria, a partir de DeepAsk (2016). Figura 6 - Valor dos subsídios da União e do FGTS, de 2009 a 2014
Fonte: elaboração própria, a partir de DeepAsk (2016). urbe. Revista Brasileira de Gestão Urbana (Brazilian Journal of Urban Management), 2018 set./dez., 10(3), 473-484 482 Alvarenga, D. N., & Reschilian, P. R. O MCMV e a produção do espaço urbano
na Região Metropolitana do Vale do
Paraíba e Litoral Norte (RMVPLN) produzem, em relação ao espaço público e à
cidade (Ferreira, 2012, p. 52). produzem, em relação ao espaço público e à
cidade (Ferreira, 2012, p. 52). Orçamento Geral da União (OGU) e o valor dos subsídios
aplicados na aquisição das unidades habitacionais
oriundos do FGTS. Observa-se um salto no valor dos
subsídios da União no caso de São José dos Campos a
partir de 2013, refletindo a contratação de unidades
habitacionais voltadas para a faixa 1, verificada na
Figura 2, totalizando 4.914 moradias para a população
de baixíssima renda. Os subsídios oriundos do OGU
representam, aproximadamente, 30% do valor total
dos financiamentos e subsídios do programa MCMV
em São José dos Campos, 25% no caso de Taubaté e
18% na cidade de Jacareí. Segundo dados da Caixa Econômica Federal (CEF,
2017), as construtoras Gafisa, MRV, Rossi, Tenda,
Rodobens, PDG, Goldfarb e Cury passaram a atuar na
RMVPLN a partir de 2009. De acordo com a Tabela 1,
o número total de unidades contratadas na faixa 1 do
programa, considerando os três municípios analisados,
é de 8.286 unidades; já nas faixas 2 e 3, o total sobe
para 22.123 unidades habitacionais contratadas em
São José dos Campos, Jacareí e Taubaté, no período
de 2009 a 2014. Os dados relativos à contratação nas
faixas 2 e 3 revelam a atuação das grandes empresas
no chamado segmento econômico, em detrimento da
moradia de interesse social. Ferreira (2012) destaca a urgência na conscientização
das construtoras e incorporadoras quanto à qualidade
da inserção urbana dos novos empreendimentos,
principalmente no acesso à rede de infraestrutura e
transportes, uma vez que, ao atuar no MCMV, o setor
privado se beneficia de importantes fundos públicos. Com relação ao déficit habitacional absoluto nos
três municípios, nota-se uma significativa redução do
déficit relativo, principalmente na cidade de Jacareí,
conforme Tabela 2. No entanto, de acordo com Vieira
(2015), o déficit habitacional relativo à faixa de renda de
zero a três salários mínimos ainda não foi equacionado
no município, uma vez que o déficit dessa faixa era
igual 3.849 unidades em 2010, e foram construídas
1.248 moradias até o ano de 2014. Por isso, a responsabilidade social de sua atividade
deve ser-lhe imputada. O MCMV e a produção do espaço urbano
na Região Metropolitana do Vale do
Paraíba e Litoral Norte (RMVPLN) Tabela 2 - São José dos Campos, Taubaté e Jacareí: relação entre o déficit habitacional e o número de unidades concluídas por meio do Programa MCMV
(2009-2014)
Município
Déficit absoluto 2010
Unidades concluídas
PMCMV
Déficit absoluto restante
Redução do déficit
relativo (%)
(Faixas 1, 2 e 3)
São José dos Campos
21.910
9.661
12.249
45
Taubaté
8.969
4.235
4.734
47
Jacareí
7.053
6.186
867
88 Tabela 2 - São José dos Campos, Taubaté e Jacareí: relação entre o déficit habitacional e o número de unidades concluídas por meio do Programa MCMV
(2009-2014) Tabela 2 - São José dos Campos, Taubaté e Jacareí: relação entre o déficit habitacional e o número de unidades concluídas por meio do Programa MCMV
(2009-2014) urbe. Revista Brasileira de Gestão Urbana (Brazilian Journal of Urban Management), 2018 set./dez., 10(3), 473-484
482/484
Tabela 2 - São José dos Campos, Taubaté e Jacareí: relação entre o déficit habitacional e o número de unidades concluídas por meio do Programa MCMV
(2009-2014)
Município
Déficit absoluto 2010
Unidades concluídas
PMCMV
Déficit absoluto restante
Redução do déficit
relativo (%)
(Faixas 1, 2 e 3)
São José dos Campos
21.910
9.661
12.249
45
Taubaté
8.969
4.235
4.734
47
Jacareí
7.053
6.186
867
88
Fonte: elaboração própria, a partir de DeepAsk (2016). 483 Financeirização da moradia e segregação socioespacial Embora o problema habitacional no Brasil demonstre
a dependência dos municípios em relação aos recursos
do governo federal, é necessário retomar alternativas
baseadas no fortalecimento da gestão pública, na
autogestão, individual ou coletiva, e na produção de
habitação de interesse social. Há de se pensar arranjos
que preveem a melhoria de habitação e núcleos
existentes, como discutem as assessorias técnicas nos
movimentos de moradia, uma vez que o financiamento
dessas melhorias atenderiam a uma demanda muito
maior do que aquela por empreendimentos novos. É preciso fortalecer a discussão na proposta de
ocupação de imóveis construídos vagos, para que se
cumpra a função social da propriedade, uma vez que
muitas vezes são unidades providas de infraestrutura
completa, como ressaltam Fix & Arantes (2009). regulação e apropriação, manteve-se inalterada. A política pública habitacional do Governo Lula
priorizou a construção de moradia como resposta
ao déficit habitacional no país, por meio da criação
do Programa MCMV. O pacote habitacional lançado
em 2009 foi adotado como medida contracíclica para
garantir empregos e crescimento econômico, após a
crise financeira de 2008. Conclusão O desenvolvimento nas áreas de telecomunicação
e de logística foi determinante para configuração de
uma relação direta e imediata do global com local,
assinalando o rápido crescimento do processo de
internacionalização do capital financeiro após a
década de 1980, no período neoliberal. Estratégias
políticas federais foram adotadas no Governo FHC,
conectando espaços produtivos nacionais aos mercados
internacionais, consolidando o papel das instituições
financeiras internacionais no país. A criação do SFI,
em 1997, incorporou a racionalidade das finanças na
organização do financiamento da política habitacional,
integrando o crédito imobiliário ao mercado de
capitais, por meio da criação de novos instrumentos
financeiros, os CRI, as CCI e as LCI. O MCMV e a produção do espaço urbano
na Região Metropolitana do Vale do
Paraíba e Litoral Norte (RMVPLN) Apesar do grande investimento direcionado ao
programa, principalmente para a faixa 1, o espaço
urbano vem se desenhando distanciado do ideal de
cidade no seu valor de uso. A reforma urbana não
ocorreu. A continuidade do processo de segregação e de
periferização na formação dos novos tecidos urbanos
reforça a cidade no seu valor de troca, evidenciando
o favorecimento aos interesses particulares, em uma
escala internacional, em detrimento do interesse público. A financeirização do espaço urbano aparece como um A financeirização do espaço urbano aparece como um A financeirização da moradia, discutida por
intermédio do número de unidades habitacionais
produzidas e dos recursos aplicados nas três faixas
do Programa MCMV, no período de 2009 a 2014, nas
cidades de São José dos Campos, Jacareí e Taubaté,
situadas na RMVPLN, revela a predominância da
produção de moradia focada no mercado, para famílias
com renda mensal de quatro a dez salários mínimos,
o então chamado “segmento econômico”. O processo
de financeirização da moradia e do solo urbano marca
o triunfo dos mercados e promove a continuidade
das dinâmicas segregadoras, especialmente para a
população de baixíssima renda, para as quais o direito
à moradia não se encontra associado à promoção do
direito à cidade. “[...] rearranjo escalar que articula, de forma
inédita, as escalas global e local no cerne do
processo de urbanização, com todas as tensões
e contradições que esse processo engendra [...]”
(Sanfelici, 2013, p. 43), “[...] rearranjo escalar que articula, de forma
inédita, as escalas global e local no cerne do
processo de urbanização, com todas as tensões
e contradições que esse processo engendra [...]”
(Sanfelici, 2013, p. 43), evidenciando o triunfo dos mercados nessa nova
ordem global e a posição do Estado como servo das
corporações multinacionais (Santos, 2014). evidenciando o triunfo dos mercados nessa nova
ordem global e a posição do Estado como servo das
corporações multinacionais (Santos, 2014). urbe. Revista Brasileira de Gestão Urbana (Brazilian Journal of Urban Management), 2018 set./dez., 10(3), 473-484 Referências Amore, C. S. (2015). “Minha Casa Minha Vida” para
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no Brasil. Revista Novos Estudos Cebrap, 35(2), 119-133. Fundação Sistema Estadual de Análise de Dados – SEADE. (2016). IMP - Informações dos Municípios Paulistas - 2016. São Paulo: SEADE. Recuperado em 11 de janeiro de 2017,
de http://www.imp.seade.gov.br Fundação Sistema Estadual de Análise de Dados – SEADE. (2016). IMP - Informações dos Municípios Paulistas - 2016. São Paulo: SEADE. Recuperado em 11 de janeiro de 2017,
de http://www.imp.seade.gov.br Vieira, I. G. (2015). Novas formas de urbanização, estudo
da tipologia de condomínios fechados para o programa
Minha Casa, Minha Vida, Jacareí, São Paulo (Dissertação
de mestrado). Instituto de Pesquisa e Desenvolvimento,
Universidade do Vale do Paraíba, São José dos Campos. Maricato, E. (2013). Financeirização da política
habitacional. In L. O. Royer (Eds.), Financeirização
da política habitacional: limites e perspectivas. São Paulo: Annablume Editora. Recuperado em 8
de janeiro de 2017, de http://annablume.com.br/
index.php?option=com_content&view=article&id= Recebido: Fev. 17, 2017
Aprovado: Jan. 29, 2018 urbe. Revista Brasileira de Gestão Urbana (Brazilian Journal of Urban Management), 2018 set./dez., 10(3), 473-484
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https://openalex.org/W2954678766
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https://europepmc.org/articles/pmc6599201?pdf=render
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English
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Building a DNA barcode library for the freshwater fishes of Bangladesh
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Scientific reports
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Md. Mizanur Rahman1, Michael Norén2, Abdur Rob Mollah1 & Sven O. Kullander We sequenced the standard DNA barcode gene fragment in 694 newly collected specimens,
representing 243 species level Operational Barcode Units (OBUs) of freshwater fishes from Bangladesh. We produced coi sequences for 149 out of the 237 species already recorded from Bangladesh. Another
83 species sequenced were not previously recorded for the country, and include about 30 undescribed
or potentially undescribed species. Several of the taxa that we could not sample represent erroneous
records for the country, or sporadic occurrences. Species identifications were classified at confidence
levels 1(best) to 3 (worst). We propose the new term Operational Barcode Unit (OBU) to simplify
references to would-be DNA barcode sequences and sequence clusters. We found one case where there
were two mitochondrial lineages present in the same species, several cases of cryptic species, one case
of introgression, one species yielding a pseudogene to standard barcoding primers, and several cases
of taxonomic uncertainty and need for taxonomic revision. Large scale national level DNA barcode
prospecting in high diversity regions may suffer from lack of taxonomic expertise that cripples the
result. Consequently, DNA barcoding should be performed in the context of taxonomic revision, and
have a defined, competent end-user. Received: 21 November 2018
Accepted: 3 June 2019
Published online: 28 June 2019 Fish and fisheries play an important role in Bangladesh’s economy, nutrition and culture. With 47 609 km2 of
inland water bodies, it is the third inland fish producing country in the world after China and India1. The annual
fish production from inland waters is estimated to be 3,496,958 mt, of which 1,163,606 mt comes from inland
open waters1. Fish is second staple food in Bangladesh and alone supplements about 60% of animal protein in the
daily dietary requirement2. Open water fishery is still the primary source of food fishes for the larger population. The Hilsa (Tenualosa ilisha) capture fishery alone contributes about 12% of the fish production1 and provides
outcome for 2.5 million people2. p
p
Bangladesh sits in between the biologically rich and diverse Indo-Burma and Eastern Himalaya regions, and
is traversed by three of Asia’s largest rivers, the Ganga, Brahmaputra, and Meghna, which reach the Bay of Bengal
in Bangladesh. Nonetheless, Bangladesh has one of the most incompletely known national freshwater fish faunas
in Asia. As with other tropical countries, diversity estimates for Bangladeshi freshwater fishes are uncertain. 1University of Dhaka, Department of Zoology, Dhaka, Dhaka, 1000, Bangladesh. 2Swedish Museum of Natural
History, Department of Zoology, SE-104 05, Stockholm, Sweden. Correspondence and requests for materials should
be addressed to S.O.K. (email: sven.kullander@nrm.se) www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports C
d
bl h
C Corrected: Publisher Correction Corrected: Publisher Correction Scientific Reports | (2019) 9:9382 | https://doi.org/10.1038/s41598-019-45379-6 Building a DNA barcode library for
the freshwater fishes of Bangladesh Md. Mizanur Rahman1, Michael Norén2, Abdur Rob Mollah1 & Sven O. Kullander Md. Mizanur Rahman1, Michael Norén2, Abdur Rob Mollah1 & Sven O. Kullander Estimates of freshwater fish species vary from 2373 to 2474, 2605, or 2676, but those numbers include migrating
and estuarine species; an estimate of riverine species only gives 104 species5. From several books and review
papers on Bangladeshi fishes4,6,7, it is evident, however, that the taxonomy used is outdated and not harmonized
with the taxonomy employed globally or even in neighbour countries. Apparently, considering the economic
importance of inland fishery and the expected richness of the fish fauna, and in the absence of an expert based
taxonomy, DNA barcoding may be an important component in biological conservation and management of bio-
diversity and fishery of Bangladeshi freshwater fishes. yi
y
gi
DNA barcoding is a tool based on the observation and premise that each species is genetically distinct — has
a unique DNA. Unique sequences of DNA from expert-identified specimens enable construction of a library of
species-specific DNA sequences, “DNA barcodes”, against which unidentified samples can be matched8,9. DNA
barcodes are useful for identification of both fresh specimens and market products such as frozen fish10,11. The
standard barcode sequence, a fragment of the mitochondrial cytochrome c subunit I gene (coi), is also frequently
used in phylogeographic and phylogenetic analyses12,13, but concerns have also been raised that the utility of bar-
coding has been overstated and that dependency on single markers may lead to deficient taxonomy14,15.i i
Here, we present the results and lessons from sequencing freshwater fishes collected in markets and natural
habitats in Bangladesh, 2014–2016, in a study strictly aimed at a complete coi-based DNA barcode reference 1University of Dhaka, Department of Zoology, Dhaka, Dhaka, 1000, Bangladesh. 2Swedish Museum of Natural
History, Department of Zoology, SE-104 05, Stockholm, Sweden. Correspondence and requests for materials should
be addressed to S.O.K. (email: sven.kullander@nrm.se) Scientific Reports | (2019) 9:9382 | https://doi.org/10.1038/s41598-019-45379-6 1 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 1. Map of collecting sites in Bangladesh, 2014–2016. A symbol may cover more than one collecting
site. The map was constructed in QGIS 2.18.1 (QGIS Development Team, 2016. QGIS Geographic Information
System. Open Source Geospatial Foundation. http://qgis.org), using free vector and raster map data made
available by Natural Earth (http://naturalearthdata.com). All maps are in the public domain (http://www. naturalearthdata.com/about/terms-of-use/). Figure 1. Map of collecting sites in Bangladesh, 2014–2016. A symbol may cover more than one collecting
site. The map was constructed in QGIS 2.18.1 (QGIS Development Team, 2016. QGIS Geographic Information
System. Open Source Geospatial Foundation. Md. Mizanur Rahman1, Michael Norén2, Abdur Rob Mollah1 & Sven O. Kullander http://qgis.org), using free vector and raster map data made
available by Natural Earth (http://naturalearthdata.com). All maps are in the public domain (http://www. naturalearthdata.com/about/terms-of-use/). library of Bangladeshi freshwater fish species, introduce a new term for species level DNA sequence clusters
interpreted as candidates for DNA barcodes: the Operational Barcode Unit (OBU). Results
h Fish specimens were obtained from 150 collecting events and additional ad hoc sampling, including both markets
and natural habitats in Bangladesh (Fig. 1). Phylogenetic and mPTP trees based on the Bayesian tree are shown
in Figs 2 and 3. OBUs/species distinguished by the mPTP analysis are listed in Table S1. Metadata for sequenced
specimens are summarized in Table S2.h p
The mPTP analysis found 243 OBUs (Table S1), representing 53 families. Most of these OBUs relate to single
species verified by morphological examination. Using a scale 3 (lowest) to 1 (highest), the species determination
expertise levels for the vouchers were estimated to be 3 in 82 OBUs, 2 in 17, and 1 in 46; 99 determinations were
classed as unqualified. i
Correcting for disagreement with the trees (Figs 2 and 3), and consideration of morphological analyses,
suggests recognition of 237 candidate species. The majority of the OBUs are uncontroversial, representing
well-known, common species. There was no indication of contamination. Five species descriptions were already
published based on the present material16–20. Two species failed sequencing (Taenioides cirratus, Johnius coitor). One probable nuclear pseudogene (NUMT)
was found in Pseudapocryptes elongatus (OBU 14). One species had two mitochondrial lineages without morpho-
logical or nuclear DNA divergence (Brachygobius nunus OBU 10–11), and one species had two distinct popula-
tions separated by highly divergent mitochondrial and nuclear sequences but without detected morphological
divergence (Danio rerio OBUs 160–161). Introgression was found in Devario anomalus (OBUs 152–153). Other
OBUs may lack morphological support, as detailed in the discussion. y
p
g
pp
Several cases of putative cryptic species were recorded, as detailed below. Here we consider as putative cryptic
species OBUs that are genetically distinct, but not reciprocally diagnosable morphologically. Several of the puta-
tive cryptic OBU pairs consist of one OBU in the Meghna River drainage and another in the Karnafuli and Sangu
River drainages. Discussion OBUs in the Bayesian tree (Fig. 2) and the mPTP analysis (Fig. 3, Table S1) were monophyletic at higher levels,
except that the trees came out unresolved or with OBUs in unexpected position, as an artefact of the wide sam-
pling (spanning the entire Actinopterygii) with several long branches without close relationship to other taxa in
the tree. This affects, e.g., Psilorhynchus (Psilorhynchidae), nested within the Cyprinidae, and Butis humeralis
(Eleotridae), within the Gobiidae). The objective of the tree-based analysis is, however, not phylogenetic relation-
ships but species delimitation by branch clusters. The mPTP analysis reported 243 “species” but there are discord-
ances with the tree. The “species” in the mPTP output includes potential or probable cases of excess taxonomic Scientific Reports | (2019) 9:9382 | https://doi.org/10.1038/s41598-019-45379-6 2 www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports/ 3
19) 9:9382 | https://doi.org/10.1038/s41598-019-45379-6
Figure 2. Phylogram of relationships of Bangladeshi fish species, based on Bayesian analysis of the standard
DNA barcode fragment of the mitochondrial coi gene. The scale bar indicates expected number of substitutions
per site. Terminals are identified by NRM or DU tissue sample collection identifiers (metadata, including
GenBank Accession Numbers, in Supplementary Table S2). Figure 2. Phylogram of relationships of Bangladeshi fish species, based on Bayesian analysis of the standard
DNA barcode fragment of the mitochondrial coi gene. The scale bar indicates expected number of substitutions
per site. Terminals are identified by NRM or DU tissue sample collection identifiers (metadata, including
GenBank Accession Numbers, in Supplementary Table S2). Figure 2. Phylogram of relationships of Bangladeshi fish species, based on Bayesian analysis of the standard
DNA barcode fragment of the mitochondrial coi gene. The scale bar indicates expected number of substitutions
per site. Terminals are identified by NRM or DU tissue sample collection identifiers (metadata, including
GenBank Accession Numbers, in Supplementary Table S2). Figure 2. Phylogram of relationships of Bangladeshi fish species, based on Bayesian analysis of the standard
DNA barcode fragment of the mitochondrial coi gene. The scale bar indicates expected number of substitutions
per site. Terminals are identified by NRM or DU tissue sample collection identifiers (metadata, including
GenBank Accession Numbers, in Supplementary Table S2). Scientific Reports | (2019) 9:9382 | https://doi.org/10.1038/s41598-019-45379-6 3 www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports/ 4
9) 9:9382 | https://doi.org/10.1038/s41598-019-45379-6
Figure 3. mPTP phylogram of Bangladeshi freshwater fish species based on the standard barcode fragment
of the mitochondrial coi gene. The scale bar indicates expected number of substitutions per site. www.nature.com/scientificreports/ www.nature.com/scientificreports/ splitting and lumping, but also a number of ghost OBUs. In the following, OBU numbers are based on the mPTP
species numbers from the analysis based on the Bayesian tree.l p
y
y
To investigate the influence of the choice of method for creating the phylogenetic tree used as indata for the
mPTP species delimitation analysis, we used two different Maximum Likelihood (ML) software, RAxML and
PHYML, to create Single Most Likely Trees, and analysed those as for the Bayesian tree. All three methods of anal-
ysis resulted in the same OBUs, with the exception of Rasbora rasbora (OBU 149) and Microphis cuncalus (OBU
25), which were recovered as single OBUs from the BI tree, but as two OBUs each from the ML trees. The genetic
variation within the two putative species was intermediate between typical intra- and inter-specific variation, the
two M. cuncalus (from Padma and Karnafuli) and three R. rasbora (from Padma and Meghna) sequences having
an uncorrected furthest pairwise-p distance of 1.7% and 1.2%, respectively. Morphological examination did not
find support for recognizing more than single species within OBUs 25 and 149.i i
pp
g
g
g
p
A GenBank BLAST search of OBUs 4–6 identified them all as Glossogobius giuris, but they represent three
genetically and morphologically distinct species. The common G. giuris morphology was represented by OBU 5,
whereas OBU 4 is more similar to G. aureus from Hong Kong. OBU 6 remains unidentified. Our identification of
OBU 4 is highly tentative.i g y
OBUs 10 and 11 represent a single species, Brachygobius nunus. Morphologically identified specimens of
Brachygobius nunus from the Turag River had distinctly different coi sequences; i.e., there were two mitochondrial
lineages within a single population of B. nunus. g
g
p p
Species of Pseudolaguvia, OBUs 57–62, showed up as six distinct lineages in the mPTP tree (Fig. 3), but got
split into seven species (Table S1), which is probably unwarranted. Unfortunately, it was not straightforward to
identify some of the included lineages with named species.h y
g
p
Three specimens of Pseudapocryptes elongatus (OBU 14) were sequenced, and all three sequences had a
unique 1 basepair (thymine, T) insertion at position 622 in the complete coi sequence (corresponding to posi-
tion 6142 in the reference genome of Parapocryptes serperaster, GenBank accession number NC_029455). Scientific Reports | (2019) 9:9382 | https://doi.org/10.1038/s41598-019-45379-6 www.nature.com/scientificreports/ This
insertion creates a TAG stop-codon and frame shift, which would render the coi protein nonfunctional, strongly
suggesting that the sequences are not of mitochondrial coi, but of a nuclear pseudogene (NUMT). Resequencing
the specimens with different primers produced the same result. There are 12 coi sequences of P. elongatus depos-
ited at GenBank, but only two, AF391394 and KT124739, are long enough to include the insertion. AF391394 is
identical to our sequences, with the exception that there is no T insertion at position 622. KT124739 contains nine
differences to our sequences, out of which four are unique insertions, but also the T insertion at position 622. It is
possible that our sequences are a chimera of the coi gene and a nuclear pseudogene, although nothing suggesting
this is apparent in the raw data. However, this is the sequence one will obtain from Bangladeshi P. elongatus when
using standard barcode primers. It is unique for the species, serving as a DNA barcode.i g
p
q
p
g
Two specimens identified morphologically as Chaca chaca were analyzed, and turn out as distinct OBUs, 81
and 82. If representing distinct species, it cannot be decided at this time which one, if either, is the Chaca chaca of
Hamilton (1822). There is one other potentially valid species described from Bengal, Chaca lophioides Cuvier &
Valenciennes, 1832. Likewise, OBU 155–156 separates two specimens of Chela cachius, but there is no morpho-
logical support for this.i g
pp
OBU 32 represents Trichopsis vittata. It is the first record of a feral population of an introduced species orig-
inating in South East Asia, which is not also an aquaculture species and the barcode is specific for the aquarium
population derived from a specific region in Thailand21.i p
pi
gh
OBUs 83–85 represent three distinct species of Olyra, of which OBU 84 identified as Olyra longicaudata
Remaining two OBUs require more analysis before a final determination can be made. g
yi
OBUs 101–102 refer to two species of Badis, which are nearly indistinguishable morphologically, but have
complementary geographical distribution and distinct coi sequences and were considered as cryptic sister
species20. g
yi
OBUs 101–102 refer to two species of Badis, which are nearly indistinguishable morphologically, but have
complementary geographical distribution and distinct coi sequences and were considered as cryptic sister
species20. Discussion The clusters
marked with red were resolved as species by the mPTP analysis. Terminals are identified by NRM or DU tissue
sample collection identifiers (metadata, including GenBank Accession Numbers, in Supplementary Table S2). Figure 3. mPTP phylogram of Bangladeshi freshwater fish species based on the standard barcode fragment
of the mitochondrial coi gene. The scale bar indicates expected number of substitutions per site. The clusters
marked with red were resolved as species by the mPTP analysis. Terminals are identified by NRM or DU tissue
sample collection identifiers (metadata, including GenBank Accession Numbers, in Supplementary Table S2). Figure 3. mPTP phylogram of Bangladeshi freshwater fish species based on the standard barcode fragment
of the mitochondrial coi gene. The scale bar indicates expected number of substitutions per site. The clusters
marked with red were resolved as species by the mPTP analysis. Terminals are identified by NRM or DU tissue
sample collection identifiers (metadata, including GenBank Accession Numbers, in Supplementary Table S2). Figure 3. mPTP phylogram of Bangladeshi freshwater fish species based on the standard barcode fragment
of the mitochondrial coi gene. The scale bar indicates expected number of substitutions per site. The clusters
marked with red were resolved as species by the mPTP analysis. Terminals are identified by NRM or DU tissue
sample collection identifiers (metadata, including GenBank Accession Numbers, in Supplementary Table S2). Scientific Reports | (2019) 9:9382 | https://doi.org/10.1038/s41598-019-45379-6 Scientific Reports | (2019) 9:9382 | https://doi.org/10.1038/s41598-019-45379-6 www.nature.com/scientificreports/ p
OBUs 109–111 refer to Pethia guganio, a little studied small cyprinid species in the Ganga and Brahmaputra
basins, but also present in the Karnafuli and Sangu drainages. The mPTP analysis recovered three OBUs initially
identified as one species. Pethia guganio is probably a species complex that cannot be resolved taxonomically here. OBU 109, however, was distinguished by the presence of barbels, not otherwise known from P. guganio.f g
y
p
g g
OBUs 126, Osteobrama cotio, and 127, O. cf. cotio, differed in coi and mt-rnr2 sequences22. Samples from the
Karnafuli and Sangu drainages were slightly different from those from the Meghna drainage, including published
sequences from the Barak River. No morphological differences were found and they might be cryptic species22. Because O. cotio is a widespread species, it should be re-analyzed with inclusion of representation of the entire
geographical distribution23. The mPTP analysis here recognizes the Meghna and Karnafuli + Sangu OBUs as two
species, for which there is no morphological support.i OBUs 126, Osteobrama cotio, and 127, O. cf. cotio, differed in coi and mt-rnr2 sequences22. Samples from the
Karnafuli and Sangu drainages were slightly different from those from the Meghna drainage, including published
sequences from the Barak River. No morphological differences were found and they might be cryptic species22. Because O. cotio is a widespread species, it should be re-analyzed with inclusion of representation of the entire
geographical distribution23. The mPTP analysis here recognizes the Meghna and Karnafuli + Sangu OBUs as two
species, for which there is no morphological support.i p
p
g
pp
OBU 136 was identified morphologically as Labeo rohita, but disagrees with the other coi sequence of L. rohita,
and is identical with GenBank sequences identified as Labeo dyocheilus and L. pangusia, with a considerably dif-
ferent morphology. It may be a case of introgression in aquaculture conditions, but L. dyocheilus and L pangusia
are large mountain species, unlikely candidates for hybridization in aquaculture. p
p
g
pp
OBU 136 was identified morphologically as Labeo rohita, but disagrees with the other coi sequence of L. rohita,
and is identical with GenBank sequences identified as Labeo dyocheilus and L. pangusia, with a considerably dif-
ferent morphology. It may be a case of introgression in aquaculture conditions, but L. dyocheilus and L pangusia
are large mountain species, unlikely candidates for hybridization in aquaculture. www.nature.com/scientificreports/ Scientific Reports | (2019) 9:9382 | https://doi.org/10.1038/s41598-019-45379-6 5 www.nature.com/scientificreports/ OBU 177 contains a single small specimen from the Sangu drainage, similar to Barilius barila in the Meghna
and Padma drainages, but assessment of its taxonomic status requires revision of Barilius. g
q
OBU 185 represents a morphologically distinct species of Lepidocephalichthys. g
q
OBU 185 represents a morphologically distinct species of Lepidocephalichthys. p
p
g
y
p
p
p
y
OBU 202 represents two morphologically distinct, but similar species of Schistura, S. sijuensis from the Garo
Hills, and a possibly undescribed species from the Sangu River. It seems likely that these two taxa may be sister
species. p
OBUs 186 and 189–190 represent Lepidocephalichthys guntea morphologically, but separate in three distinct
OBUs with tentative morphological support. OBU 189 probably represents the true L. guntea. p
g
pp
p
y
p
g
OBU 202 combines two morphologically distinct taxa incompletely separated in two drainages.i p
g
y
p
y
p
g
OBUs 222–226 suggest (Table S1) five species of Oryzias, including O. dancena, but the tree indicates only four
(Fig. 2). Only two species of Oryzias have been reported from Bangladesh24 and the Oryzias sequences maybe
excessively split in the mPTP analysis.hi y p
y
The identification of OBU 240 presents a nomenclatural problem. Authorship and name of this species is
uncertain. It was described as a new species, Parambassis ornatus by Geetakumari & Vishwanath, 201225, and
also, but with a different holotype, as P. bistigmata by Geetakumari, 201226. The latter was published in May, 2012,
but the date of publication of Geetakumari & Vishwanath’s book25 was not stated in the publication itself, only the
year. The valid name, authorship and date needs more research. yh
p
All OBUs reported here, occur in freshwater, and the majority, 2001 (Table S1), may be considered to be
exclusively or predominantly freshwater species. Classification by salinity level, however, is not straightforward. Species of the Gobiidae and Eleotridae, Oryzias, Hyporhamphus limbatus, may be common far inland, but may
also be present in coastal waters and estuaries. www.nature.com/scientificreports/ The major component of the Bangladeshi fish fauna, however, is
ostariophysan, and as such only contains freshwater species.i p y
y
p
We obtained coi sequences from 149 out of the 237 freshwater fish species already recorded from Bangladesh3,
including some euryhaline or estuarine species that are common in inland waters; and 116 out of 162 classed as
strictly freshwater. Of the 237, 31 species represented taxa erroneously recorded for the country27, or sporadic
occurrences, and several more may be considered to be marine rather than freshwater5. Nine of our sequenced
species represented the 12 species of aquaculture or aquarium releases recorded from natural habitats. We further
obtained 83 OBUs representing species that were not previously recorded for the country, including an estimated
about 30 species that may be undescribed or not yet identified. Calculations of “unknowns” are uncertain insofar
as they may represent previously misidentified species or synonyms to be re-validated.h y
y
p
p
yi
p
y
y
The present study is weak in coverage in the southwestern and western parts of Bangladesh, principally the
Sundarbans and a portion of the Ganga River and tributaries. Expectations of additional taxa from that area, are
low, however. The coverage of the Chittagong Division, including Karnafuli and Sangu Rivers and the Cox’s Bazar
region provides for considerable new ocurrences and new species16,18–20, but also strong affinity to the adjacent
western Rakhine in Myanmar, e.g., in the shared distribution of Laubuka tenella19. Most of the samples, from
the Meghna, Jamuna and Padma tributaries, reflect a common fish fauna with adjacent India, the samples from
the Pyain River, draining the Garo Hills, provide a distinct representation belonging to the Eastern Himalaya
Region28. Numerous fish species have been described from the Barak River in Manipur29, draining to the Surma
and Meghna Rivers in Bangladesh. That fauna is not reflected in our material from Bangladesh, however. Also,
the taxa shared with the Kaladan River30 are only species with very wide distribution in Bangladesh and India.h The above examples demonstrate that DNA barcoding is not trivial. Contamination, introgression, multi-
ple mitochondrial DNA lineages, cryptic species, introductions and NUMTs do occur and may be difficult to
detect. The examples in our data set are very limited, however. To this comes lack of modern revisions, and short-
age of taxonomic expertise for qualified identification of voucher specimens. www.nature.com/scientificreports/ p
p
g
pp
OBU 136 was identified morphologically as Labeo rohita, but disagrees with the other coi sequence of L. rohita,
and is identical with GenBank sequences identified as Labeo dyocheilus and L. pangusia, with a considerably dif-
ferent morphology. It may be a case of introgression in aquaculture conditions, but L. dyocheilus and L pangusia
are large mountain species, unlikely candidates for hybridization in aquaculture. g
p
y
y
q
OBU 152 is complicated, as it includes specimens of Devario anomalus with D. aequipinnatus mitochondrial
genome18. Devario coxi is here nested with D. aequipinnatus from which it was diagnosed morphologically and by
mitochondrial and nuclear genes18. Intra-OBU variation suggests inclusion of three distinct haplogroups.ih g
p
y
y
q
OBU 152 is complicated, as it includes specimens of Devario anomalus with D. aequipinnatus mitochondrial
genome18. Devario coxi is here nested with D. aequipinnatus from which it was diagnosed morphologically and by
mitochondrial and nuclear genes18. Intra-OBU variation suggests inclusion of three distinct haplogroups.ih g
p
y
y
q
OBU 152 is complicated, as it includes specimens of Devario anomalus with D. aequipinnatus mitochondrial
genome18. Devario coxi is here nested with D. aequipinnatus from which it was diagnosed morphologically and by
mitochondrial and nuclear genes18. Intra-OBU variation suggests inclusion of three distinct haplogroups. g
gg
p g
p
OBUs 160–161 represent distinct OBUs, morphologically identified as Danio rerio. The two D. rerio mitotypes
were already reported23 based on mitochondrial cytochrome b sequences and SNPs. Intraspecific variation in D. rerio needs additional analysis, but at this time there is no information to support species distinction. g
gg
p g
p
OBUs 160–161 represent distinct OBUs, morphologically identified as Danio rerio. The two D. rerio mitotypes
were already reported23 based on mitochondrial cytochrome b sequences and SNPs. Intraspecific variation in D. rerio needs additional analysis, but at this time there is no information to support species distinction. y
pp
p
OBUs 164–165, 171, 181–183, 187–88, 191–192 may represent cryptic species, or overlooked distinct spe-
cies, or even populations of the same species excessively split by mPTP, but no indication was found of species
differences. y
pp
p
OBUs 164–165, 171, 181–183, 187–88, 191–192 may represent cryptic species, or overlooked distinct spe-
cies, or even populations of the same species excessively split by mPTP, but no indication was found of species
differences. Scientific Reports | (2019) 9:9382 | https://doi.org/10.1038/s41598-019-45379-6 www.nature.com/scientificreports/ Very few of the South Asian fish
genera or species groups have been revised by specialists, e.g., Psilorhynchus31, Lepidocephalichthys32, Oryzias24,
Paracanthocobitis33,34, and Sperata35. ,
p
Our results provide relevant aspects on large-scale/country-wide DNA barcoding, end-use of barcodes, and
limits of application of the DNA barcode concept, as detailed below.h The original expectations of DNA Barcoding were relatively modest: establishment of a database of distinct
vouchered reference sequences against which unidentified sequences could be compared, and which would pro-
vide a name (identification) of the organism from which the unidentified sample was taken8,15. coi, however, has
assumed additional roles, namely for building phylogenetic trees, and for species discovery15. Studies using DNA
barcodes as a tool for species discovery15,36 were called “molecular parataxonomy” by Collins & Cruickshank15,
but are perhaps better referred to as barcode or DNA prospecting. The principle of species recognition in DNA
prospecting is based on the barcode gap convention8,15, and not on a species concept or a prior hypothesis of
diagnostic characters or phylogenetic relationships8. The barcode gap, however, is a distance measure that only
shows probability of reproductive isolation between two a priori determined samples. Consequently, we prefer
the term suggested here, Operational Barcode Unit (OBU), which is akin to the ‘species-like units’ of Collins &
Cruickshank15, for raw standard barcode coi sequences, with the caveat that identity may be the result of intro-
gression. Dissimilar coi sequences indicate reduced interbreeding, which to differing degrees may also occur
between demes or populations, not only between species, considered as independent evolutionary lineages37.i p p
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OBUs can be referred to without the need for relation to a particular species, but need specialist verification
to serve as markers for species. The expectation of species delimitation methods is that they should provide
automatic species recognition8,37, but a coi sequence distinct from others or not, is not a species marker, but only
exhibiting levels of sequence divergence that uggestive species status38. As exemplified in our trees, units delin-
eated by mPTP may conflict with species identification in several aspects, primarily dependent on the expertise
of the determiner, but also reflecting introgression and sibling species, species concepts37, and availability of a
taxonomic platform, requiring taxonomic validation using independent criteria. Scientific Reports | (2019) 9:9382 | https://doi.org/10.1038/s41598-019-45379-6 6 www.nature.com/scientificreports/ DNA barcoding projects frequently report cryptic species as well as discovery of previously unrecognized
species in fishes39,40. www.nature.com/scientificreports/ Commonly, the analysis stops there, and the recognition of cryptic species in DNA barcoding
remains a by-product of molecular prospecting39. Putative cryptic species may represent widely different evolu-
tionary processes, however41. Species may be considered cryptic because of insufficient morphological analysis,
i.e., a shortcoming of morphological analysis and thus a temporary state of identification (provisionally cryptic42). Mitochondrial genome substitution due to hybridization in a particular population may also be mistaken for
cryptic species. Consequently, “cryptic OBUs” require taxonomic assessment, and evolutionary analysis, and in
the meantime remain ghost OBUs. Whereas many of our sequences cannot be linked to a particular species, only
few cases of sibling or cryptic species were demonstrated in cyprinids and badids so far18,20,22, and the unidentified
OBUs should not be assumed to represent cryptic species or undescribed species in the absence of taxonomic
analysis. Nation-wide or regional barcoding surveys, have been proposed to accelerate DNA barcode coverage43,
and some have been attempted, such as the one reported here. Another project was started in 2013 to barcode
Swedish vertebrates44. Despite Sweden’s relatively species-poor and fully known vertebrate fauna, this barcode
project does still not contain all species of vertebrates in Sweden (78.1% barcoded), or even fishes (72% bar-
coded)44. It seems unlikely that similar projects in species-rich tropical countries will be successful in achieving
completeness. DNA barcodes are taxonomic statements. Each OBU translated to a species name expresses infor-
mation that requires taxonomic expertise, sequencing and sampling skills, permanent voucher repositories, and
constant revision of the taxonomic background. It also takes competence in using a proposed barcode, because
similar OBUs may have different evolutionary explanations. We therefore consider that DNA barcoding should
be done on a taxonomic, and not a geographical, or national basis.i g
g p
Fish DNA barcodes serve a purpose, e.g., in the fishery industry for control of product authenticity and ori-
gin and for clearing potentially harmful imports, as explicit in several projects10,11,43,45,46. Hence, trade and con-
sumption species are priority targets, and the fishery and biohazard agencies are most likely the institutions that
have the means to sample and blast sequences against openly available digital sequence providers such as BOLD
and GenBank, but which do not maintain a private DNA barcode library. In the context of such use, the overall
critical factor is that users have an unknown tissue to check against a barcode library. Methods Sampling. Fish specimens were collected using a 5 mm mesh size beach seine, or, for commercial species, by
purchase directly from fishermen or at local fish markets. Specimens were obtained from 150 collecting events
and additional ad hoc sampling including both markets and natural habitats (Fig. 1). The result was 3278 whole
specimens yielding 2514 tissue samples (whole specimens or fin clips) with considerable redundance. Based on
morphological criteria and representation of different river basins, 697 samples from Bangladesh were selected
for sequencing. coi sequencing failed in two the two specimens svailable of Johnius coitor (Sciaenidae) and the
single specimen of Taenioides cirratus Gobiidae), resulting in 692 specimens for analysis.ii g
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Whole fish or fin clips were stored in 95% ethanol at −80 °C. Fin-clipped whole specimens and excess spec-
imens for morphological analyses were fixed in 10% formalin and eventually transferred to 70% ethanol for
permanent storage. Tissue samples and voucher specimens were deposited in the fish collection of the Swedish
Museum of Natural History (NRM), and the Department of Zoology, University of Dhaka (DU). Taxonomic identification. Specimens were sorted and identified morphologically before sequencing. Identification was made by using comparative material identified by experts in the NRM collection, specialist
publications, and monographs. Several experts assisted in determinations based on high-resolution photographs,
or with comments on taxonomic status. The FISH-BOL collaborator’s protocol9 provides a graded classification of determination quality, which we
employ here, with the modification that we report confidence per specimens in the respective OBU, and not per
OBU or individual sequence, and disregard their levels 4–5 which do not produce a species determination at all. “Level 1: highly reliable identification-specimen identified by (1) an internationally recognized authority of
the group, or (2) a specialist that is presently studying or has reviewed the group in the region in question”.iii The FISH-BOL collaborator’s protocol9 provides a graded classification of determination quality, which we
employ here, with the modification that we report confidence per specimens in the respective OBU, and not per
OBU or individual sequence, and disregard their levels 4–5 which do not produce a species determination at all. www.nature.com/scientificreports/ The role of the end user
is commonly underestimated or ignored in attempts at DNA barcoding, which then becomes barcode or DNA
prospecting without any explicit end user.hi p
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This might mean less stress on taxonomic hair-splitting over the taxonomy of small-size fish species, which is
relevant for many of the species in our analysis. In southern Asia, however small-size fishes are a significant com-
ponent of the nutrition47, and identification protocols for small-size fish species may be relevant for monitoring
purposes.ti In conclusion, we have lifted some of the curtain to Bangladeshi fish coi sequences in the present report, but
also exposed some of the challenges and critical aspects of DNA barcoding from a taxonomic perspective. It sug-
gests that the decisive component of DNA barcoding, are end-user capacity, and taxonomic revision dealing with
identification, nomenclature, species delimitation, introgression, multiple mitochondrial lineages, and more, We
have been successful in generating numerous coi sequences and correlate them with species, but it turns out that
was only the start of the barcoding effort. Scientific Reports | (2019) 9:9382 | https://doi.org/10.1038/s41598-019-45379-6 Methods The cleaned PCR product was sent to Macrogen (Amstelween, The Netherlands) for sequencing. Reads were
assembled and proofread in Geneious version 1049, and a GenBank BLAST (megablast) search of the GenBank nr
database was used as a first screening for misidentified or contaminated sequences. To detect and visualize con-
taminations and problems with identification, sequences were aligned in Geneious and phylogenetic hypotheses
were constructed in MrBayes version 3.250, with the following settings: GTR + I + G model, 15 million genera-
tions, with the first 25% discarded as burn-in, then sampled every 1000 generations. Convergence was checked
with Tracer51. Sequences which passed quality control were uploaded to the Barcode of Life Database (BOLD52 and pub-
lished to GenBank53. A total of 694 barcode-compliant sequences were produced on material from Bangladesh. An additional two sequences from Bangladeshi specimen sequences were not barcode compliant, due to being
pseudogenes (NUMTs). Species delimitation. OBU detection and delimitation by multi-rate Poisson Tree Process method was
investigated based on the Bayesian tree, using mPTP version54, with the following settings: 15 separate runs,
minimum branch length estimated from alignment (0.0053), one lambda for the coalescent, starting with random
species assignments, 40 million generations, the first 20 milllion generations discarded as burn-in, then sampled
every 50 thousand generations. Convergence was checked by examining mPTP’s log files. i
Single Most Likely Trees were constructed using the RAxML55 and PHYML56 plug-ins for Geneious. RAxML
analysis was performed with GTR + G model, rapid hill climbing algorithm, seven starting trees, parsimony
random seed 1, and Maximum Likelihood search convergence criterion. PHYML analysis was performed with
GTR + I + G model, proportion of invariable sites and gamma estimated in analysis, NNI topology search. The
mPTP analysis was performed as for the Bayesian. Terminology. Gene names and symbols follow ZFIN nomenclature conventions57, except that we use here
the universally common synonym coi for the mt-co1). We reserve the term DNA barcode for a ~655 bp Folmer
region coi sequence from a vouchered specimen identified as a particular species, i.e., quality levels 1–39. We use
barcoding/barcoded for the process/accomplishment of establishing a DNA barcode for a particular species. We use the short coi for the standard DNA barcode part of the mt-co1 gene unless stated otherwise. We do not
consider coi sequences as necessarily representing species or even taxonomic units. Each such sequence needs
further analysis to assess it status. Methods “Level 1: highly reliable identification specimen identified by (1) an internationally recognized authority of g
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“Level 2: identification made with high degree of confidence at all levels-specimen identified by a trained iden-
tifier who had prior knowledge of the group in the region or used available literature to identify the specimen”.iii i
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Level 3: (modified) “identification made with high confidence to genus but less so to species—specimen iden-
tified by (1) a trained identifier who was confident of its generic placement but did not substantiate their species
identification using the literature”. Scientific Reports | (2019) 9:9382 | https://doi.org/10.1038/s41598-019-45379-6 7 www.nature.com/scientificreports/ Dataset limitation. The dataset reported is limited to the data assembled by the project, 2014–2016. DNA barcode sequencing. Approximately 1 mm3 tissue was taken from the alcohol preserved fish or fin
clip, and DNA extracted using either a Kingfisher Duo (Thermo Scientific) DNA extraction robot, with recom-
mended protocol, or DNEasy Blood & Tissue Kit (QIAGEN) spin columns, with recommended protocol.h p
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(Q
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The standard barcode sequence, 655 bp from the 5′ end of coi, was sequenced with the primers Fish
Fish-F2, Fish-R1, Fish-R248 PCR was performed using illustra PuReTaq RTG PCR Beads (GE Healthcare), with 2 µL DNA extract, 0.5 µL
of each primer and adding water for a 25 µL reaction PCR cycling was: 94 °C 5′, 35 * (54 °C 30″, 72 °C 30″), 72 °C
7′. In a few cases where molecular and morphological analyses conflicted (i.e., where there was reason to suspect
hybridization or that the coi sequence was a nuclear pseudogene), an additional mitochondrial gene (mt-rnr2)
and a nuclear gene rag1 exon 3) were sequenced. g
g
q
mt-rnr2 was sequenced using the primers 16S_arLm2 (CCTCGCCTGTTTACCAAAAACA) and 16S_brHm
(CTCCGGTCTGAACTCAGATCACGT), with PCR cycling as for COI except with 57 °C annealing tempera-
ture. rag1 exon 3 was sequenced using the primers R1_Dan1f (TGGCCATAAGGGTMAACAC) and R1_4078r
(TGAGCCTCCATGAACTTCTGAAGRTAYTT), with PCR cycling as for coi. (
)
y
g
The PCR product was cleaned by adding 5 μL of a mix of 20% Exonuclease I (EXO) and 80% FastAP
Thermosensitive Alkaline Phosphatase (Fermentas) to each 25 μl reaction, and incubated at 37 °C for 30 minutes,
then heated to 80 °C for 15 minutes. Methods We propose the neutral term OBU (Operational Barcode Unit), as descriptor
of presumed species level terminals found in an analysis of coi using multiple sequences, or a single unidentified
specimen. Ideally, an OBU will be found eventually to represent a distinct species, and its sequence serve as a
DNA barcode. We designate as ghost OBU a distinct standard DNA barcode sequence, or group of standard DNA
barcode sequences, that cannot be identified as pertaining to any known species. Ghost OBUs may represent
unidentified species, new species, NUMTs, within-species haplogroups, or bad sequence reads. Ethics statement. Specimens were already available in museum collections, purchased from fishermen or at
markets; or collected in the wild using a beach seine or hand net and euthanized through immersion in buffered
tricaine-methanesulphonate (MS 222) until cessation of opercular movements plus an additional 30 minutes,
following the protocol in permits from the Swedish Environmental Protection Agency (dnr 412-7233-08 Nv) and
the Stockholm Ethical Committee of the Swedish Board of Agriculture (dnr N 85/15). Collecting in Bangladesh
was conducted under a permit to the University of Dhaka, and approval of the Ethical Review Committee, Faculty
of Biological Sciences, University of Dhaka. Data Availability y
All sequence and associated voucher data are available from BOLD and GenBank. Voucher metadata are available
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7. The Zebrafish Information Network, https://wiki.zfin.org/display/general/ZFIN+Zebrafish+Nomenclature+Conventions (2018) Acknowledgements g
Research on Bangladeshi freshwater fishes was supported by the project “Genetic characterization of freshwater
fishes in Bangladesh using DNA barcodes”, funded by the Swedish Research Council, contract D0674001 to Sven
Kullander and Abdur Rob Mollah. We thank several colleagues for helpful comments on this manuscript, and
for support in identification of specimens: Ralf Britz, Kevin Conway, Ng Heok Hee, Maurice Kottelat; and the
Chairman of the Department of Zoology, University of Dhaka, for logistic support. Author Contributions S.O.K. initiated the project, acquired funding, managed project administration, specimen curation and internal
reports, performed the taxonomic analyses, and wrote the main part of the manuscript. M.N. developed
methods of analysis, generated and analyzed molecular sequences, curated tissue samples, and participated
in the writing of the final manuscript. M.M.R. contributed specimens, organized fieldwork, participated in
generation of sequences, taxonomic determinations, curation of specimens, and made substantial contributions
to the manuscript. A.R.M. Participated in funding acquisition, fieldwork, and logistics. All authors reviewed the
manuscript. Scientific Reports | (2019) 9:9382 | https://doi.org/10.1038/s41598-019-45379-6 Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-019-45379-6 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. 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 © The Author(s) 2019 Scientific Reports | (2019) 9:9382 | https://doi.org/10.1038/s41598-019-45379-6 10
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Traveltime Analysis of VSP Seismograms in a Horizontal Transversely Isotropic Medium: A Physical Modeling Result
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Terrestrial, atmospheric and oceanic sciences/Terrestrial, atmospheric, and oceanic sciences
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TAO, Vol. 12, No. 4, 685-693, December 2001 TAO, Vol. 12, No. 4, 685-693, December 2001 1 Institute of Applied Geophysics, Institute of Seismology, National Chung Cheng University, Min-hsiung
Chia-yi, Taiwan, ROG
2Department of Applied Physics, National Chiayi University, Min-hsiung, Chia-yi, Taiwan, ROG
*Corresponding author address: Prof. Young-Fa Chang, Institute of Applied Geophysics, Institute of
Seismology, National Chung Cheng University, Min-hs'1ung, Chia-yi 621, Taiwan, ROG
E-mail: seichyo@eq.ccu.edu.tw NOTES AND CORRESPONDENCE
Traveltime Analysis of VSP Seismograms in a Horizontal
Transversely Isotropic Medium: A Physical Modeling Result
Young-Fa Chang 1 • * and Chih-Hsiung Chang2 (Manuscript received 7 November 2000, in final form 30 August 2001) (Manuscript received 7 November 2000, in final form 30 August 2001) ABSTRACT In order to investigate the effects of anisotropy in a VSP survey, the
multi-component VSP data for observing the qP-, qSH- and qSV-waves
were collected with two different source-to-well offsets in an elastic trans
versely isotropic medium (TIM) with a horizontal symmetry axis (HTI). The traveltimes of the events were then computed using two different
algorithms: first with elastic constants of the medium, and second with "iso
tropic velocities". Comparing the traveltimes of computations with the re
corded data, a very clear and important consequence can be derived. For
strongly anisotropic media (the anisotropy for the qP- and qS-waves are
25% and 29%, respectively, in our laboratory work), the effects of anisot
ropy increase with increasing source-to-well offset. As the source-to-well
offset is small compared with the depth of the receiver ( offseUdepth $ 0.2
in our work), the effects of anisotropy can be ignored. However, in process
ing a large source-to-well offset VSP survey ( offseUdepth :2: 0.35 in this study),
special attention must be paid on the effects of anisotropy. 1. INTRODUCTION Much of the earth's crust appears to be more or less anisotropic (Crarnpin 1981; Crampin
and Lovell 1991; Helbig 1993), making seismic wave propagation more complicated than in
isotropic media. Increasing demand for fossil fuels and minerals has encouraged geophysicists
to explore deep and small reservoirs that could be possibly missed by traditional techniques. 685 TAO, Vol. 12, No. 4, December 2001 686 Therefore, seismic exploration techniques are being extended from simple isotropic models to
complex anisotropic models in order to construct the geological structures in more detail p
p
g
g
Among seismic exploration techniques, the vertical seismic profile (VSP) is the most
powerful one for investigation of anisotropy and the lithology around the well can be clearly
studied. Shear-wave splitting in the near-surface late Tertiary shale is clear in both crosshole
and VSP data (Winterstein and Paulsson 1990). Recorded as the components in the principal
anisotropic axis, the shear-waves of different polarities reveal different velocities and reflec
tion coefficients (Lynn and Thomsen 1990). The effects of singularities in sedimentary basins
were observed in examining the shear-wave splitting in multi-offset VSPs at a borehole site in
the Paris Basin (Bush and Crampin 1991). Shear-wave splitting was observed on 3-component
seismograms from earthquakes of 5 to 18 km depths in the crystalline basement beneath the
Los Angeles basin (Li et al. 1994). Wide-aperture walkaway VSP data acquired through trans
versely isotropic horizontal layers can be used to determine the qP-wave phase-slowness sur
face (Sayers 1997). Multi-component VSPs may reveal a strong vertical birefringence near the
surface (MacBeth et al. 1997). However, due to geological complexity and the limitation of sources and receivers, the
anisotropic characteristics of the earth can not be easily identified or quantified from field
data, but they can be observed in physical modeling. Ebrom et al. (1990) used a wave propaga
tion experiment in a fractured medium to show that the moveout curves for a survey line
oriented parallel to the fractures are hyperbolic, but those for a survey line oriented perpen
dicular to the fractures are non-hyperbolic. Cheadle et al. (1991) used industrial phenolite to
study the propagations of seismic waves in an orthorhombic anisotropic medium. Brown et al. (1991) showed that the effect of the shear-wave window and the variation of the hyperbolic
NMO parameter with offset are clearly observed in ultrasonic waves propagation through an
orthorhombic medium. Chang et al. 1. INTRODUCTION (1994) clearly demonstrated the phenomena of shear
wave splitting in a transversely isotropic medium (TIM). Chang and Gardner (1997) investi
gated the effects of subsurface fractures in a three-layer medium and observed that the hori
zontal moveout velocity decreases from the strike direction toward the transverse direction of
the fractures. In this paper, we study the effects of anisotropy on VSP data using physical models. The
observed anisotropic traveltimes in a strongly TIM (phenolite) with a horizontal symmetry
axis (HTI) are compared with its theoretical anisotropic traveltimes and the isotropic traveltimes
for two different source-to-well geometries. 2. EXPERIMENTS A pair of
small transducers of 0.25 inch diameter was used as source and receiver (Panarnetrics A133S
for detecting in-line particle motion, and Ultran SWC18-5 for detecting off-line and perpen
dicular particle motion). The source-transducer emits an ultrasonic pulse into the medium and
the echo is detected by the receiver-transducer. The oscilloscope (Tektronix TDS 420) re
ceives the data and converts the analogic data into digital data. A personal computer is used to
record and process the digital data. The bulk density of phenolite is 1.4 g/cm3• The elastic constants of the phenolite normal
ized by the density are A11=15.8, A13=5.0, A33=8.7, A44=2.2, and A66=4.5 (106 m'/s') (Chang
and Chang 2001a). The experimental error of velocity in the experiment is within 1 %. The
velocity-anisotropies ((Vmax-Vmin)/Vmax) of the qP- and qS-waves for the phenolite are
25% and 29%, respectively. 2. EXPERIMENTS Vertical fractures caused by the horizontal pressure or tension force of the earth are com
monly seen in the geological strata, giving the strata HTI for the seismic waves. In this study,
a strongly anisotropic phenolite, composed of layers of paper bonded with phenolic resin, was
used to simulate a HTI stratum. This phenolite is transversely isotropic because the paper is
made up of no preferred direction. The configuration of the model, apparatus and arrangement
of the measurements are shown in Fig. 1. The pulser/receiver (Panarnetrics 5058PR) emits high pulse voltage to excite the transducer, 687 Young-Fa Chang & Chih-Hsiung Chang z
. (qP)
Unit: cm
""k: Source
\J : Receiver
PC
(qSH)
4
WELL
Fig. 1. Configuration of the physical modeling,
arrangement of the sources and
receiver, apparatus and directions used
in this study. and then receives and amplifies the weak signals. An orthogonal coordinate was adopted to
describe the polarizations of the waves (Fig. 1). The Z-axis is vertical. The X- and Y-axes are
horizontal and are perpendicular and parallel to the vertical fractures, respectively. A pair of
small transducers of 0.25 inch diameter was used as source and receiver (Panarnetrics A133S
for detecting in-line particle motion, and Ultran SWC18-5 for detecting off-line and perpen
dicular particle motion). The source-transducer emits an ultrasonic pulse into the medium and
the echo is detected by the receiver-transducer. The oscilloscope (Tektronix TDS 420) re
ceives the data and converts the analogic data into digital data. A personal computer is used to
record and process the digital data. z
. (qP)
Unit: cm
""k: Source
\J : Receiver
PC
(qSH)
4
WELL
Fig. 1. Configuration of the physical modeling,
arrangement of the sources and
receiver, apparatus and directions used
in this study. z
. (qP)
Unit: cm
""k: Source
\J : Receiver
PC
(qSH)
4
WELL : Source
: Receiver Fig. 1. Configuration of the physical modeling,
arrangement of the sources and
receiver, apparatus and directions used
in this study. Fig. 1. Configuration of the physical modeling,
arrangement of the sources and
receiver, apparatus and directions used
in this study. and then receives and amplifies the weak signals. An orthogonal coordinate was adopted to
describe the polarizations of the waves (Fig. 1). The Z-axis is vertical. The X- and Y-axes are
horizontal and are perpendicular and parallel to the vertical fractures, respectively. 3.RESULTS The sources were located on the top surface and the receivers were located in the well. The length and time have been rescaled to simulate field seismic data, and the length and time
scale factors are 10000:1. Two source-to-well offsets, 100 and 500 m, were used. The receiv
ers were located between 100 and 1450 m depth with an interval of 50 m. Since the transducers
are small relative to the propagation distance of the wave, the transducers can be considered as
points. As the dominant polarizations are in the Z direction, the dominant events that are the 688 TAO, Vol. 12, No. 4, December 2001 qP-waves in this profile can be observed. When the dominant polarizations are in the Y and X
directions, the dominant events that are the qSH- and qSV-waves, respectively, can be observed. The VSPs with 100 m source-to-well offset for qP-, qSH- and qSV-waves are shown in Figs. 2, 3 and 4, respectively. And the VSPs with 500 m source-to-well offset for qP-, qSH- and
qSV-waves are shown in Figs. 5, 6 and 7, respectively. The exact phase velocities of the body waves in a homogeneous TIM can be derived from
the equations of motion. However, the group velocity can not be derived for a wave radiated
from a point source. But it can be obtained from the phase velocity using a differential opera
tor (Thomsen 1986). The solid lines in Figs. 2-7 are the theoretical traveltimes for the waves
propagation in the phenolite with group velocities. To view the effects of the anisotropy on the
propagation of elastic body waves in a HTI, the traveltimes of the elastic body waves propaga
tion with isotropic velocities were computed for comparison. The isotropic velocities are the
qP-, qSH-, and qSV-waves velocities in the Z-axis direction; they are 4000, 2100 and 1500 ml
s, respectively. The dashed lines in Figs. 2-7 show the traveltimes of the waves computed with
the isotropic velocities. The phenomenon of velocity cusps for qSV-waves in a HTI can be
expected to show up in Fig.7 as the receivers are 400-500 m in depth by the solid line. 4. DISCUSSION AND CONCLUSIONS The P-waves are easier to generate and detect than the S-waves in both field and laboratory. Therefore, the qP-waves can be easily and clearly observed in the experimental data (Figs. 2 Depth (m)
100 '
300
500
700
900
1100
1300
F {
'·
90
90
'
-
"
3. á::,. 9P,.,,
qi--es
180
180
¿À
ÁÂ Â
270
270
,o-
"';,
HI
360
360
G
'o
ms
ms Depth (m)
100 '
300
500
700
900
1100
1300
F {
'·
90
90
'
-
"
3. á::,. 9P,.,,
qi--es
180
180
¿À
ÁÂ Â
270
270
,o-
"';,
HI
360
360
G
'o
ms
ms
Fig. 2. The Z-Z component VSP with 100 m source-to-well offset. The domi
nant events in the profile are the qP-waves. The solid line is the theoreti
cal traveltimes of the qP-waves and the dashed line is the traveltimes for
the P-waves propagation using an isotropic velocity. Fig. 2. The Z-Z component VSP with 100 m source-to-well offset. The domi
nant events in the profile are the qP-waves. The solid line is the theoreti
cal traveltimes of the qP-waves and the dashed line is the traveltimes for
the P-waves propagation using an isotropic velocity. Fig. 2. The Z-Z component VSP with 100 m source-to-well offset. The domi
nant events in the profile are the qP-waves. The solid line is the theoreti
cal traveltimes of the qP-waves and the dashed line is the traveltimes for
the P-waves propagation using an isotropic velocity. Young-Fo Chang & Chih-Hsiung Chang 689 ms
Depth (m)
100
300
500
700
900
1100
1300
200
400
600
ms
Fig. 3. The Y-Y component VSP with 100 m source-to-well offset. The domi
nant events in the profile are the qSH-waves. The solid line is the theo
retical traveltimes of the qSH-waves and the dashed line is the traveltimes
for the SH-waves propagation using an isotropic velocity. ms
Depth (m)
100
300
500
700
900
1100
1300
200
400
600
ms Fig. 3. The Y-Y component VSP with 100 m source-to-well offset. The domi
nant events in the profile are the qSH-waves. The solid line is the theo
retical traveltimes of the qSH-waves and the dashed line is the traveltimes
for the SH-waves propagation using an isotropic velocity. 200
400
600
800
100
ms
Depth (m)
100
300
500
- ,_ à'
E ;>-,
"
-
, ___
D ... "_ --. 4. DISCUSSION AND CONCLUSIONS -
-.. 9Si; :-..,
,,,,
"
qye>s
700
900
1 100
1300
I
l)J1
--â...,-
""'-
,,_
reflected qP-waves
'
"e-
,
·-
-
.. ---¾
. '
>
r )>
'-;-... > ,,>
"'-
>
200
400
600
800
100
ms 200
400
600
800
100
ms
Depth (m)
100
300
500
- ,_ à'
E ;>-,
"
-
, ___
D ... "_ --. -
-.. 9Si; :-..,
,,,,
"
qye>s
700
900
1 100
1300
I
l)J1
--â...,-
""'-
,,_
reflected qP-waves
'
"e-
,
·-
-
.. ---¾
. '
>
r )>
'-;-... > ,,>
"'-
>
200
400
600
800
100
ms
Fig. 4. The X-X component VSP with 100 m source-to-well offset. The qSV
waves are weak and can only be observed in the shallow traces. The
reflected qP-waves are the qP-waves reflected from the face opposite to
that the receivers located. The solid line is the theoretical traveltimes of
the qSV-waves and the dashed line is the traveltimes for the SV-waves
propagation using an isotropic velocity. Fig. 4. The X-X component VSP with 100 m source-to-well offset. The qSV
waves are weak and can only be observed in the shallow traces. The
reflected qP-waves are the qP-waves reflected from the face opposite to
that the receivers located. The solid line is the theoretical traveltimes of
the qSV-waves and the dashed line is the traveltimes for the SV-waves
propagation using an isotropic velocity. 690 TAO, Vol. 12, No. 4, December 2001 140
240
340
440
540
ms
Depth (m)
100
300
500
-
B
qPß '""
Wq"Yes
'
C-
--
'. -
700
c -
; ,
-
,c,, '--;:-c
900
1100
1300
c
140
240
;:-.... ""'» c'
JC
)<
340
.. "
• -
........ . , •'
¼
"'>c
)
c
..... 440
>
'• >
tf:i:_
a'les
540
ms
Fig. 5. The Z-Z component VSP with 500 m source-to-well offset. The domi
nant events in the profile are the qP-waves. The reflected qP-waves are
the qP-waves reflected from the bottom of the model. The solid line is
the theoretical traveltimes of the qP-waves and the dashed line is the
traveltimes for the P-waves propagation using an isotropic velocity. 140
240
340
440
540
ms
Depth (m)
100
300
500
-
B
qPß '""
Wq"Yes
'
C-
--
'. 4. DISCUSSION AND CONCLUSIONS -
700
c -
; ,
-
,c,, '--;:-c
900
1100
1300
c
140
240
;:-.... ""'» c'
JC
)<
340
.. "
• -
........ . , •'
¼
"'>c
)
c
..... 440
>
'• >
tf:i:_
a'les
540
ms Fig. 5. The Z-Z component VSP with 500 m source-to-well offset. The domi
nant events in the profile are the qP-waves. The reflected qP-waves are
the qP-waves reflected from the bottom of the model. The solid line is
the theoretical traveltimes of the qP-waves and the dashed line is the
traveltimes for the P-waves propagation using an isotropic velocity. Depth (m)
100
300
ms
500
700
I I I
qP½waves
900
1100
1300
340
540
740
940
ms
Fig. 6. The Y-Y component VSP with 500 m source-to-well offset. The domi
nant events in the profile are the qSH-waves. The solid line is the theo
retical traveltimes of the qSH-waves and the dashed line is the traveltimes
for the SH-waves propagation using an isotropic velocity. Depth (m)
100
300
ms
500
700
I I I
qP½waves
900
1100
1300
340
540
740
940
ms Fig. 6. The Y-Y component VSP with 500 m source-to-well offset. The domi
nant events in the profile are the qSH-waves. The solid line is the theo
retical traveltimes of the qSH-waves and the dashed line is the traveltimes
for the SH-waves propagation using an isotropic velocity. Young-Fa Chang & Chih-Hsiung Chang 691 340
540
740
940
ms
Depth (m)
100
300
500
700
900
1100
1300
-. -
. t·-
, ' ¹
q-L.I, I
t
,
es
"
<; ,_.....__,.. reflected qP
1
1
"'-... ... ºwaves
....... ¸
A·--. '
''-
."-
'-"'
-
""
'
.lI -rt
::--
.---. ''-
•
r-
. . '
'-
:-,
340
540
740
940
ms
Fig. 7. The X-X component YSP with 500 m source-to-well offset. The qSY
waves are too obscure to be observed. The reflected qP-waves are the
qP-waves reflected from the face opposite to that the receivers located. The solid line is the theoretical traveltimes of the qSY-waves and the
dashed line is the traveltimes for the SY-waves propagation using an
isotropic velocity. 340
540
740
940
ms
Depth (m)
100
300
500
700
900
1100
1300
-. -
. t·-
, ' ¹
q-L.I, I
t
,
es
"
<; ,_.....__,.. reflected qP
1
1
"'-... ... 4. DISCUSSION AND CONCLUSIONS ºwaves
....... ¸
A·--. '
''-
."-
'-"'
-
""
'
.lI -rt
::--
.---. ''-
•
r-
. . '
'-
:-,
340
540
740
940
ms Fig. 7. The X-X component YSP with 500 m source-to-well offset. The qSY
waves are too obscure to be observed. The reflected qP-waves are the
qP-waves reflected from the face opposite to that the receivers located. The solid line is the theoretical traveltimes of the qSY-waves and the
dashed line is the traveltimes for the SY-waves propagation using an
isotropic velocity. and 5). The qSH-waves are irreverent to qP- and qSY-waves. Hence, qSH-waves can be seen
in Figs. 3 and 6; however, they are blurrier than qP-wave YSPs, and the small amplitude and
high frequency converted qP-waves arrive before the qSH-waves. Although the same trans
ducers are used for measuring the qSH- and qSY-waves, with a 90 degree difference in
polarization, the qSY-waves show an obstacle in the recorded data (Figs. 4 and 7). The qSY
waves are generally noisier than the qSH-waves in the ultrasonic transmission experiments. This is because the attenuation of medium for the qSY-wave is stronger than the qSH-wave,
and the qSY-wave is somewhat like to convert to qP-wave. Although the qSY-waves are
obscure and are contaminated by the direct qP-waves and converted qP-waves, they still can
be recognized in the shallow traces of Fig. 4; but in Fig. 7 they are too weak to be observed. In
Figs. 2-6, the good agreement between the theoretical (solid lines) and observed traveltimes
confirms that the events we observed are the quasi body waves propagating with the group
velocity. The isotropic arrivals advanced those of the anisotropic arrivals (in Figs. 2-3 and 5-6)
is because that the isotropic velocities (P- and S-waves) used in this study are the maximum
velocities of the qP- and qSH-waves. But the isotropic velocity of the SY-waves is not the
maximum velocity of the qSY-wave (Figs. 4 and 7). and 5). The qSH-waves are irreverent to qP- and qSY-waves. Hence, qSH-waves can be seen
in Figs. 3 and 6; however, they are blurrier than qP-wave YSPs, and the small amplitude and
high frequency converted qP-waves arrive before the qSH-waves. Although the same trans
ducers are used for measuring the qSH- and qSY-waves, with a 90 degree difference in
polarization, the qSY-waves show an obstacle in the recorded data (Figs. 4 and 7). 4. DISCUSSION AND CONCLUSIONS The qSY
waves are generally noisier than the qSH-waves in the ultrasonic transmission experiments. This is because the attenuation of medium for the qSY-wave is stronger than the qSH-wave,
and the qSY-wave is somewhat like to convert to qP-wave. Although the qSY-waves are
obscure and are contaminated by the direct qP-waves and converted qP-waves, they still can
be recognized in the shallow traces of Fig. 4; but in Fig. 7 they are too weak to be observed. In
Figs. 2-6, the good agreement between the theoretical (solid lines) and observed traveltimes
confirms that the events we observed are the quasi body waves propagating with the group
velocity. The isotropic arrivals advanced those of the anisotropic arrivals (in Figs. 2-3 and 5-6)
is because that the isotropic velocities (P- and S-waves) used in this study are the maximum
velocities of the qP- and qSH-waves. But the isotropic velocity of the SY-waves is not the
maximum velocity of the qSY-wave (Figs. 4 and 7). 0: When the source,to-well offset is small (offset/depth :0:: 0.2 in our work), it can be seen
that the curves of the predicted arrivals of the quasi body waves in our modeling HTI fit the
ones that were computed using the isotropic velocities (Figs. 2-4), especially for depths greater 692 TAO, Vol. 12, No. 4, December 2001 than 500 m. The departure between these curves becomes smaller as the receiver runs deeper. As the source-to-well offset is increased (offset/depthö 0.35 in this study) (Figs. 5-7) or the
depth of the small offset VSPs is reduced (Figs. 2-4 ), deviation between the curves of arrivals
that are respectively computed from the elastic constant and isotropic velocities is also increased. The conclusion thus can be drawn that the idea of isotropic velocity can be confidently applied
to the processing of VSP data .when the offset of the source to the well is small relative to the
depth of the receiver. For a relatively large offset, the effects of anisotropy must be taken into
account in the processing of VSP data. Otherwise, the VSP data can be erroneously processed
and interpreted. Acknowledgment This research was financially supported by the National Science Council
under the contract no. NSC 89-2116-M-194-004. Acknowledgment This research was financially supported by the National Science Council
under the contract no. NSC 89-2116-M-194-004. B., and L.A. Thomsen, 1990: Reflection shear-wave data collected near the princi- REFERENCES Brown, R. J., D. C. Lawton, and S. P. Cheadle, 1991: Scaled physical modelling of anisotro
pic wave propagation: Multioffset profiles over an orthorhombic medium. Geophys. J. Int., 107, 693-702. Bush, I., and S. Crampin, 1991: Paris Basin VSPs: case history establishing combinations of
fine-layer (or lithologic) anisotropy and crack anisotropy from modelling shear
wavefields near point singularities. Geophys. J. Int., 107, 433-477. Chang, C. H., G. H.F. Gardner, and J. A. McDonald, 1994: A physical model of shear-wave
propagation in a transversely isotropic solid. Geophysics, 59, 484-487. Chang, C. H., and G. H.F. Gardner, 1997: Effects of vertically aligned subsurface fractures
on seismic reflections: A physical model study. Geophysics, 62, 245-252. Chang, Y. F., and C. H. Chang, 2001a: Laboratory results for the features of body wave
propagation in a transversely isotropic medium. Geophysics, in press. Cheadle, S. P., R. J. Brown, and D. C. Lawton, 1991: Orthorhombic anisotropy: A physical
seismic modeling study. Geophysics, 56, 1603-1613. Crampin, S., 1981: A review of wave motion in anisotropic and cracked elastic-media. Wave
motion, 3, 343-391. Crampin, S., and J. H. Lovell, 1991: A decade of shear-wave splitting in the earth's crust:
what does it mean? what use can we make of it? and what should we do next ? Geophys. J. Int., 107, 387-407. Ebrom, D. A., R.H. Tatham, K. K. Sekharan, J. A. McDonald, and G. H.F. Gardner, 1990:
Hyperbolic traveltime analysis of first arrivals in an azimuthally anisotropic medium:
A physical modeling study. Geophysics, 55, 185-191. Helbig, K., 1993: Simultaneous observation of seismic waves of different polarization indi
cates subsurface anisotropy and might help to unravel its cause. J. Applied Geophysics,
30, 1-24. Li, Y. G., T. L. Teng, and T. L. Henyey, 1994: Shear-wave splitting observations in the north
ern Los Angles basin, southern California. Bull. Seism. Soc. Am., 84, 307-323. Lynn, H.B., and L.A. Thomsen, 1990: Reflection shear-wave data collected near the princi- Young-Fa Chang & Chih-Hsiung Chang pal axes of azimuthal anisotropy. Geophysics, 55, 147-156. MacBeth, C., X. Y. Li, X. Zeng, D. Cox, and J. Queen, 1997: Processing of a nine-component
near-offset VSP for seismic anisotropy. Geophysics, 62, 676-689. Young-Fa Chang & Chih-Hsiung Chang 693 MacBeth, C., X. Y. Li, X. Zeng, D. Cox, and J. Queen, 1997: Processing of a nine-component
near-offset VSP for seismic anisotropy. Geophysics, 62, 676-689. MacBeth, C., X. Y. Li, X. Zeng, D. Cox, and J. Queen, 1997: Processing of a nine-component
near-offset VSP for seismic anisotropy. Geophysics, 62, 676-689. MacBeth, C., X. Y. Li, X. Zeng, D. Cox, and J. Queen, 1997: Processing of a nine-component
near-offset VSP for seismic anisotropy. Geophysics, 62, 676-689. Sayers, C. M., 1997: Determination of anisotropic velocity models from walkaway VSP data
acquired in the presence of dip. Geophysics, 62, 723-729. Sayers, C. M., 1997: Determination of anisotropic velocity models from walkaway VSP data
acquired in the presence of dip. Geophysics, 62, 723-729. Sayers, C. M., 1997: Determination of anisotropic velocity models from walkaway VSP data
acquired in the presence of dip. Geophysics, 62, 723-729. Thomsen, L., 1986: Weak elastic anisotropy. Geophysics, 51, 1954-1966. Thomsen, L., 1986: Weak elastic anisotropy. Geophysics, 51, 1954-1966. Winterstein, D. F., and B. N. P. Paulsson, 1990: Velocity anisotropy in shale determined from
crosshole seismic and vertical seismic profile data. Geophysics, 35, 470-479. Winterstein, D. F., and B. N. P. Paulsson, 1990: Velocity anisotropy in shale determined from
crosshole seismic and vertical seismic profile data. Geophysics, 35, 470-479.
|
https://openalex.org/W2070752776
|
https://www.biodiversitylibrary.org/partpdf/72470
|
English
| null |
On the Existence of Rudimentary Antlers in the Okapi
|
Proceedings of the Zoological Society of London
| 1,907
|
public-domain
| 3,134
|
* For explanation of the Plates, see p. 134. 26
PROF
E
RAY
LA 26
PROF
E
RAY
LA eb. 5, 126 RAY
LANKESTER
ON 3. On
the
Hxistence
of
Rudimentary
Antlers
in
the
Okapi. By
E. Ray
Lanxester,
M.A.,
D.Sc.,
LL.D.,
F.B.S.,
F.Z.8.,
Director
of
the
British
Museum
(Natural
His-
tory). |
| (Plates
VI.
&
VII.*
and
Text-figures
49-55.) We know a great deal more as to the horns of the Okapi than
was the case when I communicated my description of that animal
to the Society in 1901, and founded the genus Okapia. The
two
skulls
sent
home
by
Sir
Harry
Johnston—the
first
seen
in
Europe—were
hornless,
and
it
was
at
first
a
matter
of
doubt as to whether the Okapi wasa hornless Giraffid, or whether
the
male
possessed
horns
whilst
these
two
skulls
were
the
one
immature and the other that of a hornless female. During
the
printing
of
my
memoir
additional
specimens
were
received
in
Brussels,
and
were
transmitted
to
Dr. Forsyth
Major
in
London
for
study
and
description. I
saw
in
Dr. Forsyth
Major’s
possession
a
fine
adult
Okapi
skull
which
had
a
pair
of
well-developed bony cones rising each by a broad base from the
frontals,
of
which
they
appeared
to
form
part. No
suture
was
visible. An
outline
of
this
skull
was
published
in
my
memoir
by
kind
permission
of
Dr. Forsyth
Major. I
also
was
able
to
examine
and
to
mention
the
existence
of
a
curious
structure
discovered by that gentleman in regard to these ossicusps ; and I
described
it
in
the
following
terms
:—‘“‘
The
fine
bony
cones
three
inches
long,
which
have
made
their
appearance
in
the Text-fig. 49. Text-fig. 49. Drawing of a fore and aft section through the tip of the ossicone of an adult Okapi
in the collection of the Museum of the Congo Independent State. The section
and drawing were made by Dr. Forsyth Major. Th
ti
h
th
t
ti
f t
fi
f
th
f
i t
th The section shows the penetration of transverse fissures from the surface into the
interior of the horn-tip. p
a, dense ivory-like bone; 6, posteriorly-placed transverse fissure; c, more anterior
transverse fissure (marking off and presumably about to cut off and detach an
anterior seement or plate of bone as a rudimentary “ antler ”) Brussels
skull,
show
no
suture
at
their
base,
nor
any
indication
of origin as separate cap-like structures. For all that one can see
they may be direct outgrowths of the frontal bone itself. Curiously ici
LON
eae
anvils Left. |
Right. Left. Right. H.Grénvold
dei
London
Stereoscopic
Co
PLOT
N=
ene
S
Ol
Oi
i Left. Right. Right. Right. H.Grénvold 4
Co
imp- 4
Co
imp- ‘0 the uncertainty which exists as to the origin of skin and skull which
»s sent home from Africa as belonging to one individual, whereas they
in certain cases belong to distinct individuals, it is still doubtful as to
wuetner tne female Okapi has or has not in the adult condition a small knob-like
protuberance of the integument, separable from the subjacent bone and representing
the horn of the male. PLOT
N=
ene
S
Ol
Oi
i eas
LOO,
see
valle G.M
Woodward
del. London
Stereoscopic
Co
imp. G.M
Woodwa 1907. RUDIMENTARY
ANTLERS
IN
THE 127 enough,
the
point
and
posterior
margin
of
the
bony
cone
are
polished
as
though
it
had
protruded
through
the
skin
like
a
cervine antler. The point is separated by a suture from the rest
of the ‘ ossicone,’ forming a small terminal cap of bone a third of
an
inch
in
depth. This
curious
structure,
as
well
as
a
possible
second suture a little lower down the ossicone, was pointed out to
me
by
Dr. Forsyth
Major. These
appearances
will
be
figured
in
that
gentleman’s
memoir
on
the
Brussels’
specimens.”
This
is
the
first
and
so
far
the
only
published
notice
of
the
antler-like
tips
of
the
Okapi’s
horns. The
figure
prepared
by
Dr. Major
of
the section made by him through the end of this Okapi’s ossicone
is
reproduced
in
the
text-figure
here
appended
(text-fig. 49). Dr. Major
does
not
himself
propose
to
publish
anything
further
at present on the Okapi, and the little drawing has been placed
in my hands by him. A tracing of it was also kindly sent to me
by M. Fraipont, of Liége. The
further
history
of
our
knowledge
of
the
horns
of
the
Okapi
has
been
complicated
by
the
arrival
in
Europe
of
various
specimens,
concerning
the
sex
of
which
either
erroneous
informa-
tion or none at all has been given by the natives from whom the
specimens
were
obtained. Thus
Dr. Forsyth
Major
was
led
to
suppose
that
the
female
Okapi
has
a
small
unattached
ossicone,
some two inches in length, when adult, but he subsequently came
to the conclusion that this supposed female was in reality a young
male. In
‘
La
Belgique
Coloniale,’
No. 21,
May
1902,
Dr. Forsyth
Major wrote :—“ L’Okapi posséde deux cornes frontales, recouvertes
dune peau velue, plus petites, de forme conique et presque verti-
cales
chez
la
femelle;
plus
grandes,
dirigées
obliquement
en
arriére et en peu triangulaires chez le male.” subsequent
date
Dr. Forsyth
Major
came
to
the
conclu-
t the specimen supposed to be a female possessing small
sicones,
was
in
reality
a
young
male
(Proc. Zool. Soe. ». * One of these broad-skulled specimens has, however, been found to possess a pair
of completely detached bony ossicones of minute size embedded in the integument.
The specimen is a little older (as indicated by the dentition) than Sir Harry John-
ston’s larger individual (that mounted in the British Museum), but is not quite
adult. It belongs to Messrs. Rowland Ward. I give here figures of the minute
ossicone (text-figs. 50 & 51). PLOT
N=
ene
S
Ol
Oi
i 339), and that the female Okapi is hornless, whilst the
ie
possesses
“horns”
which
make
their
appearance
as
conical
structures,
ossifying
independently
of
the
sub-
nes
(as
in
the
Giraffe)
and
becoming
firmly
ankylosed
mtal
bone
in
the
adult—a
boss-like
upgrowth
of
which
0 the structure of the complete horn. s little room for doubt that this is the true account of
r, though we still are in want of full information as to
icters
of
the
adult
female
Okapi*. In
a
subsequent
vation I shall be able to give more precisely the characters
» types of skull, supposed to be that of the horn-bearing
the
hornless
female,
respectively. The
skulls
carrying
sed or merely loosely-attached bony cone on the frontals At
a
subsequent
date
Dr. Forsyth
Major
came
to
the
conclu-
on that the specimen supposed to be a female possessing small
*
ossicones,
was
in
reality
a
young
male
(Proc. Zool. Soe. ». 339), and that the female Okapi is hornless, whilst the
ie
possesses
“horns”
which
make
their
appearance
as
conical
structures,
ossifying
independently
of
the
sub-
nes
(as
in
the
Giraffe)
and
becoming
firmly
ankylosed
mtal
bone
in
the
adult—a
boss-like
upgrowth
of
which
0 the structure of the complete horn. s little room for doubt that this is the true account of
r, though we still are in want of full information as to
icters
of
the
adult
female
Okapi*. In
a
subsequent
vation I shall be able to give more precisely the characters
» types of skull, supposed to be that of the horn-bearing
the
hornless
female,
respectively. The
skulls
carrying
sed or merely loosely-attached bony cone on the frontals 128 b. 5, RAY
LANKESTER
ON (supposed to be those of males) are longer and narrower than the
equally large or larger skulls devoid of any bony cones in connec-
tion with the frontals (supposed to be those of females) *. What-
ever
opinion
is
held,
or
whatever
decision
may
be
ultimately
arrived at in regard to these two types of skull, it is the fact that
they are very distinct from one another and that all the Okapi skulls
which I have examined can be definitely assigned to one or the Text-fig. 50. a. Text-fig. 50. PLOT
N=
ene
S
Ol
Oi
i Rudimentary free ossicone of hemispherical shape from the skin overlying the frontal
bossed region of the skull of an Okapi of the broad-skulled type—sub-adult
(deciduous molars very much worn, premolars not yet visible ; third lower molar
in use on both sides, fifth cusp shows slight wear). a, natural size; 6, enlarged. Text-fig.
51. Section of the ossicusp drawn in text-fig. 50, to show the incomplete ossification. Section of the ossicusp drawn in text-fig. 50, to show the incomplete ossification. other
of
these
two
types. There
is
no
third
form
known. The
two types may perhaps be best distinguished as O. johrestoni (the
name
I
gave
to
the
broad
hornless
sub-adult
skull
accompanying 1907. RUDIMENTARY
ANTLERS
IN
THE 129 the
skin
sent
home
by
Sir
Harry
Johnston)
and
0. liebrechtsi,
the name given by Dr. Forsyth Major to the more elongate and
narrow
type
of
skull,
which
is
that
usually
provided
with
bony
cones
attached
to
or
ankylosed
with
the
frontal
bones. It
is
important
to
note
that
Dr. Major
figures
a
skull
(Proc. Zool. Soe. loc. cit. p. 423) which is hornless and is regarded by him
as that of a female of the elongate type, O. liebrechtsi. I hope
shortly
to
publish
some
measurements
and
outlines
of
these
two
types
of
skull. I
have
examined
three
of
the
UO. johnstoni-type,
and
five
of
the
O. liebrechtsi-type. Though
there
is
considerable
variation in the number and breadth of the white stripes on the
fore and hind limbs of the skins of Okapi received in this country
(including
the
excellent
specimens
obtained
independently
by
Major
Powell
Cotton
and
by
Captain
Boyd
Alexander
from
widely
separated
localities,
the
former
from
the
Ituri
Forest,
the
latter
from
the
Welle
River),
I
have
seen
no
evidence
that
a
different
striping of the skin is associated with the difference of skull-form. On
the
contrary,
there
is
positive
evidence
that
the
striping
of
the
skin
is
very
nearly
identical
(though
no
two
specimens
are
exactly alike) in animals which possessed the liebrechtsi form of skull
with
that
exhibited
by
the
mounted
specimen
(0. johnstoni)
with
hornless
skull,
sent
home
by
Sir
Harry
Johnston,
figured
by
me,
and
now
in
the
British
Museum. Nevertheless,
it
is
true
that
direct and convincing evidence is as yet wanting for the conclusion
that O. liebrechtsi is merely the male of O. johnstoni. y
j
When
I
had
an
opportunity
(in
1904)
of
examining
the
fine
skin of the adult (supposed) male Okapi, presented by the Congo
State
to
the
Museum
of
Paris,
which
is
set
up
in
the
publie
gallery
there,
I
was
especially
anxious
to
note
the
state
of
the
horn-tips. Proc.
Zoou.
Soc.—1907, * Since reading this paper, I have been kindly permitted by the authorities of the
Royal Scottish Museum to examine the horns of one of the specimens above referred
to by making a section of the tip of the horn. The piece cut out has been drawn
and then carefully replaced and cemented in position, so that no injury is done to the
specimen. The skull lent to me by the Royal Scottish Museum is that of which I had
already drawn the horn-tips in figs. 1 to 8, Plate VI., before it had passed from
the possession of Messrs. Rowland Ward & Co. The sections drawn on an
enlarged scale in text-figs. 52 & 53 explain themselves. It is seen that the grooves
or fissures visible on the surface do not extend very deeply, but that there is evidence
of resorptive activity in the form of certain branching canal-like structures lying
deeply within the bony matter, which have probably been excavated by resorptive
ingrowths from the soft surface tissues. Text-fig.
51. I
found
that
they
were
represented
in
the
mounted
specimen
and
were
seen
projecting
through
the
skin
which
clothed
the
‘‘ossicone”
up
to
a
limit
of
about
half-an-inch
from
the
tip. From
this
level
the
dense
bony
matter
was
naked. It
showed
in
each
horn
two
fine
transverse
grooves,
as
10
the
ossicone
examined
and
sliced
by
Dr. Forsyth
Major. This
went
far
to
prove
that
the
condition
noted
by
him
was
not
exceptional
or
morbid,
and
accordingly
I
have
examined
the
ossicones of other specimens of adult male Okapis, as opportunity
occurred. Several
skins
and
imperfect
skeletons
have
been
received in London by dealers in zoological specimens, and I am
especially
indebted
to
Messrs. Rowland
Ward
&
Co. for
the
opportunity
of
examining
the
ossicones
of
four
adult
Okapis. Of
two
of
these
individuals
I
have
had
the
ossicones
drawn
(PI. VI.) so as to show the free termination from different points
of
view. The
two
other
specimens
examined
by
me
presented
the same remarkable appearances as those figured, and as shown
by the Paris Okapi, but I was unable to procure carefully drawn
figures
of
them. Thus,
including
the
Brussels
skull
examined
by
Dr. Forsyth
Major,
I
have
ascertained
the
existence
of
these
transverse
grooves
or
fissures
in
six
adult
male
Okapis. I
have 9 Zoou. Soc.—1907,
No. IX. 9 b. 5, 130 RAY
LANKESTER
ON also
evidence
of
their
existence
in
a
plaster
cast
of
another
specimen
which
passed
through
the
hands
of
Messrs. Rowland
Ward & Co. An
examination
of
the
figures
given
in
Plate
VI. shows
that
in all four ossicones (the right and left of two adult male Okapis)
the free terminal region is smooth and polished, forming a cap of
about half an inch in length, whilst this region is followed by a
rougher
substance,
furrowed
on
the
surface. The
polished
region
projects
beyond
the
skin,
the
rougher
region
is
clothed
by
the
living
integument. In
all
there
are
very
deep
horizontal
fissures
in
the
polished
material
of
the
“cap.”
These
fissures
are
some-
what
irregular
in
form,
and
it
is
impossible
without
making
a
section
(which
I
had
not
permission
to
do)*
through
the
solid
material to ascertain their depth. They are of the same nature as
those shown in the text-figure in section (text-figure 49, p. 126). Text-fig.
51. I think there can be little doubt that these transverse fissures
are
caused
by
the
ingrowth
of
the
living
tissue
after
the
pro-
trusion of the dense polished cap, so as to cut off the protruding
portion and provide for its breaking off—Just as an antler is cut
off
and
prepared
for
disruption
in
the
Cervidee. A
small
conical
piece is thus thrown off from the end of the horn or ossicusp, and
may be regarded as a rudimentary or minute “antler.” But the process of discarding these minute points or antlers in
the Okapi appears to differ from what occurs in the Cervide, not
only in the minute size of the discarded segments, but in the fact
that
the
preparation
for
the
breaking
off
a
second
(and
even
a
third) segment takes place before the first piece has been got rid
of. The
living
tissue
having
absorbed
the
bony
matter
by
a
horizontal
ingrowth
and
having
created
a
transverse
break
in
the
continuity of the osseous substance (see text-figure), recedes for a
distance of a sixth of an inch or less, and then again penetrates
inwards,
forming
a
new
horizon
of
disruption;
and
from
the
appearance of the specimens figured in Plate VI., especially figs. 4
and 5, it seems that this process of the recession of the living
investment
of
the
horn-tip
and
the
subsequent
ingrowth
of
the
living
tissue,
may
be
again
repeated
before
the
most
anterior
piece is broken off, so that these horizontal fissures are visible on
the
surface
of
the
horn-tip,
following
one
another
somewhat
irregularly. 1907. 131 RUDIMENTARY
ANTLERS
IN
THE Text-fig. 52. Post. A. B. Text-fig. 52. Text-fig. 52. Text-fig. 52. Post B. A. Lankester, E. Ray. 1907. "On the Existence of Rudimentary Antlers in the
Okapi." Proceedings of the Zoological Society of London 1907, 126–135. https://doi.org/10.1111/j.1096-3642.1907.tb01804.x. View This Item Online: https://www.biodiversitylibrary.org/item/97670
DOI: https://doi.org/10.1111/j.1096-3642.1907.tb01804.x
Permalink: https://www.biodiversitylibrary.org/partpdf/72470 A. The diagram C shows the direction im which certain cuts have been made in the
left osseous horn (ossicone or ossicusp) of the Edinburgh Okapi (also illustrated
in P). VI. figs. 1 to 8). The diagram C shows the direction im which certain cuts have been made in the
left osseous horn (ossicone or ossicusp) of the Edinburgh Okapi (also illustrated
in P). VI. figs. 1 to 8). )
g
)
a, anterior border; b, posterior border.’ L, left side; R, right side. d
i
h
i
h
l i
f h
f
f h
bi )
g
)
a, anterior border; b, posterior border.’ L, left side; R, right side. A i
d
i
h
i
h
l i
f h
f
f h
bi p
g
A isa drawing three times the natural size of the cut surface of the bisected horn,
the bisection being effected in a plane erected on the line ab of the diagram C. It shows the eating in of the transverse fissures into the dense bony substance,
and a number of irregular spaces and fissures which are probably cavities due to
re-sorption of the bone. B is a drawing of the other half of the same bisected
horn-tip. A isa drawing three times the natural size of the cut surface of the bisected horn,
the bisection being effected in a plane erected on the line ab of the diagram C. It shows the eating in of the transverse fissures into the dense bony substance,
and a number of irregular spaces and fissures which are probably cavities due to
re-sorption of the bone. B is a drawing of the other half of the same bisected
horn-tip. Text-fig. 53. Drawing of three times the natural size of the surface of a section obtained by
cutting half of the same bisected horn-tip through a plane erected on the line R
of the diagram, separating the shaded from the unshaded area. The penetration
of the transverse fissures is shown. The cavities m, ”, 0 correspond to the
transverse fissures labelled m, n, 0 in fig. 5 of Plate VI., representing the same
specimen
before
it
was
divided. A9? A9? This file was generated 8 August 2024 at 17:38 UTC Holding Institution Smithsonian Libraries and Archives Copyright & Reuse Copyright & Reuse
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W282090071.txt
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http://www.bioinformation.net/010/97320630010555.pdf
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en
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Erratum in Jayapalan & Natarajan, The role of CDK5 and GSK3B kinases in hyperphosphorylation of microtubule associated protein tau (MAPT) in Alzheimer’s disease
|
Bioinformation
| 2,014
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cc-by
| 120
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open access
www.bioinformation.net
Erratum
Volume 10(8)
Erratum: A numerical error in methodology section under Sequence
retrieval and modeling side heading
Bioinformation - 2013 Dec 9(20): 1023-1030
The role of CDK5 and GSK3B kinases in
hyperphosphorylation of microtubule associated protein
tau (MAPT) in Alzheimer’s disease
Saranya Jayapalan & Jeyakumar Natarajan*
Data Mining and Text Mining Laboratory, Department of Bioinformatics, Bharathiar University, Coimbatore-641046, India
Erratum in:
Bioinformation - 2014 Aug 30 10(8): 555 [Numerical error corrected - On page 1024
(Methodology section: Sequence retrieval and modeling), in second column, in line3 and line18
the entry for length of MAPT sequence currently read as “358” It should read “352”]
ISSN 0973-2063 (online) 0973-8894 (print)
Bioinformation 10(8): 555 (2014)
555
© 2014 Biomedical Informatics
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W2798665765.txt
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https://link.springer.com/content/pdf/10.1007/JHEP11(2018)094.pdf
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en
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Unitarity of the box diagram
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The Journal of high energy physics/The journal of high energy physics
| 2,018
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cc-by
| 7,431
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Published for SISSA by
Springer
Received: September 26, 2018
Accepted: November 8, 2018
Published: November 14, 2018
Roji Piusa and Ashoke Senb
a
Perimeter Institute for Theoretical Physics,
Waterloo, ON N2L 2Y5, Canada
b
Harish-Chandra Research Institute, HBNI,
Chhatnag Road, Jhusi, Allahabad 211019, India
E-mail: rpius@perimeterinstitute.ca, sen@hri.res.in
Abstract: The complete proof of cutting rules needed for proving perturbative unitarity
of quantum field theories usually employs the largest time equation or old fashioned perturbation theory. None of these can be generalized to string field theory that has non-local
vertices. In arXiv:1604.01783 we gave a proof of cutting rules in string field theory, which
also provides an alternative proof of cutting rules in ordinary quantum field theories. In
this note we illustrate how this works for the box diagram of φ4 field theory, avoiding the
contributions from anomalous thresholds.
Keywords: Scattering Amplitudes, String Field Theory
ArXiv ePrint: 1805.00984
Open Access, c The Authors.
Article funded by SCOAP3 .
https://doi.org/10.1007/JHEP11(2018)094
JHEP11(2018)094
Unitarity of the box diagram
Contents
1 Introduction and summary
1
2 The issues
2
3 Unitarity of the box diagram
3
1
14
Introduction and summary
Cutkosky’s diagrammatic analysis shows that the discontinuities of a Feynman diagram
across the ‘normal threshold’ singularities produce the result needed for unitarity of the
S-matrix [1, 2]. However typically a Feynman diagram possesses many other Landau singularities e.g. anomalous thresholds at one loop [3, 4] and more complicated singularities
at higher loop, and there are discontinuities associated with these singularities as well. A
recent discussion on these may be found in [5, 6]. For this reason the standard approach
to proving the cutting rules needed for unitarity makes use of indirect methods e.g. the
largest time equation [7, 8] or old fashioned perturbation theory [9] based on time ordered
diagrams. A recent analysis along similar line, suitable for dealing with vertices with finite
number of time derivatives, can be found in [10].
Unfortunately these approaches are not suitable for proving the cutting rules for the
Feynman diagrams arising in string field theory, since the vertices are non-local, not only in
space but also in time, involving exponentials of quadratic functions of momenta. For this
reason in [11] we developed a different approach to proving the cutting rules in such theories
based on direct analysis of Feynman diagrams.1 Although originally developed for string
field theory, this approach also gives an alternative, diagrammatic proof of cutting rules in
ordinary quantum field theories. Another approach to proving unitarity in local theories
by directly dealing with momentum space Feynman diagrams was suggested in [13]. We
suspect that this approach is closely related to the one described in [11], but the precise
relation is not clear at present.
Since [11] gave an iterative proof of the cutting rules to all orders in perturbation
theory, the analysis was necessarily somewhat abstract. In this paper we complement the
analysis of [11] by showing how the method works for establishing the cutting rules for the
box diagram that appears in the computation of one loop eight point amplitude in φ4 field
theory. We emphasize however that the purpose of this note is not to prove unitarity of the
1
There have been two recent papers [6, 12] on unitarity of non-local field theories of the kind that arise
in string field theory, but both seem to only focus on the analysis of discontinuity across normal thresholds.
–1–
JHEP11(2018)094
4 Unitarity of tree diagrams
2
The issues
In this section we shall briefly discuss the issues that plague the proof of unitarity directly
in momentum space. The singularities of a Feynman diagram are associated with Landau
singularities where the integrand has poles due to certain number of internal propagators
going on-shell and furthermore the integration contour over loop momenta are pinched, i.e.
it is not possible to move away from the poles by deforming the integration contour in the
complex loop momentum plane. At such singularities, the integral typically has a branch
cut, leading to a discontinuity of the amplitude across the branch cut. In [1] Cutkosky gave
a general formula for computing the discontinuity across a given threshold singularity. If
a certain number of propagators go on-shell at a singularity, then the discontinuity in the
amplitude from the corresponding branch cut is computed by replacing the i/(−k 2 −m2 +i)
factor in each of these propagators by 2πδ(−k 2 − m2 ).
Let us consider a singularity where the on-shell internal propagators are such that
together they can be interpreted as an intermediate state in the original amplitude, e.g. if
the set consists of the propagators carrying momenta ` and pA + pB − ` in figure 1. (We are
assuming that the incoming particles come from the left and the outgoing particles move to
the right.) Branch points associated with such singularities are known as normal thresholds.
In this case the discontinuity computed from Cutkosky’s formula can be regarded as a
product of two on-shell amplitudes, integrated over the phase space of the intermediate
states. Therefore this looks similar to a contribution to −i T † T = −i T † |nihn|T that is
needed for unitarity of the T-matrix — related to the S-matrix via S = 1 − i T . Such
contributions are usually represented as cut diagrams, where the cut is a single line that
divides the diagram into a left half and a right half, with the cut propagators representing
the on-shell propagators.
While this goes a long way towards proving unitarity of the theory, there are some
missing ingradients:
1. A given Feynman diagram may have singularities other than normal threshold, e.g.
if the propagators carrying momenta `, ` + pC and pA − ` in figure 1 were on-shell at
the singularity. Such singularities are known as anomalous thresholds. Cutkosky’s
–2–
JHEP11(2018)094
box diagram for which there are many derivations. The goal is to illustrate how the iterative
all order diagrammatic proof of the cutting rules given in [11] works for the box diagram.
The rest of the paper is organized as follows. In section 2 we discuss some general
issues that arise while trying to prove unitarity of amplitudes written as momentum space
integrals. In section 3, which is the main body of the paper, we show how the method
developed in [11] is used to prove cutting rules for the box diagram of φ4 field theory. This
proof assumes the validity of cutting rules for connected and disconnected tree diagrams.
For completeness, in section 4 we give a proof of cutting rules for tree diagrams, again by
making use of the general method described in [11]. We shall use mostly plus signature for
the space-time metric and denote a D dimensional momentum as (`0 , ~`) where `0 denote
the time component and ~` denotes the spatial components.
formula can still be used to compute the discontinuity across such branch points; however in this case the on-shell states cannot collectively be regarded as an intermediate
state in the sum −i T † |nihn|T .
2. In computing −i T † T we need to reverse the signs of i in the propagators of the
amplitude to the right of the cut so that it represents a matrix element of T † . This
does not follow from Cutkosky’s formula for discontinuity.
3
Unitarity of the box diagram
R
Usually in φ4 theory with interaction term −(λ/4!) dD x φ4 one takes the propagator of
momentum k to be −i/(k 2 + m2 − i) and the vertex to be −iλ/4!. Furthermore in the
computation of the T-matrix we have an overall factor of i. However, as in [11], we shall
use a slightly different but equivalent convention where for computation of the T-matrix
we use the following rules:
1. The propagator of momentum k is given by 1/(−k 2 − m2 + i).
2. The vertex is given by λ/4!.
3. For each loop integral we have a factor of i.
4. If the diagram has nc disconnected components then we have a factor of i1−nc .
P
We shall drop the overall momentum conserving delta function (2π)D δ (D) ( i pi ), associated with each connected component, from the expressions for the amplitudes. The
–3–
JHEP11(2018)094
While for any specific graph one can do a more detailed analysis taking into account all
these effects, the general proof of unitarity based on this approach becomes cumbersome.
These problems were overcome in [7, 8] where a different proof of unitarity was given based
on the ‘largest time equation’. An alternative proof was given in [9] based on the old
fashioned perturbation theory. However unlike Cutkosky’s original analysis, which did not
depend on the detailed structure of the vertices as long as they do not introduce additional
singularities at finite momentum, the analysis of [7–9] requires working in coordinate space
where at least for the time coordinate the propagator and vertices are expressed in the
position space instead of the momentum space. Unfortunately for string field theory,
for which the vertices are exponentials of quadratic functions of momenta, there is no
convenient representation of the vertices in the coordinate space. Therefore the analysis
of [7–9] do not apply.
This difficulty was overcome in [11] that expressed T − T † as a sum over cuts diagrams
by working directly in momentum space. Furthermore the part of the contribution to the
right of the cut was shown to be hermitian conjugated, representing a contribution to T † .
This method is well-suited for string field theory and other non-local theories, but also for
ordinary quantum field theories with local vertices, giving an alternative proof of unitarity.
In the next two sections we shall illustrate how this works for the box diagram and tree
diagrams in φ4 theory.
pB
&
pD
.
↑ (pA − `)
↑ (` + pC )
`→
pC
Figure 1. The box diagram of the eight point amplitude in φ4 theory. The external momenta
pA , pB , pC , pD entering at the four vertices are taken to be positive if ingoing.
space-time dimension D = d + 1 will be chosen such that the box diagram of figure 1 has
no ulltra-violet divergence. This requires D ≤ 7.
With this convention the contribution of the box diagram shown in figure 1 to the
T-matrix is given by,
Z
i 4
dD `
I(pA , pB , pC , pD ) = λ
{−`2 − m2 + i}−1 {−(` + pC )2 − m2 + i}−1
2
(2π)D
{−(pA − `)2 − m2 + i}−1 {−(pA + pB − `)2 − m2 + i}−1 .
(3.1)
pA , pB , pC and pD denote net external momenta entering the vertices. In this convention
an outgoing particle will have negative p0 . Since each of pA , pB , pC and pD receives
contribution from two incoming or outgoing external states, they can be space-like or timelike and arbitrarily large in magnitude. Therefore all the singularities that can appear in
the box diagram can be present here. In particular by taking pA to be a large time-like
momentum we can mimick the case of a massive external particle above the threshold of
production of a pair of φ particles — this is the situation in which the anomalous threshold
is commonly discussed.2
Our goal will be to compute the quantity
D ≡ I(pA , pB , pC , pD ) − I(−pA , −pB , −pC , −pD )∗ ,
(3.2)
that represents a contribution to T − T † . We write down the expression for I(−pA , −pB ,
−pC , −pD )∗ by taking the complex conjugate of (3.1) and changing the signs of all the
external momenta. Making a change of variables ` → −` in the resulting expression,
2
In fact we do not even need to assume in our analysis that the external momenta are on-shell.
–4–
JHEP11(2018)094
%
pA
(pA + pB − `) →
we have
Z
i
dD `
I(−pA , −pB , −pC , −pD )∗ = − λ4
{−`2 − m2 − i}−1 {−(` + pC )2 − m2 − i}−1
2
(2π)D
{−(pA − `)2 − m2 − i}−1 {−(pA + pB − `)2 − m2 − i}−1 .
(3.3)
This gives
i 4
λ
2
Z
dd `
(2π)d
Z
d`0
{−`2 − m2 + i}−1 {−(` + pC )2 − m2 + i}−1
2π
{−(pA − `)2 − m2 + i}−1 {−(pA + pB − `)2 − m2 + i}−1
+{−`2 − m2 − i}−1 {−(` + pC )2 − m2 − i}−1
2
2
{−(pA − `) − m − i}
−1
2
2
−1
{−(pA + pB − `) − m − i}
.
(3.4)
Unitarity of the S-matrix demands that T −T † must be equal to −i T † T . This translates
to the cutting rules which tell us that D is given by the sum over all cuts of the box diagram,
with the following rules for evaluating a cut diagram:
1. A cut must divide the diagram into the left half and the right half, with the convention
that the incoming particles come from the left and the outgoing particles travel to
the right.
2. A cut propagator corresponds to the replacement:
P (k) ≡
−k 2
1
− m2 + i
⇒
Pc (k) ≡ −2 π i δ(−k 2 − m2 ) θ(k 0 ) ,
(3.5)
where k denotes the momentum flowing along the propagator from the left side of
the cut to the right side. The −i factor in the expression for Pc may seem unfamiliar,
but in our convention this combines with the factor of i from loop integral to give
the correct integration measure over the phase space.
3. The part of the amplitude to the right of the cut is replaced by its hermitian conjugate
— involving complex conjugation and reversal of the signs of all external momenta.
4. Cut on an external line has no effect.
5. If a cut diagram has nL disconnected components on the left of the cut and nR
disconnected components on the right of the cut, then it should be multiplied by an
additional factor of (−1)nR −1 . This factor is needed to ensure that cutting rules lead
to the unitarity relation T − T † = −i T † T [11].
We shall first prove that for fixed ~` in (3.4), the contribution to D from the `0 integral
vanishes unless the integration contour is pinched between two singularities. 3 For this we
3
As in [11], we shall only allow deformations of loop energy integration contour into the complex plane,
but keep the integration contours for spatial components of the loop momenta always along the real axes.
–5–
JHEP11(2018)094
D=
D R = A − A∗ ,
(3.6)
Z
Z
i 4
dd `
d`0
A= λ
{−`2 − m2 + i}−1 {−(` + pC )2 − m2 + i}−1
d
2
2π
(2π)
R
{−(pA − `)2 − m2 + i}−1 {−(pA + pB − `)2 − m2 + i}−1 ,
(3.7)
where
and A∗ is obtained from A by
1. replacing R by R0 ,
2. reversing the signs of all the external momenta, and
3. complex conjugation.
We shall prove that DR is given by the sum over cuts of the contributions to A. The
full cutting rule is then obtained by adding the contributions from each small region R of
this type.
In this section we shall prove the cutting rule for DR assuming that it holds for all tree
diagrams — including disconnected ones. This analysis will follow closely the one given
in section 5.2.3 of [11] for one vertex irreducible diagrams. In the next section we shall
describe the proof of cutting rules for connected and disconnected tree diagrams.
Now for ~` ∈ R a certain number of propagators become nearly on-shell when `0 takes
the value where its integration contour is nearly pinched. Since for small R, `0 lies within
4
For string field theory the `0 integration contour for both terms have their ends fixed at ±i∞ [11].
However a similar cancellation occurs for these contours as well. In this case bad behaviour in some
directions at ∞ prevents us from deforming the contours to ∞, but the relevant contours can be deformed
to each other.
–6–
JHEP11(2018)094
deform the `0 integral to ∞ in the lower half plane for the first term inside the square
bracket in (3.4) and to ∞ in the upper half plane for the second term in the square bracket
in (3.4), picking up residues from the poles that the contour passes through during the
deformation. Since the poles of the first term are complex conjugates of the poles in the
second term, we pick residues from exactly the same set of poles with i replaced by −i.
Furthermore in the first term the poles are traversed in the clockwise direction while in
the second term the poles are traversed in the anti-clockwise direction. As long as there
are no nearby poles, we can set = 0 while evaluating these residues. In this case their
contributions exactly cancel. This argument breaks down if the contours are pinched since
the residues diverge as → 0, and we have to carefully take the limit to see if there is any
left-over contribution after we combine the results of the two terms. 4
The pinch singularities occur when a pair of poles in the `0 plane approach the integration contour from the opposite sides. Therefore in the ~` space they occur on a subspace
of codimension 1 or higher (if more than two poles approach the same point). We shall call
this the pinched subspace. We shall focus on the integration over a small neighborhood R
of the pinched subspace. R is a (D − 1) dimensional space, i.e. of codimension zero. We
denote by R0 the image of R under ~` → −~`, and consider the quantity
3
pA − `
4
`
1
2
pA + pB − `
` + pC
a small range at the pinch, we can assign definite signs to the energies carried by each
internal propagator at the pinch. We shall now associate with the region R a reduced
diagram that is obtained from the original diagram by shrinking to points all propagators
that are not nearly on-shell near the pinch. Furthermore we shall draw the nearly on-shell
propagators such that energy flows from left to right near the pinch. For definiteness, and
to consider a situation of maximal complexity, we shall consider a region R for which all
four internal propagators are nearly on-shell at the pinch, and5
`0 > 0,
`0 + p0C > 0,
p0A − `0 > 0,
p0A + p0B − `0 > 0 ,
(3.8)
at the pinch. The corresponding reduced diagram is shown in figure 2. We have dropped
the external legs from this diagram to avoid cluttering. We also number the propagators
carrying momenta `, ` + pC , pA − ` and pA + pB − ` by 1, 2, 3 and 4 respectively. It is easy
to see that the corresponding integral for A∗ , after making a change of integration variable
`0 → −`0 , will be pinched at the same value of `0 .
Let us denote by P (k) the propagator with momentum k:
P (k) ≡
−k 2
1
1
1
p
p
=
,
2
− m + i
k 0 − ~k 2 + m2 + iε k 0 + ~k 2 + m2 − iε
where ε is positive for positive . Therefore we can express (3.7) as
Z
Z
i 4
dd `
d`0
A= λ
P (`) P (` + pC ) P (pA − `) P (pA + pB − `) .
d
2
2π
R (2π)
(3.9)
(3.10)
Since the pinch is assumed to occur at the positive values of `0 , `0 + p0C , p0A − `0 and
p0A + p0B − `0 , the relevant poles of the propagators that take part in pinching the contour,
are at
q
q
0
` = ~`2 + m2 − iε,
`0 = −p0C + (~` + p~C )2 + m2 − iε,
q
q
`0 = p0A − (~
pA − ~`)2 + m2 + iε, `0 = p0A + p0B − (~
pA + p~B − ~`)2 + m2 + iε . (3.11)
5
A similar analysis can be carried out for all other reduced diagrams.
–7–
JHEP11(2018)094
Figure 2. The reduced diagram of figure 1 associated with R when at the pinch all propagators
are nearly on-shell and the energies carried by the propagators lie in the range (3.8). The internal
lines are drawn in a way so that at the pinch energy flows from the left to the right along each of
the propagators. Since four propagators become on-shell at the pinch, the pinched subspace has
comension 4, and R is a small neighborhood of this codimension 4 subspace.
Note that at the pinch the poles from the propagators 1 and 2 are in the lower half `0 plane
while the poles from the other propagators are in the upper half `0 plane. Therefore while
deforming the `0 contour to the lower half plane, we shall pick up residues from the poles
of the propagators 1 and 2 at the pinch. For this reason the set {1, 2} will play a special
role in our analysis.
We now define
P 0 (k) ≡
1
1
p
p
= P (k) − Pc (k) ,
k 0 − ~k 2 + m2 − iε k 0 + ~k 2 + m2 − iε
(3.12)
Pc (k) ≡ −2 π i δ(−k 2 − m2 ) θ(k 0 ) ,
(3.13)
is the cut propagator. It follows from (3.12) that
P (`)P (`+pC ) = P 0 (`)P 0 (`+pC )+Pc (`)P (`+pC )+P (`)Pc (`+pC )−Pc (`)Pc (`+pC ) . (3.14)
Using this in (3.10) we can express A as
b + A(1) + A(2) − A(12) ,
A=A
(3.15)
where
b = i λ4
A
2
Z
i 4
λ
2
Z
i
= λ4
2
Z
i
= λ4
2
Z
A(1) =
A
(2)
(12)
A
Z
R
dd `
(2π)d
Z
R
dd `
(2π)d
Z
R
dd `
(2π)d
Z
R
dd `
(2π)d
d`0 0
P (`) P 0 (` + pC ) P (pA − `) P (pA + pB − `) ,
2π
(3.16)
d`0
Pc (`) P (` + pC ) P (pA − `) P (pA + pB − `) ,
2π
(3.17)
d`0
P (`) Pc (` + pC ) P (pA − `) P (pA + pB − `) ,
2π
(3.18)
d`0
Pc (`) Pc (` + pC ) P (pA − `) P (pA + pB − `) .
2π
(3.19)
In writing (3.17)–(3.19) we have used the notation of [11] in which A(i1 ···in ) is obtained
from A by replacing the i1 , · · · in -th propagators by cut propagators. The diagrammatic
representations of A(1) , A(2) and A(12) have been shown in figures 3–5 with the thick vertical
line across a propagator representing a cut propagator Pc . We emphasize that these are
not yet cut diagrams as the cut does not divide the graph into a left half and a right half,
and we neither reverse the momenta not complex conjugate any part of the graph. Instead
these should be regarded as tree amplitudes since a cut propagator can be regarded as a
pair of incoming and outgoing lines with identical momentum. By a similar manipulation
we can express A∗ as
b∗ + A(1)∗ + A(2)∗ − A(12)∗ .
A∗ = A
(3.20)
b the relevant poles in the `0 plane,
We now note that in the expression (3.16) for A
responsible for the pinch in the original amplitude A, are all in the upper half plane, since
–8–
JHEP11(2018)094
where
3
3
4
4
=
1
1
2
2
1
Figure 3. Representation of A(1) . On the left hand side the vertical line through propagator 1
represents that it is a cut propagator. On the right hand side this is made explicit by replacing the
cut propagator by a pair of external lines — one outgoing and one incoming.
4
3
JHEP11(2018)094
3
4
=
1
2
2
1
2
Figure 4. Representation of A(2) .
3
4
3
4
1
2
=
1
2
1
2
Figure 5. Representation of A(12) .
in the locations of the poles of the integrand, the signs of the iε in the first line of (3.11)
are reversed. Therefore the `0 contour is not pinched, and by our previous argument,
b−A
b∗ = 0 .
A
(3.21)
On the other hand, since A(1) , A(2) and A(12) are tree amplitudes, and since we are assuming
that cutting rules hold for tree amplitudes, A(1) − A(1)∗ , A(2) − A(2)∗ and A(12) − A(12)∗ are
given by sum over cuts of the tree diagrams. This gives, in the notation of [11]:
(1)
(1)
(1)
A(1) − A(1)∗ = A1 + A2 + A12 ,
(12)
A(12) − A(12)∗ = A1
(12)
+ A2
(2)
(2)
(2)
A(2) − A(2)∗ = A1 + A2 + A21 ,
(12)
(12)
+ A12 + A21 .
(3.22)
(i ···i )
Here Aj11···jmn for 1 ≤ ik ≤ 2, 1 ≤ jk ≤ 2 represents sum over all cuts of A(i1 ···in ) satisfying
the following properties:
1. The cut can be viewed as a cut of the original graph contributing to the amplitude A.
–9–
2. The cut crosses the j1 , · · · jm ’th propagators in the set {1, 2} and possibly other
propagators outside the set {1, 2}.
(i ···i )
(1)
(12)
A2 = A2
,
(2)
(12)
A 1 = A1
,
(1)
(12)
A12 = A12 ,
(2)
(12)
A21 = A21 .
(3.23)
These identities can be verified by explicitly examining the equalities of figures 7 and 13, 9
and 12, 8 and 14, and figures 11 and 15. Using (3.15), (3.20), (3.21), (3.22) and (3.23) we
now get
(1)
(1)
(1)
(2)
(1)
(2)
(2)
(2)
(12)
A − A∗ = A1 + A2 + A12 + A1 + A2 + A21 − A1
= A1 + A2 .
(12)
− A2
(12)
(12)
− A12 − A21
(3.24)
The diagrammatic representation of the right hand side, given by the sum of the left
hand sides of figures 6 and figure 10, is shown in figure 16. We now see that this is
precisely given by the sum of all possible cuts of the reduced diagram shown in figure 2. In
particular possible contributions from anomalous thresholds, represented by figure 7 and
figure 9, cancel with the contributions from figures 13 and 12 and do not appear in the
final expression. These cancellations are special cases of the general results described in
eqs. (5.26)–(5.33) of [11].
We end this section with a few remarks:
1. Our analysis automatically includes other reduced diagrams that are obtained by
contracting one or more propagators in figure 2. We simply have to set to zero
all terms where the corresponding propagator is replaced by a cut propagator. For
example if we take the triangle diagram obtained by contracting the propagator 3 in
figure 2, the final answer for A − A∗ will include sum over only the second and third
diagrams in figure 16.
2. This does not cover all the cases however. An example is shown in figure 17 where
at the pinch poles on the same side come from non-adjacent propagators. We can
analyze this by repeating the analysis, with the role of the momenta ` and ` + pC
in (3.14) now played by ` and ` − pA − pB . The rest of the analysis proceeds as before,
– 10 –
JHEP11(2018)094
On the other hand Ai11j n describes sum over cuts of A(i1 ···in ) which pass through the
j-th and i1 -th propagators in the set {1, 2} and possibly other propagators outside the
set {1, 2}, but which are not regular cuts of the original amplitude A since, viewed in the
context of the original graph, the i1 -th propagator carries energy across the cut in the
wrong direction. Explicit diagrammatic representation of all the terms on the right hand
side of (3.22) has been given in figures 6–15. In particular figures 8, 11, 14 and 15 describe
(1)
(2)
(12)
(12)
contributions to A12 , A21 , A12 and A21 respectively. As is clear from the right hand
sides of these figures, these are perfectly good cuts of the tree amplitude A(i1 ···in ) , even
though the left hand sides of these figures show that they are not valid cuts of A.
We shall now write down a few identities following from the simple observation that
a propagator cut twice has the same expression as the propagator cut once, since the cut
passing through an external line has no effect. Therefore we have
=
+
+
(1)
=
+
+
(1)
Figure 7. Contributions to A2 . Unless cancelled, this would represent contributions from anomalous threshold.
with the role of the set {1, 2} played by the set {1, 4}. The final result for A − A∗ ,
(1)
(4)
(14)
according to the general result of [11], will be given by A1 + A4 + A14 , which is
(12)
simply the sum over all cuts of figure 17. (The corresponding contribution A12 was
not present in the previous example since figure 2 has no cut that passes through
both propagators 1 and 2.)
3. In our analysis we have assumed that for a given R, the `0 contour has a single pinch
point to which two or more poles approach. We can also have more than one pinch on
the `0 contour, with two or more poles approaching each pinch point. Since after we
factorize each propagator as in (3.9), each denominator factor is linear in `0 and has
a single zero, different denominator factors must be responsible for different pinches.
Therefore we can divide the denominators into different sets, with the first set S1
responsible for the first pinch, the second set S2 responsible for the second pinch and
so on. Different pinch points will have different reduced diagrams associated with
them, since the list of singular propagators and the direction of energy flow through
these propagators will depend on the pinch.
– 11 –
JHEP11(2018)094
Figure 6. Contributions to A1 . The figure on the left hand side expresses it as a sum of cut
diagrams of the original graph, with the propagator 1 replaced by the cut propagator. The right
hand side shows this as a sum of cuts of a tree diagram in which the propagator 1 is replaced
by a pair of incoming and outgoing lines. We have not shown the external states of the original
amplitude A in any of the diagrams.
=
(1)
+
=
+
(2)
Figure 9. Contributions to A1 .
+
=
+
(2)
Figure 10. Contributions to A2 .
=
(2)
Figure 11. Contributions to A21 .
– 12 –
JHEP11(2018)094
Figure 8. Contribution to A12 . In the left hand side the cut seems to cross the propagator 1 of
the original diagram in the reverse direction so that it appears as if the energy of the propagator
1 flows from the right of the cut to the left of the cut. However since the propagator 1 is already
on-shell, the correct representation of the diagram is on the right hand side where it is represented
as the cut of a tree diagram. In this representation there is nothing unusual.
+
=
+
+
.
=
+
(12)
Figure 13. Contributions to A2
.
=
(12)
Figure 14. Contributions to A12 .
=
(12)
Figure 15. Contributions to A21 .
– 13 –
JHEP11(2018)094
(12)
Figure 12. Contributions to A1
+
+
+
Figure 16. Complete contribution to A − A∗ .
`
−pC − `
2
4
pA − `
` − pA − pB
Figure 17. A reduced diagram in which non-adjacent propagators 1 and 4 have poles on the same
side at the pinch. At the pinch the energy flows from left to right in each of the propagators. In
the figure the momenta are labelled so that they flow from left to right.
We can now carry out the analysis by first treating the product of denominators in
b is still
the set S1 as in (3.14). The main difference will be that now the `0 contour in A
pinched due to the other set of denominators belonging to S2 , S3 , · · · etc. The other
terms contain delta functions that force `0 to be at the first pinch and therefore the
denominators in the other sets remain finite. These terms can be analyzed as before.
b by decomposing the second set of denominators, belonging to
We can now analyze A
the set S2 , as in (3.9) and repeat the analysis. We continue this till we reach a stage
where we have a sum of terms where in one term the contour is not pinched (the
b and in the other terms the delta-function fixes `0 at a pinch. The final
analog of A)
result will then be given by the sum of cuts of all the reduced diagrams corresponding
to all the pinches.
4
Unitarity of tree diagrams
Since the proof of cutting rules for the box diagram assumed the validity of cutting rules
for connected and disconnected tree diagrams, we shall prove the cutting rules for tree
diagrams in this section. The analysis is a straightforward application of sections 5.2.2
and 5.3 of [11].
We begin with the simple diagram shown in figure 18(a) with p0 > 0. Its expression is
given by
A = λ2 (−p2 − m2 + i)−1 .
– 14 –
(4.1)
JHEP11(2018)094
3
1
p→
p→
(a)
(b)
U
U
P
P
D
D
(b)
(a)
Figure 19. (a) Schematic representation of a generic connected tree diagram in which the blobs
U and D themselves are connected tree diagrams. (b) Example of such a generic connected tree
diagram.
Therefore
A − A∗ = λ2 (−p2 − m2 + i)−1 − λ2 (−p2 − m2 − i)−1 = λ2 (−2π i) δ(−p2 − m2 ) . (4.2)
On the other hand the cut diagram shown in figure 18(b) has the same expression. (We
can drop the θ(p0 ) term from the cut propagator since p0 has been chosen to be positive.)
This proves the cutting rule for figure 18(a).
Next we shall prove the cutting rules for any connected tree amplitude assuming that
it holds for all connected tree amplitudes with at least one less vertex. For this we follow
closely the analysis of section 5.2.2 of [11] of ‘one vertex reducible’ diagrams. Let P be any
vertex of the amplitude to which at least two internal lines are connected. Then the general
form of the diagram can be represented as in figure 19(a), with each of the blobs U and
D describing some connected tree diagram. If AU and AD are the amplitudes associated
– 15 –
JHEP11(2018)094
Figure 18. (a) A tree diagram in φ4 theory with six external lines and one internal propagator.
In drawing this we have made an exception to our conventions and have drawn the external lines.
The internal line has energy p0 flowing from the left to the right. (b) Cut diagram of (a).
with the tree diagrams U and D respectively, then the full amplitude A is given by6
A = λ−1 AU AD ,
(4.3)
where the λ−1 factor accounts for the fact that both AU and AD includes a factor of λ
from the vertex P whereas in A we have only one factor of λ from the vertex. Therefore
A − A∗ = λ−1 {AU AD − A∗U A∗D } .
(4.4)
AU − A∗U = ∆U L + ∆U R ,
AD − A∗D = ∆DL + ∆DR .
(4.5)
Using (4.5) and some trivial rearrangement of terms we can express (4.4) as
A − A∗ = λ−1 {A∗U ∆DL + ∆U L ∆DL + ∆U L A∗D − ∆U R ∆DR + AU ∆DR + ∆U R AD } . (4.6)
This can be verified e.g. by expressing both sides in terms of A∗U , A∗D , ∆U R , ∆U L , ∆DR
and ∆DL . The diagrammatic representations of the six terms in (4.6) have been shown in
figure 20. Special attention should be paid to the minus sign of the fourth term on the right
hand side of (4.6). This is compatible with the fourth term in figure 20 due to the (−1)nR −1
factor that multiplies each cut diagram, nR being the number of disconnected components
on the right of the cut. If we denote by nU R and nDR the number of disconnected components on the right of the cut in U and in D, then the product of the cut diagrams of
U and D carries a factor of (−1)nU R −1+nDR −1 . On the other hand the fourth cut diagram
of figure 20 carries a factor of (−1)nU R +nDR −1 . The two differ by a sign showing that the
fourth diagram of figure 20 is indeed given by −λ−1 ∆U R ∆DR .
We now note that the six cut diagrams of figure 20 exhaust all possible cuts of the
diagram 19(a). This shows that A − A∗ is indeed given by the sum over all cut diagrams
of A in accordance with the cutting rules.
Finally we turn to the proof of cutting rules for disconnected tree diagrams following
the analysis of section 5.3 of [11]. Again the proof will proceed via induction, namely we
shall prove the result assuming that it holds for diagrams with less number of vertices. For
this let us suppose that the graph contains two pieces B and C that are disconnected from
each other. This has been shown in figure 21. B and C themselves may be disconnected
graphs, but each will contain less number of vertices and therefore satisfy cutting rules.
6
Note that some of the external lines of U at P are internal lines of D and some of the external lines of
D at P are internal lines of U . Therefore they are generically off-shell. This does not have any effect on
our analysis since the validity of cutting rules does not require the external lines to be on-shell.
– 16 –
JHEP11(2018)094
Now since AU and AD are themselves connected tree amplitudes with less number of
vertices than A, AU − A∗U and AD − A∗D are given by sum over cut diagrams of U and D.
We divide each of these cut diagrams into two classes: ∆U R and ∆U L will denote the sum
over cut diagrams of U for which the cut passes on the left and right of P respectively,
and similarly ∆DR and ∆DL will denote the sum over cut diagrams of D for which the cut
passes on the left and right of P respectively. Therefore we have
'$
'$
'$
'$
'$
'$
U
U
U
U
U
U
&%
&%
&%
&%
&%
&%
'$
'$
'$
'$
'$
'$
D
D
D
D
D
D
&%
&%
&%
&%
&%
&%
#
B
"!
#
C
"!
Figure 21. Schematic representation of a tree amplitude with at least two disconnected components. The blobs B and C represent tree amplitudes which themselves may have additional
disconnected components.
#
#
B
B
# # # # #
B
B
B
B
B
"!
"!
"!
"!
"!
"!
"!
#
#
# # # # #
C
C
C
C
C
C
C
"!
"!
"!
"!
"!
"!
"!
(a)
(b)
(c)
(d)
(e)
(f)
(g)
Figure 22. Diagrammatic representation of the seven terms in (4.8). These can also be interpreted
as cuts of figure 21.
Denoting by B and C the expressions for the amplitudes associated with the graphs B and
C, we have the full amplitude A given by
A = −i B C ,
(4.7)
where the factor of −i arises due to the fact that the total number of disconnected components of A is equal to the sum of the number of disconnected components of B and of C, and
– 17 –
JHEP11(2018)094
Figure 20. Diagrammatic representation of the six terms in (4.6). These can also be interpreted
as cuts of figure 19(a).
therefore due to the (i)1−nc factor in the expression for the amplitude with nc disconnected
components, the product B C has one more factor of i compared to A. This gives
A − A∗ = −i (B C + B ∗ C ∗ )
= −iB ∗ C − iB C ∗ + i (B − B ∗ ) (C − C ∗ ) + i (B − B ∗ ) C ∗ − i(B − B ∗ ) C
+i B ∗ (C − C ∗ ) − i B (C − C ∗ ) .
(4.8)
Acknowledgments
We would like to thank Alok Laddha and Terry Tomboulis for useful discussions. The
research of R.P. was supported in part by Perimeter Institute for Theoretical Physics. Research at Perimeter Institute is supported by the Government of Canada through Industry
Canada and by the Province of Ontario through the Ministry of Research and Innovation.
This research of A.S. was supported in part by the J. C. Bose fellowship of the Department
of Science and Technology, India.
Open Access. This article is distributed under the terms of the Creative Commons
Attribution License (CC-BY 4.0), which permits any use, distribution and reproduction in
any medium, provided the original author(s) and source are credited.
References
[1] R.E. Cutkosky, Singularities and discontinuities of Feynman amplitudes, J. Math. Phys. 1
(1960) 429 [INSPIRE].
[2] M. Fowler, Introduction to Momentum Space Integration Techniques in Perturbation Theory,
J. Math. Phys. 3 (1962) 936.
[3] S. Mandelstam, Unitarity Condition Below Physical Thresholds in the Normal and
Anomalous Cases, Phys. Rev. Lett. 4 (1960) 84 [INSPIRE].
[4] R.E. Cutkosky, Anomalous Thresholds, Rev. Mod. Phys. 33 (1961) 448.
[5] R. Zwicky, A brief Introduction to Dispersion Relations and Analyticity, in Proceedings of
Quantum Field Theory at the Limits: from Strong Fields to Heavy Quarks (HQ 2016),
Dubna Russia (2016), pg. 93 [arXiv:1610.06090] [INSPIRE].
[6] P. Chin and E.T. Tomboulis, Nonlocal vertices and analyticity: Landau equations and general
Cutkosky rule, JHEP 06 (2018) 014 [arXiv:1803.08899] [INSPIRE].
– 18 –
JHEP11(2018)094
The second step is the result of trivial algebraic manipulation. The seven terms in (4.8)
can be diagrammatically represented as the seven cut diagrams shown in figure 22. The
additional minus signs in the contributions from figure 22(c), (d) and (f), given respectively
by the third, fourth and sixth terms in (4.8), account for the fact that if nR denotes the
number of disconnected components to the right of the diagram, then the sum of nR − 1
for the component diagrams differ from (nR − 1) of the full diagram by 1.
We now note that the seven terms in figure 22 are in one to one correspondence with
the cuts of A. This proves the validity of the cutting rule for the disconnected amplitude A.
[7] M.J.G. Veltman, Unitarity and causality in a renormalizable field theory with unstable
particles, Physica 29 (1963) 186 [INSPIRE].
[8] G. ’t Hooft and M.J.G. Veltman, Diagrammar, NATO Sci. Ser. B 4 (1974) 177.
[9] G.F. Sterman, An Introduction to quantum field theory, Cambridge University Press,
Cambridge U.K. (1993).
[10] E.T. Tomboulis, Causality and Unitarity via the Tree-Loop Duality Relation, JHEP 05
(2017) 148 [arXiv:1701.07052] [INSPIRE].
[12] F. Briscese and L. Modesto, Cutkosky rules and perturbative unitarity in Euclidean nonlocal
quantum field theories, arXiv:1803.08827 [INSPIRE].
[13] D. Anselmi, Algebraic cutting equations, Annals Phys. 394 (2018) 294 [arXiv:1612.07148]
[INSPIRE].
– 19 –
JHEP11(2018)094
[11] R. Pius and A. Sen, Cutkosky rules for superstring field theory, JHEP 10 (2016) 024
[Erratum ibid. 1809 (2018) 122] [arXiv:1604.01783] [INSPIRE].
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https://link.springer.com/content/pdf/10.1007%2FJHEP03%282018%29079.pdf
|
English
| null |
Chiral algebras in Landau-Ginzburg models
|
The Journal of high energy physics/The journal of high energy physics
| 2,018
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cc-by
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Published for SISSA by
Springer Published for SISSA by
Springer Published for SISSA by
Springer Received: January 22, 2018
Accepted: March 2, 2018
Published: March 13, 2018 Received: January 22, 2018
Accepted: March 2, 2018
Published: March 13, 2018 Received: January 22, 2018
Accepted: March 2, 2018
Published: March 13, 2018 JHEP03(2018)079 Open Access, c⃝The Authors.
Article funded by SCOAP3. Mykola Dedushenko Mykola Dedushenko
Joseph Henry Laboratories, Princeton University,
Princeton NJ, 08540, U.S.A. 3
Landau-Ginzburg models 3.1
Quasihomogeneous case 3.1.1
The OPE in the cohomology 3.1.2
Classical and quantum chiral algebra 3.1.3
Non-abelian global symmetries 4
N = (2, 2) models 4
N = (2, 2) models 5
Examples
5.1
Degenerate examples
5.2
N = 2 minimal models
5.2.1
The Ak+1 series
5.2.2
D and E series of minimal models 5
Examples 5
Examples 5.1
Degenerate examples 5.2
N = 2 minimal models
5.2.1
The Ak+1 series 5.2
N = 2 minimal models 5.2.1
The Ak+1 series 5.2.2
D and E series of minimal models 6
Discussions and further directions
39 6
Discussions and further directions 1
Introduction Contents 1
Introduction 2
N = (0, 2) theories
3
2.1
Conventions and some generalities
3
2.2
Supercurrent multiplet and RG invariance
5
2.2.1
General case
5
2.2.2
Emergent conformal invariance in the cohomology
8
2.2.3
OPE in the cohomology
10
2.3
Chiral algebras of superconformal theories
10
2.4
The operator cohomology and the superspace
12
2.4.1
Classical and quantum observables
12
2.4.2
Classical observables and the cohomology
12
2.4.3
The cohomology of Q+ in F0 and of D+ in bF0
14 2
N = (0, 2) theories 2.1
Conventions and some generalities 2.2
Supercurrent multiplet and RG invariance
2.2.1
General case
2.2.2
Emergent conformal invariance in the cohomology
2.2.3
OPE in the cohomology JHEP03(2018)079 2.3
Chiral algebras of superconformal theories 2.4.2
Classical observables and the cohomology
b 2.4.3
The cohomology of Q+ in F0 and of D+ in bF0 E-mail: dedushenko@gmail.com E-mail: dedushenko@gmail.com Abstract: Chiral algebras in the cohomology of the Q+ supercharge of two-dimensional
N = (0, 2) theories on flat spacetime are discussed. Using the supercurrent multiplet, we
show that the answer is renormalization group invariant for theories with an R-symmetry. For N = (0, 2) Landau-Ginzburg models, the chiral algebra is determined by the operator
equations of motion, which preserve their classical form, and quantum renormalization of
composite operators. We study these theories and then specialize to the N = (2, 2) models
and consider some examples. Keywords: Conformal and W Symmetry, Conformal Field Theory, Extended Supersym-
metry, Field Theories in Lower Dimensions ArXiv ePrint: 1511.04372 Open Access, c⃝The Authors. Article funded by SCOAP3. Open Access, c⃝The Authors. Article funded by SCOAP3. https://doi.org/10.1007/JHEP03(2018)079 https://doi.org/10.1007/JHEP03(2018)079 Contents
1
Introduction
1
2
N = (0, 2) theories
3
2.1
Conventions and some generalities
3
2.2
Supercurrent multiplet and RG invariance
5
2.2.1
General case
5
2.2.2
Emergent conformal invariance in the cohomology
8
2.2.3
OPE in the cohomology
10
2.3
Chiral algebras of superconformal theories
10
2.4
The operator cohomology and the superspace
12
2.4.1
Classical and quantum observables
12
2.4.2
Classical observables and the cohomology
12
2.4.3
The cohomology of Q+ in F0 and of D+ in bF0
14
3
Landau-Ginzburg models
14
3.1
Quasihomogeneous case
17
3.1.1
The OPE in the cohomology
20
3.1.2
Classical and quantum chiral algebra
22
3.1.3
Non-abelian global symmetries
24
4
N = (2, 2) models
25
5
Examples
29
5.1
Degenerate examples
29
5.2
N = 2 minimal models
30
5.2.1
The Ak+1 series
30
5.2.2
D and E series of minimal models
37
6
Discussions and further directions
39 1
Introduction Two-dimensional theories with N = (0, 2) supersymmetry have been attracting attention
over the last couple of decades. A motivation largely came from their potential phenomeno-
logical relevance for heterotic string compactifications, which require the internal theory
to be an N = (0, 2) SCFT. But these theories are interesting and rich quantum field the-
ories by themselves, which makes them a good object to study and apply various physical
ideas. Thinking in that direction, gauge theories are of course of particular importance in – 1 – theoretical physics and deserve attention in various dimensions and with various amounts
of supersymmetry. But besides that, N = (0, 2) gauged linear sigma models are known
to be a useful tool to construct N = (0, 2) SCTFs, and hence heterotic string vacua, as
infrared (IR) fixed points of the renormalization group (RG) flow (see [1, 2] or just [3] and
references therein). Recently, the dynamics of two-dimensional N = (0, 2) supersymmetric gauge theories,
both abelian and non-abelian, have seen an increasing interest, especially due to devel-
opments in [4–6]. At the same time, more basic models of N = (0, 2) interacting matter
without gauge fields, sometimes referred to as N = (0, 2) Landau-Ginzburg (LG) mod-
els, have been studied, some references being [2, 7, 8]. One can think of starting from
an N = (0, 2) LG model with global flavor symmetries and then gauging these global
symmetries to obtain an N = (0, 2) gauge theory. Therefore, it might be beneficial for
certain questions to first study properties of LG models and then ask what happens to
these properties after gauging. JHEP03(2018)079 The property we want to study in this paper is the chiral algebra in the cohomology
of one of the supercharges. The supercharges Q+ and Q+ of the (0, 2) theory satisfy: {Q+, Q+} = 2P++,
(1.1) (1.1) where P++ is the right-moving translation generator. Since Q
2
+ = 0, one can study its
cohomology, and the above equation implies that it is holomorphic, or, in terms of light-
cone coordinates x±± (in Lorenzian signature), the cohomology depends non-trivially only
on x−−, while differentiation with respect to x++ annihilates cohomology classes. This
observation was first made in [9] and then in [10] used to elucidate some properties of
N = (2, 2) LG models and their IR fixed points. 1
Introduction Then, part of the analysis from [10] was
extended to N = (0, 2) gauge theories in [11]. Chiral algebras of N = (0, 2) half-twisted sigma models were studied to some extent
in the literature due to their connection with the theory of chiral differential operators. In particular, the perturbative approach was developed in [12] and [13], and some non-
perturbative aspects were studied in [14] and [15]. There was also a number of papers on
topological rings (which are finite sectors of chiral algebras in N = (0, 2) theories), some
examples being [16–19]. However, it seems that systematic analysis of chiral algebras in
N = (0, 2) LG models and gauge theories has not been performed yet. Our goal is to make
a small step in this direction. In this paper we first study some general properties of N = (0, 2) theories on R1,1. Then we restrict to a certain class of models, namely LG models, and later consider LG
models with N = (2, 2) supersymmetry and give some examples. An important question about chiral algebras of N = (0, 2) theories is how they behave
under the RG flow. It turns out that in theories with the R-symmetry chiral algebra is
an RG invariant, while it is not completely clear whether this is the case for more general
theories without R-symmetry. The RG invariance underlies all approaches to extract some
useful information about the CFT in the IR, such as, for example, in [10] and [11]. It seems
that this has never been proved in the literature though. – 2 – We study RG invariance of the chiral algebra on general grounds using the N =
(0, 2) supercurrent multiplet described in [20]. If the theory has an R-symmetry, the
supercurrent multiplet becomes what is called the R-multiplet. In such a situation, only
using manipulations with the R-multiplet, we show that there is a stress-energy tensor in
the cohomology. This fact underlies the finding of [11] that the stress-energy tensor in the
cohomology is not spoiled by anomaly if and only if the R-symmetry is non-anomalous. So we obtain conformal symmetry in the chiral algebra. It is interesting to note that this
stress-energy tensor is the one of the half-twisted N = (0, 2) theory. 2
N = (0, 2) theories In this section we discuss some general aspects of two-dimensional (0, 2)-supersymmetric
theories and their chiral algebras. 1
Introduction Since the stress-energy
tensor in cohomology is identified with the left-moving stress-energy tensor of the CFT in
the IR, we can say the following: in N = (0, 2) theories with the R-symmetry, the RG flow
from the UV to the IR performs a half-twist. JHEP03(2018)079 Having conformal symmetry in the cohomology is a very strong restriction. It turns out
that because of it, the chiral algebra cannot depend on any dimensionful constants. This
fact allows one to argue that for the LG models, the chiral algebra is in fact tree level exact,
and the OPE of the cohomology classes can be computed using the free field correlators. This makes chiral algebra a potentially powerful tool for obtaining exact results. We should
note, however, that in this discussion we assume that there are no non-perturbative effects. This seems a reasonable assumption for LG models on a topologically trivial space with a
topologically trivial target only. In section 2 of this paper we discuss general aspects of N = (0, 2) theories, namely the
supercurrent multiplet and its ambiguities, conformal invariance in the cohomology and
its implications for the OPE. We also discuss what is the chiral algebra of SCFT’s and
relate it to the notion of a chiral ring of [21]. We then discuss the superspace technique to
describe the Q+ cohomology. In section 3 we discuss general properties of N = (0, 2) LG models and specify to the
quasi-homogeneous superpotentials. We then review the statement that the OPE of the
cohomology classes can be computed using the free theory and argue that the chiral algebra
is tree level exact (but still different from the classical algebra due to the singularities one
encounters in defining composite operators). In section 4 we specialize to N = (2, 2)
supersymmetry, and in section 5 we discuss a few examples, focusing on the Ak+1 series of
N = 2 minimal models. We then conclude in section 6 and mention some further directions. 2
N = (0, 2) theories 2
N = (0, 2) theories 2.1
Conventions and some generalities The two-dimensional theories with (0, 2) supersymmetry are characterized by the existence
of two conserved supercharges Q+ and Q+ of positive (or right-handed) chirality acting on
the Hilbert space of the theory. They satisfy: Q2
+ = Q
2
+ = 0,
{Q+, Q+} = 2P++,
(2.1) +
+
{Q+, Q+} = 2P++,
(2.1) (2.1) – 3 – – 3 – where 2P++ = P0 + P1 is a light-cone momentum. The standard geometric realization
of supersymmetry is to consider the superspace R2|2 with bosonic coordinates x0, x1 and
fermionic coordinates θ+ and θ
+. Superfields are distributions on this superspace taking
values in operators acting on the Hilbert space. The supercharges Q+ and Q+ act on
operators (and therefore on superfields) by commutators, and the geometric realization of
this action is through the differential operators: Q+ =
∂
∂θ+ + iθ
+
∂
∂x++ ,
Q+ = −∂
∂θ
+ −iθ+
∂
∂x++ ,
(2.2) (2.2) JHEP03(2018)079 so that for an arbitrary superfield F, we have [Q+, F]± = Q+F, where [. . . ]± denotes a
graded commutator. These operators obviously satisfy the required relation [Q+, Q+] =
−2i
∂
∂x+ . We also have another pair of differential operators on R2|2, D+ and D+, given by: so that for an arbitrary superfield F, we have [Q+, F]± = Q+F, where [. . . ]± denotes a
graded commutator. These operators obviously satisfy the required relation [Q+, Q+] =
−2i
∂
∂x+ . We also have another pair of differential operators on R2|2, D+ and D+, given by: D+ =
∂
∂θ+ −iθ
+
∂
∂x++ ,
D+ = −∂
∂θ
+ + iθ+
∂
∂x++ ,
(2.3) (2.3) for which the key property is that they anticommute with Q+ and Q+ and hence can be
used in constructing supersymmetric Lagrangians. for which the key property is that they anticommute with Q+ and Q+ and hence can be
used in constructing supersymmetric Lagrangians. We also adopt the convention in which hermitian conjugation reverses the order of
fermions, that is (θ1θ2)† = θ2θ1. The basic superfields are The basic superfields are The basic superfields are 1) Chiral superfields satisfying D+Φ = 0. The component expansion contains a complex
scalar φ and a left spinor ψ+: 1) Chiral superfields satisfying D+Φ = 0. 3) Real superfield. 2.1
Conventions and some generalities The component expansion contains a complex
scalar φ and a left spinor ψ+: Φ = φ + iθ+ψ+ −iθ+θ
+∂++φ
(2.4) (2.4) The antichiral superfield satisfies D+Φ = 0 and is given by: The antichiral superfield satisfies D+Φ = 0 and is given by: Φ = φ + iθ
+ψ+ + iθ+θ
+∂++φ. (2.5) (2.5) 2) Fermi superfields satisfying D+Λ = E(Φ), where E(Φ) is a chiral superfield con-
structed as a holomorphic function of basic chiral superfields. The component ex-
pansion contains a right-handed spinor λ and an auxiliary field G: 2) Fermi superfields satisfying D+Λ = E(Φ), where E(Φ) is a chiral superfield con-
structed as a holomorphic function of basic chiral superfields. The component ex-
pansion contains a right-handed spinor λ and an auxiliary field G: 2) Fermi superfields satisfying D+Λ = E(Φ), where E(Φ) is a chiral superfield con-
structed as a holomorphic function of basic chiral superfields. The component ex-
pansion contains a right-handed spinor λ and an auxiliary field G: Λ = λ + θ+G −iθ+θ
+∂++λ −θ
+E(Φ),
(2.6) (2.6) where E itself has to be expanded in components. The opposite chirality Fermi
superfield satisfies D+Λ = −E(Φ) and is given by: where E itself has to be expanded in components. The opposite chirality Fermi
superfield satisfies D+Λ = −E(Φ) and is given by: Λ = λ + θ
+G + iθ+θ
+∂++λ −θ+E(Φ). (2.7) (2.7) – 4 – If U is a real superfield, it can always be thought of as a real part of some chiral
superfield (not necessarily a local one; also we will allow for superfields which are chiral
only on-shell). We will denote the imaginary part of this chiral superfield by eU. Then
U + ieU is chiral on-shell and U −ieU is antichiral. The relation between U and eU is: D+ eU = iD+U,
D+ eU = −iD+U,
(2.8) +
+
D+ eU = −iD+U,
(2.8) (2.8) up to equations of motion. This eU is defined up to a term which is constant on-shell. 1Our conventions are different from [20]. 2.1
Conventions and some generalities If
the component expansion of U is JHEP03(2018)079 U = u + iθ+χ+ + iθ
+χ+ + θ+θ
+∂++v,
(2.9) (2.9) where we wrote the highest component as a derivative of some function v, then the com-
ponent expansion of eU is: where we wrote the highest component as a derivative of some function v, then the com-
ponent expansion of eU is: eU = v + θ+χ+ −θ
+χ+ −θ+θ
+∂++u,
(2.10) (2.10) again up to terms which vanish on equations of motion. e again up to terms which vanish on equations of motion. e Note that if we want components of U and eU to be local operators, then U cannot be
an arbitrary local real superfield. Its highest component, written as ∂++v above, should
be a derivative of a local field. Only in such a case v above is also local and hence eU is
also the local superfield. 2.2
Supercurrent multiplet and RG invariance 2.2.1
General case The general N = (0, 2) multiplet containing the stress-energy tensor and the supersymme-
try current was described in [20]. It is referred to as the supercurrent multiplet. It consists
of real superfields S++, T−−−−and a complex superfield W−satisfying:1 ∂−−S++ = D+W−−D+W−,
D+T−−−−= ∂−−W−,
D+T−−−−= ∂−−W−,
D+W−= C,
(2.11) (2.11) D+W−= C,
(2.11) where C is a complex constant (a space-filling brane current). The component expansions
which solve these constraints are: S++ = j++ −2iθ+S+++ −2iθ
+S+++ −2θ+θ
+T++++,
W−= −S+−−−iθ+
T++−−+ i
2∂−−j++
−θ
+C + iθ+θ
+∂++S+−−,
W−= −S+−−+ iθ
+
T++−−−i
2∂−−j++
−θ+C −iθ+θ
+∂++S+−−,
T−−−−= T−−−−−θ+∂−−S+−−+ θ
+∂−−S+−−+ 1
2θ+θ
+∂2
−−j++. ( (2.12) 1Our conventions are different from [20]. – 5 – – 5 – Applying constraints (2.11) to these expansions implies conservation of S+ (the supersym-
metry current), conservation of T (the stress-energy tensor) and symmetry of T: Applying constraints (2.11) to these expansions implies conservation of S+ (the supersym-
metry current), conservation of T (the stress-energy tensor) and symmetry of T: ∂++S+−−+ ∂−−S+++ = 0,
∂++T±±−−+ ∂−−T±±++ = 0,
T++−−−T−−++ = 0. (2.13) (2.13) Quite naturally, constraints (2.11) do not determine the supercurrent multiplet uniquely. There are two types of ambiguities which preserve both the conservation laws and the form
of equations (2.11). One ambiguity corresponds to improvement transformations: Quite naturally, constraints (2.11) do not determine the supercurrent multiplet uniquely. There are two types of ambiguities which preserve both the conservation laws and the form
of equations (2.11). One ambiguity corresponds to improvement transformations: JHEP03(2018)079 S++ →S++ + [D+, D+]U,
W−→W−+ ∂−−D+U,
W−→W−+ ∂−−D+U,
T−−−−→T−−−−+ ∂2
−−U,
(2.14) (2.14) where U is an arbitrary real scalar superfield. These transformations do not change con-
served charges. Another ambiguity corresponds to the possibility of modifying the supercurrent multi-
plet by another conserved current (say, corresponding to some flavor symmetry), satisfying
an additional requirement of locality which will be explained in a moment. If we have
another conserved superspace current I±±, that is a pair of real superfields satisfying: ∂−−I++ + ∂++I−−= 0,
(2.15) (2.15) then we can use it to shift the supercurrent multiplet, i.e. define a new multiplet: S++ →eS++ = S++ + I++,
W−→f
W−= W−+ i
2D+I−−,
W−→f
W−= W−−i
2D+I−−,
T−−−−→eT−−−−= T−−−−+ 1
2∂−−eI−−. (2.16) (2.16) Note that in the last equation we use eI−−, a real superfield related to I−−as in (2.8). 2.2.1
General case That is, eI−−is such that I−−+ ieI−−is chiral. The new superfields eS++, f
W−and eT−−−−
will also satisfy the constraints (2.11). However, most conserved charges will be shifted by
this transformation. Note that for the above transformation to make sense in a local QFT,
both I±± and eI−−have to be local, so there is an extra requirement on I±± that not only
it has to be a conserved local superspace current, but also eI−−has to be local. In the cases
of interest for us, this will actually be the case. Note that in the last equation we use eI−−, a real superfield related to I−−as in (2.8). That is, eI−−is such that I−−+ ieI−−is chiral. The new superfields eS++, f
W−and eT−−−−
will also satisfy the constraints (2.11). However, most conserved charges will be shifted by
this transformation. Note that for the above transformation to make sense in a local QFT,
both I±± and eI−−have to be local, so there is an extra requirement on I±± that not only
it has to be a conserved local superspace current, but also eI−−has to be local. In the cases
of interest for us, this will actually be the case. – 6 – One can easily read offthe action of Q+ on various components of the supercurrent
multiplet, and we are interested in the following: One can easily read offthe action of Q+ on various components of the supercurrent
multiplet, and we are interested in the following: {Q+, S+++} = −i
T++++ + i
2∂++j++
,
{Q+, S+−−} = i
T++−−−i
2∂−−j++
, {Q+, S+++} = −i
T++++ + i
2∂++j++
,
{Q+, S+−−} = i
T++−−−i
2∂−−j++
,
{Q+, T++++} = ∂++S+++,
{Q+, T++−−} = −∂++S+−−,
{Q+, T−−−−} = −∂−−S+−−. (2.17) {Q+, T++++} = ∂++S+++, JHEP03(2018)079 {Q+, T−−−−} = −∂−−S+−−. (2.17) (2.17) We see that neither component of the stress-energy tensor is annihilated by Q+, so com-
ponents of T by itself do not represent any Q+-cohomology classes. However, certain
relations hold in the cohomology, in particular T++−−−i
2∂−−j++ is Q+-exact. If we define
the “virial current” Vµ as: We see that neither component of the stress-energy tensor is annihilated by Q+, so com-
ponents of T by itself do not represent any Q+-cohomology classes. However, certain
relations hold in the cohomology, in particular T++−−−i
2∂−−j++ is Q+-exact. 2.2.2
Emergent conformal invariance in the cohomology In the previous subsection we considered a general N = (0, 2) theory in 2d, which a priori
did not have any R-symmetries. The lowest component j++ of the superfield S++ did not
satisfy any conservation laws and, moreover, was not even accompanied by j−−. As was
noted in [20], if we restrict to the case C = 0 and W−= i
2D+R−−, where R−−is another
real superfield (and also relabel S++ by R++), we get what is called an R-multiplet. The
equation relating S++ and W−becomes simply ∂−−R++ + ∂++R−−= 0, so the lowest
component j−−of R−−together with j++ form a conserved R-current. So we have: R−−= j−−−2iθ+S+−−−2iθ
+S+−−−2θ+θ
+T++−−,
(2.23) (2.23) JHEP03(2018)079 with ∂++j−−+ ∂−−j++ = 0. In this situation, it becomes possible to define a new stress-
energy tensor: with ∂++j−−+ ∂−−j++ = 0. In this situation, it becomes possible to define a new stress-
energy tensor: eT++++ = T++++ + i
2∂++j++,
eT++−−= T++−−−i
2∂−−j++,
eT−−−−= T−−−−−i
2∂−−j−−,
(2.24) (2.24) which is also symmetric and conserved (by virtue of the conservation of j), but also it
satisfies: which is also symmetric and conserved (by virtue of the conservation of j), but also it
satisfies: eT++−−= {Q+, . . . },
{Q+, eT−−−−} = 0. (2.25) eT++++ = {Q+, . . . },
eT++−−= {Q+, . . . },
eT−−−−̸= {Q+, . . . },
{Q+, eT−−−−} = 0. (2.25) eT++++ = {Q+, . . . },
eT−−−−̸= {Q+, . . . }, (2.25) This procedure for N = (0, 2) theories is known as a half-twisting. The above relations
demonstrate that when it can be performed, one has the full 2d conformal invariance in
the cohomology of Q+: the cohomology class represented by eT−−−−plays the role of the
holomorphic2 stress-energy tensor. It also ensures that the Q+-cohomology is invariant
under the RG flow. The RG invariance of the chiral algebra implies that it carries a useful
information about the IR fixed point. Let us also take a closer look at the ambiguities of the supercurrent multiplet in
the presence of R-symmetry. The improvement transformations are determined by a real
superfield U though (2.14), which tells us how R++, W−and T−−−−are improved. 2.2.1
General case If we define
the “virial current” Vµ as: V−−= 0,
V++ = ij++,
(2.18) (2.18) then we have: T µ
µ = ∂µVµ −{Q+, 4iS+−−},
(2.19) (2.19) which looks like condition for an effective scale-invariance [22], with the effective current
for constant dilatations given by dµ = xνTνµ −Vµ. This current is “almost conserved”: ∂µdµ = {Q+, . . . }. (2.20) (2.20) The current dµ itself is not Q+-closed. Even though dµ is not precisely conserved, only up
to Q+-exact terms, we still can try to define a “charge” D corresponding to this current. If we have a local operator O(0) inserted at the origin, we define the action of D on this
operator as follows. Pick a contour C enclosing O(0) and define: [D, O(0)] =
I
C
⋆d(x)O(0) =
I
C
dxµϵµνdν(x)O(0). (2.21) (2.21) This definition is clearly contour-dependent, since dµ(x) is not conserved. As we deform
the contour a bit, [D, O(0)] changes by [∂µdµ(x), O] integrated over the area swept by the
deformation of the contour. But ∂µdµ(x) is Q+-exact, so if O(0) is Q+-closed, the change
in [D, O(0)] under the contour deformation is Q+-exact. This means that [D, O(0)] is
well-defined up to a Q+-exact piece when it acts on Q+-closed operators. Moreover, one
can check that: [D, Q+] = Q+,
(2.22) (2.22) which shows that D maps Q+-closed operators into Q+-closed operators. So we conclude
that D is a well-defined operator in the cohomology. It generates scale-transformations
there. Since D is not Q+-closed itself, we can say that scale transformations act as outer
automorphisms in the cohomology. – 7 – 2To be more precise, we should Wick rotate to the Euclidean signature in order to have holomorphy. 2.2.2
Emergent conformal invariance in the cohomology On
R−−it acts by: R−−→R−−−2∂−−eU,
(2.26) (2.26) where the relation between U and eU is as in (2.8), that is where the relation between U and eU is as in (2.8), that is U = u + iθ+χ+ + iθ
+χ+ + θ+θ
+∂++v,
eU = v + θ+χ+ −θ
+χ+ −θ+θ
+∂++u. (2.27) (2.27) For an improvement transformation of the R-multiplet to make sense, we have to assume
that both U and eU are local superfields. In view of the comment we made before, this For an improvement transformation of the R-multiplet to make sense, we have to assume
that both U and eU are local superfields. In view of the comment we made before, this
2To be more precise, we should Wick rotate to the Euclidean signature in order to have holomorphy. 2To be more precise, we should Wick rotate to the Euclidean signature in order to have holomorphy. – 8 – restricts the class of allowed U. While for a general supercurrent multiplet the improvem
ent transformations were parametrized by an arbitrary local real superfield U, for the R-
multiplet they are parametrized by such a local real superfields U that eU is also local. Thus the R-multiplet allows a smaller class of improvements then a general supercurrent
multiplet. This is not surprising after all. For the general supercurrent multiplet, only
the stress-energy tensor and the supersymmetry currents are conserved, so improvements
should only preserve their conservation. In the R-multiplet, on the other hand, we also have
the conserved R-current, so preserving its conservation (and the R-charge value) restricts
the class of allowed improvements. In terms of component currents, the improvement transformation is: JHEP03(2018)079 j−−→j−−−2∂−−v,
T++−−→T++−−−∂++∂−−u, j++ →j++ + 2∂++v,
j−−→j−−−2∂−−v,
T++++ →T++++ + ∂2
++u,
T++−−→T++−−−∂++∂−−u,
T−−−−→T−−−−+ ∂2
−−u,
S+++ →S+++ + i∂++χ+,
S+−−→S+−−−i∂−−χ+,
S+++ →S+++ −i∂++χ+,
S+−−→S+−−+ i∂−−χ+. (2.28) S+−−→S+−−−i∂−−χ+,
S+−−→S+−−+ i∂−−χ+. (2.28) (2.28) As expected, this transformation does not spoil conservation of any of these currents. It does not shift values of any conserved charges either. Also, it is easy to check that
components eT++++ and eT++−−of the half-twisted stress-energy tensor are shifted by Q+-
exact terms. On the other hand, eT−−−−is shifted by ∂2
−−(u + iv), which is, being the
lowest component of chiral superfield U + ieU, is Q+-closed but generally is not Q+-exact. Therefore, there is a family of possible holomorphic stress tensors in the Q+-cohomology,
corresponding to different improvements. 3Even if the operator representing the cohomology class in the full theory is not left-moving, the class
it represents is left-moving. Since Lorenz-invariance of the full theory induces Lorenz-invariance in the
cohomology, one indeed can use the argument made in the text. 2.2.3
OPE in the cohomology This is possible only for p = 0, that is
µ should not be there. All dependence on dimensionful coupling constants of the original supersymmetric
theory will therefore be hidden in the Q+-exact term. This simple observation will be
helpful later. It will imply that one can turn offall dimensionful couplings for the OPE
computation. In the models we are going to study this will mean that it is enough to
compute OPE in the free theory. 2.2.3
OPE in the cohomology If we have two operators O1 and O2 representing nontrivial Q+-cohomology classes, we
can consider their OPE. On very general grounds we have: O1(x++, x−−)O2(0, 0) =
X
n,m
(x++)n(x−−)mOn,m(0, 0). (2.33) (2.33) Now recall that the operator ∂++ acts trivially in the cohomology, that is if O1 is Q+-
closed, then ∂++O1 is Q+-exact, and thus so is ∂++O1(x++, x−−)O2(0, 0). Acting with
∂++ on the right-hand side then gives a Q+-exact answer, that is: Now recall that the operator ∂++ acts trivially in the cohomology, that is if O1 is Q+-
closed, then ∂++O1 is Q+-exact, and thus so is ∂++O1(x++, x−−)O2(0, 0). Acting with
∂++ on the right-hand side then gives a Q+-exact answer, that is: JHEP03(2018)079 X
n,m
n(x++)n−1(x−−)mOn,m(0, 0) = [Q+, . . . ]. (2.34) (2.34) This implies that all terms except those with n = 0 are Q+-exact. If the cohomology
classes represented by Oi have scaling dimensions hi, we can then write: O1(x++, x−−)O2(0, 0) =
X
k
1
(x−−)h1+h2−hk Ok(0, 0) + [Q+, . . . ]. (2.35) (2.35) Note also that, since in the cohomology we have left-movers only, scaling dimensions and
spins coincide.3 This, in particular, implies an obvious conclusion that no dimensionful
constants can appear in the OPE of the cohomology classes. Any dimensionful constant
will have non-trivial dimension but trivial spin, and therefore its appearance will either
break scaling or Lorentz-invariance of the OPE. Indeed, if we have some dimensionful
parameter µ, then in the expression: Note also that, since in the cohomology we have left-movers only, scaling dimensions and
spins coincide.3 This, in particular, implies an obvious conclusion that no dimensionful
constants can appear in the OPE of the cohomology classes. Any dimensionful constant
will have non-trivial dimension but trivial spin, and therefore its appearance will either
break scaling or Lorentz-invariance of the OPE. Indeed, if we have some dimensionful
parameter µ, then in the expression: O1(x++, x−−)O2(0, 0) =
X
k
µp
(x−−)∆Ok(0, 0) + [Q+, . . . ],
(2.36) (2.36) scaling invariance implies ∆= h1 + h2 −hk −h(µ)p, where h(µ) is the dimension of
while Lorentz invariance implies ∆= h1 + h2 −hk. This is possible only for p = 0, that
µ should not be there. scaling invariance implies ∆= h1 + h2 −hk −h(µ)p, where h(µ) is the dimension of µ,
while Lorentz invariance implies ∆= h1 + h2 −hk. 2.2.2
Emergent conformal invariance in the cohomology Another ambiguity, namely shifting by the superspace current I±±, works in a straight-
forward way: R++ →R++ + I++,
R−−→R−−+ I−−,
T−−−−→T−−−−+ 1
2∂−−eI−−. (2.29) (2.29) If we denote the components of I±± by: If we denote the components of I±± by: I±± = i±± −2iθ+I+±± −2iθ
+I+±± −2θ+θ
+H++±±,
(2.30) (2.30) and introduce a local operator h−−such that and introduce a local operator h−−such that ∂++h−−= H++−−,
(2.31) ∂++h−−= H++−−, (2.31) then the shifting transformation in components works as: then the shifting transformation in components works as: j±± →j±± + i±±,
S+±± →S+±± + I+±±,
S+±± →S+±± + I+±±,
T++±± →T++±± + H++±±,
T−−−−→T−−−−−∂−−h−−. (2.32) (2.32) This ambiguity will naturally arise in a later discussion. This ambiguity will naturally arise in a later discussion. – 9 – 2.3
Chiral algebras of superconformal theories For superconformal theories, the N = (0, 2), d = 2 super-Poincare algebra of symmetries
is enhanced to Vir ⊕g
SVir, where Vir denotes the left-handed Virasoro algebra (generated
by the holomorphic stress-energy tensor) and the g
SVir denotes the right-handed N = 2 – 10 – super-Virasoro algebra (generated by the corresponding anti-holomorphic currents). The
left-handed algebra might be enlarged to the super-Virasoro as well (or even some larger
W-algebra) if we have more symmetries on the left, but it graded-commutes with the N = 2
Virasoro on the right in any case. Let us restrict to the NS sector of the g
SVir. The operators Q+ and Q+ can be identified
as eG+
−1/2 and eG−
−1/2 respectively — two of the fermionic generators of g
SVir (we put tildes
on g
SVir and on its generators to emphasize that this is an anti-holomorphic algebra). In
a conformal case, we have the radial quantization Hilbert space H, and we assume that it
has an inner product, such that eG−
1/2 =
eG+
−1/2
†
is a special supersymmetry generator. Part of the super-Virasoro algebra relations are: JHEP03(2018)079 { eG−
−1/2, eG+
−1/2} = 2eL−1,
{ eG+
−1/2, ( eG+
−1/2)†} ≡{ eG+
−1/2, eG−
1/2} = 2eL0 −eJ0. (2.37) (2.37) Recall that in conformal case we have a state-operator correspondence. Therefore, instead
of computing the operator cohomology, we can equivalently ask for the cohomology of
eG+
−1/2 acting on the Hilbert space H. The second equation in (2.37) shows that, by the
standard Hodge theory argument, this cohomology can be identified with the kernel of
2eL0 −eJ0. Also, in a unitary theory, it shows that 2eL0 −eJ0 ≥0. Now, every state in the Hilbert space is built by acting with eL−n, eJ−n, eG+
−α, eG−
−α, n, α >
0 on a superconformal primary state. It is easy to see that all these operators except eG+
−1/2
increase the eigenvalue of 2eL0−eJ0, while eG+
−1/2 does not change it. Therefore, if the primary
state has 2eL0 −eJ0 > 0, then all states in its superconformal family have 2eL0 −eJ0 > 0
and thus do not contribute to the cohomology. On the other hand, if some primary state
|∆⟩has zero eigenvalue of 2eL0 −eJ0, then so does eG+
−1/2|∆⟩, while other states in the same
conformal family have 2eL0 −eJ0 > 0. 2.3
Chiral algebras of superconformal theories But (2eL0 −eJ0)|∆⟩= 0 and the second equation
of (2.37) imply that eG+
−1/2|∆⟩= 0. Therefore, in such a case there is just one non-trivial
state in the superconformal family which contributes to the cohomology — the primary
state itself. This way we prove that in the NS sector of a unitary N = (0, 2) superconformal
theory there is an isomorphism: H(H, eG+
−1/2)
≃{Primaries of ]
SVir with 2eL0 −eJ0 = 0}
= {|ψ⟩∈H : eLn|ψ⟩= eJn|ψ⟩= eG+
α−1|ψ⟩= eG−
α |ψ⟩= (2eL0 −eJ0)|ψ⟩= 0, n, α > 0}. H(H, eG+
−1/2) ≃{Primaries of ]
SVir with 2eL0 −eJ0 = 0}
= {|ψ⟩∈H : eLn|ψ⟩= eJn|ψ⟩= eG+
α−1|ψ⟩= eG−
α |ψ⟩= (2eL0 −eJ0)|ψ⟩= 0, n, α > 0}. (2.38) Notice that these are what is usually called the chiral primaries with respect to ]
SVir. In
fact, this is essentially the construction of [21] applied to N = (0, 2) theories. In the
N = (2, 2) case, [21] describe the chiral ring of the N = (2, 2) model by studying the set of
(anti)chiral primaries both with respect to the left- and the right-moving super-Virasoro
algebras. For the N = (0, 2) theories, we have in (2.38) only the chiral primary condition
with respect to the right-moving super-Virasoro algebra. For that reason, the object we – 11 – get is not just the chiral ring: it involves holomorphic OPEs as part of its structure and is
usually referred to as the W-algebra, or also chiral algebra. Another remark is that for N = (2, 2) theories, the chiral algebra that we study
encodes the (c, c) and (a, c) rings of [21] as a part of its structure. Indeed, by considering
the subspace of H(H, eG+
−1/2) annihilated by 2L0 −J0, where L0 and J0 are from the left-
moving SVir algebra, we get the space {|ψ⟩∈H : (2L0 −J0)|ψ⟩= (2eL0 −eJ0)|ψ⟩= 0},
which is the space of chiral primaries with respect to both SVir and ]
SVir, and therefore
gives rise to the (c, c) ring under the OPE. Analogously, picking the subspace annihilated
by L0 + J0, we get the (a, c) ring. 2.3
Chiral algebras of superconformal theories JHEP03(2018)079 One consequence of this is that in N = (2, 2) theories, the (anti)chiral primaries, which
form the (c, c) or (a, c) rings of the theory, always show up in the chiral algebra as primaries
of the left-moving ]
SVir. In the simplest cases they will generate the whole chiral algebra,
but as we will see later, there might be other primary operators in the algebra, which are
not simply elements of the (c, c) or (a, c) ring. 2.4.1
Classical and quantum observables In the models we are going to study later in this paper, the chiral algebra will turn out to
be tree-level exact. As we will argue, no loop corrections will contribute to the cohomology. However, despite our usual intuition that “tree level” means “classical”, it is important to
understand that the quantum chiral algebra in the Q+-cohomology is not the same as the
classical one. The distinction comes from the way we multiply operators. In classical field theory, to multiply fields we use the usual point-wise multiplication
of functions on space-time. In quantum theory, even at the tree level, we should subtract
singularities which appear when different operators collide, which for example gives the
usual notion of normal ordering in CFT. It might happen (and it will happen in concrete examples) that the classical composite
operator is Q+-closed, but the singular part we need to subtract to define the quantum
operator is not Q+-closed. This subtlety should be taken into account when computing the
chiral algebra of the theory. But still, as a step in this direction, it is useful to understand
the structure of the classical chiral algebra first. 2.4.2
Classical observables and the cohomology Let us introduce the space of classical observables F and the space of classical superob-
servables bF. We will sometimes refer to a generic field as φ and to a generic superfield as
Φ. Both of these spaces classically carry the structures of supercommutative algebras. Definition 1. F is a supercommutative algebra of polynomials of fields φ and their deriva-
tives ∂n
−−∂m
++φ whose coefficients are analytic functions on a space-time, modulo classical
equations of motion. In other words, F = Cω(M)[. . . , φ, ∂n
−−∂m
++φ, . . . ]/I, where Cω(M)
denotes analytic functions on M, and I denotes an ideal generated by the equations of
motion and all their derivatives. – 12 – If the classical equations of motion do not depend on space-time coordinates explicitly
(only through the coordinate-dependence of the generating fields), we can introduce: Definition 2. F0 is a subalgebra of F of observables which do not depend on a space-time
point explicitly. In other words, it is generated by the same fields and their derivatives as
F (and also modulo equations of motion), but the coefficients are taken to be just complex
numbers rather than functions. There are straightforward superspace analogs of these: Definition 3. bF is a supercommutative algebra of polynomials of superfields Φ and their
bosonic and super-derivatives ∂n
−−∂m
++Dp
+D
q
+Φ whose coefficients are analytic functions on
superspace, modulo classical superspace equations of motion. JHEP03(2018)079 If the superspace equations of motion do not include any explicit dependence on
a superspace point, i.e. if they have the form of a polynomial of generating fields
∂n
−−∂m
++Dp
+D
q
+Φ with complex coefficients, we again can define a subalgebra: Definition 4. bF0 is a subalgebra of bF of superobservables which do not depend on a space-
time point explicitly. In other words, it is generated by the same superfields and their
derivatives as bF (and also modulo superspace equations of motion), but the coefficients are
taken to be just complex numbers rather then functions. Our goal is to compute the cohomology of Q+ acting on F in the situation when the
equations of motion do not depend on the superspace point explicitly. The first observation
is that the operator Q+ only acts on the generating fields of the algebra F, it does not
act on the c-number functions which can possibly multiply these fields. Proprosition 2. For any a ∈F0 there exists a unique A ∈bF0 such that a = A
. The problem which we are addressing is to find the cohomology of Q+ in F0. That
is, the classes of fields a ∈F0 which satisfy [Q+, a] = 0, modulo those a for which a =
[Q+, b], b ∈F0. Now from the Proposition 2, we know that there exist A, B ∈bF0, such that
a = A| and b = B|. The equation [Q+, a] = 0 implies then Q+A| = 0. There is a small subtlety here which shows why it is correct to look for the cohomology
of D+ rather than Q+: D+ acts on bF0 by definition, while Q+ = D+ +2iθ+∂+ does not, as
it introduces an explicit dependence on θ (therefore Q+ acts from bF0 to a bigger space bF). However, we can write: D+A| = Q+A| = 0. But D+A ∈bF0, so we can apply Proposition
1 and conclude that D+A = 0. Analogously a = [Q+, b] implies A = D+B. This proves the Proprosition 3. The cohomology of Q+ in F0 (denoted H(F0)) is isomorphic to the
cohomology of D+ in bF0 (denoted H( bF0)). The isomorphism H( bF0) →H(F0) is defined
by taking the lowest component of the superfield. This proposition shows why in the rest of the paper we are going to study the coho-
mology of D+. 2.4.3
The cohomology of Q+ in F0 and of D+ in b
F0 2.4.3
The cohomology of Q+ in F0 and of D+ in b
F0 Take an arbitrary A ∈bF0. A is some general superfield, and it can be expanded into
components with respect to the Grassmann coordinates. The most basic property it satis-
fies is that the supersymmetry transformations of its components are encoded in the way
differential operators Q+ and Q+ act on it. This follows simply from the fact that this
holds for the generating superfields from which A is constructed and the fact that we do
not allow explicit dependence on the superspace coordinates in the algebra bF0. So we have: [Q+, A] = Q+A,
(2.42) (2.42) JHEP03(2018)079 and the same for Q+. Supersymmetry relates all components of A and it is straightforward
to see that: prosition 1. If the lowest component A
of the superfield A ∈bF0 vanishes, then A = 0. Proprosition 1. If the lowest component A
of the superfield A ∈bF0 vanishes, then A = 0. The algebras F0 and bF0 are related in an obvious way: any element of F0 can be found
as a component of some superfield in bF0. In particular, we can always find a superfield A
which contains a given element a ∈F0 as its lowest component. Moreover, supersymmetry
defines this A uniquely, so: roprosition 2. For any a ∈F0 there exists a unique A ∈bF0 such that a = A
. 2.4.2
Classical observables and the cohomology This means that
it is enough to compute the cohomology of Q+ acting on F0. To be more rigorous, we can
introduce operators of multiplication by xµ called m(xµ): ∀O ∈F, m(xµ)O = xµO,
(2.39) (2.39) and notice that they commute with Q+. Then we can introduce a bigrading on F by saying
that an explicit factor of (x0)n(x1)m has degree (n, m). After this it becomes obvious that H(F) ≃
M
n,m≥0
Hn,m(F),
(2.40) (2.40) where the bar over the right hand side means that we should actually consider a completion
of this space with respect to some norm, because we have to allow infinte sums (series) to
account for the possibility of having analytic functions as coefficients. As we mentioned, Q+ does not act in any way on xµ, and because of that: Hn,m(F) ≃H(F0). (2.41) (2.41) Therefore, from now on we will only study the cohomology in F0, which of course only
makes sense when the equations of motion do not depend on the superspace point explicitly. Therefore, from now on we will only study the cohomology in F0, which of course only
makes sense when the equations of motion do not depend on the superspace point explicitly. – 13 – 3
Landau-Ginzburg models The N = (0, 2) Landau-Ginzburg (LG) model is described by a set of chiral superfields
Φi, i = 1 . . . n and Fermi superfields Λa, a = 1 . . . m. The action is (we assume summation
over repeated indices, even if they both appear upstairs or downstairs; sometimes we will
write the sum sign explicitly to avoid possible confusion): S = 1
π
Z
d2xd2θ
−i
2Φ
i∂−−Φi −1
2Λ
aΛa
+ 1
π
Z
d2xdθ+ΛaJa(Φ)|θ
+=0 + h.c.,
(3.1) (3.1) – 14 – where in general D+Λa = Ea(Φ) and P
a Ea(Φ)Ja(Φ) = 0. The classical superspace
equations of motion are: D+∂−−Φ
i = iΛ
a ∂Ea
∂Φi −2iΛa ∂Ja
∂Φi ,
D+∂−−Φi = −iΛa ∂E
a
∂Φ
i + 2iΛ
a ∂Ja
∂Φ
i ,
D+Λ
a = −2Ja(Φ),
D+Λa = 2Ja(Φ). D+∂−−Φ
i = iΛ
a ∂Ea
∂Φi −2iΛa ∂Ja
∂Φi ,
D+∂−−Φi = −iΛa ∂E
a
∂Φ
i + 2iΛ
a ∂Ja
∂Φ
i ,
D+Λ
a = −2Ja(Φ),
D+Λa = 2Ja(Φ). (3.2) D+Λa = 2Ja(Φ). (3.2) (3.2) JHEP03(2018)079 The supersymmetry currents of this theory are: The supersymmetry currents of this theory are: The supersymmetry currents of this theory are: S+++ = i
2ψi
+∂++φ
i,
S+++ = −i
2ψ
i
+∂++φi,
S+−−= i
2λaE
a(φ) −iλ
aJa(φ),
S+−−= iλaJa(φ) −i
2λ
aEa(φ). (3.3) (3.3) It is not hard to find a superfield S++ such that S+++ =
i
2D+S++| and S+++ =
−i
2D+S++|: S++ = 1
2D+ΦiD+Φ
i. (3.4) (3.4) If we also introduce: W−= i
2Λ
aEa −iΛaJa,
T−−−−= ∂−−Φi∂−−Φ
i + i
2Λa∂−−Λ
a −i
2∂−−ΛaΛ
a,
(3.5) (3.5) then we find that: then we find that: ∂−−S++ = D+W−−D+W−,
D+T−−−−= ∂−−W−,
D+W−= 0. (3.6) +
D+W−= 0. (3.6) (3.6) We see that these are precisely the relations (2.11) of the N = (0, 2) d = 2 supercurrent,
and moreover, the component expansions of S++, W−and T−−−−, written as in (2.12),
include the supersymmetry currents (3.3). Therefore, we have described the supercurrent
multiplet of the theory (3.1). In a generic situation, it is not an R-multiplet, because there
are no R-symmetries. We see that these are precisely the relations (2.11) of the N = (0, 2) d = 2 supercurrent,
and moreover, the component expansions of S++, W−and T−−−−, written as in (2.12),
include the supersymmetry currents (3.3). 3
Landau-Ginzburg models Therefore, we have described the supercurrent
multiplet of the theory (3.1). In a generic situation, it is not an R-multiplet, because there
are no R-symmetries. The algebra
bF0 is a supercommutative algebra freely generated by superfields
Φi, Φ
i, Λa, Λ
a and their derivatives (with respect to ∂−−, ∂++, D+ and D+ applied arbi-
trary number of times) modulo the relations. The relations are: the ones that follow from
{D+, D+} = 2i∂++ and D2
+ = D
2
+ = 0, the chirality conditions D+Φi = 0, D+Λa = Ea(Φ)
and the superspace equations of motion as written above. All differential corollaries of the
relations should also be included as relations of course. – 15 – It is not too hard to find a set of independent generators G, so that all the relations will
be taken into account and we will have simply bF0 ≃C[G], a polynomial algebra generated
by those generators. It is not too hard to find a set of independent generators G, so that all the relations will
be taken into account and we will have simply bF0 ≃C[G], a polynomial algebra generated
by those generators. We will now find this G. First of all, due to the chirality conditions, Φi can only appear
with the D+ derivative (moreover, with at most one, because D2
+ = 0), and Φ
i — with
D+. The chirality condition for Λa allows to replace D+Λa by Ea(Φ), while the equation
of motion D+Λa = −2Ja(Φ) allows to replace D+Λa by an expression without derivatives. Therefore it is enough to consider only bosonic derivatives acting on Λa. However the
simple relation: JHEP03(2018)079 2i∂++Λa = {D+, D+}Λa = D+D+Λa + D+D+Λa = D+Ea(Φ) + 2D+Ja(Φ)
(3.7) (3.7) shows that ∂++ derivatives acting on Fermi superfields can also be removed. Therefore,
in the generating set G, it is enough to include only ∂n
−−Λa and ∂n
−−Λ
a, with n ≥0,
and the appropriate derivatives of bosonic chiral superfields. By appropriate derivatives
of bosonic chiral superfields we mean the following. First, we need to include ∂n
−−Φi and
∂n
−−Φ
i with n ≥0. D+Φi and D+Φ
i should also be included, but there is no need to
include expressions like D+∂n
−−Φi, because, as equations of motion for Φi show, D+∂−−Φi
and D+∂−−Φ
i can be replaced by expressions without derivatives. 3
Landau-Ginzburg models Expressions like ∂n
++Φi,
D+∂n
++Φi and their complex conjugates have to be included, they cannot be reduced to
expressions without derivatives. Finally, there is no need to include both ∂++ and ∂−−
derivatives because of: 2i∂++∂−−Φ
i = D+D+∂−−Φ
i = D+
iΛ
a ∂Ea
∂Φi −2iΛa ∂Ja
∂Φi
. (3.8) (3.8) So, to summarize, we write the generating set explicitly: G =
∂n
−−Φi, ∂n
−−Φ
i, ∂n
++Φi, D+∂n
++Φi, ∂n
++Φ
i, D+∂n
++Φ
i, ∂n
−−Λa, ∂n
−−Λ
a, n ≥0
. (3.9) G =
∂n
−−Φi, ∂n
−−Φ
i, ∂n
++Φi, D+∂n
++Φi, ∂n
++Φ
i, D+∂n
++Φ
i, ∂n
−−Λa, ∂n
−−Λ
a, n ≥0
. (3.9)
To emphasize once again, we claim that: G =
∂n
−−Φi, ∂n
−−Φ
i, ∂n
++Φi, D+∂n
++Φi, ∂n
++Φ
i, D+∂n
++Φ
i, ∂n
−−Λa, ∂n
−−Λ
a, n ≥0
. (3.9) G =
∂n
−−Φi, ∂n
−−Φ
i, ∂n
++Φi, D+∂n
++Φi, ∂n
++Φ
i, D+∂n
++Φ
i, ∂n
−−Λa, ∂n
−−Λ
a, n ≥0
. (3.9)
To emphasize once again, we claim that: To emphasize once again, we claim that: To emphasize once again, we claim that: To emphasize once again, we claim that: bF0 ≃C[G]. (3.10) (3.10) Using the relations satisfied by the fields, it is not hard to describe the action of D+ in
terms of the generators in G. We have: Using the relations satisfied by the fields, it is not hard to describe the action of D+ in
terms of the generators in G. We have: D+(∂n
−−Φi) = 0,
D+(∂n
−−Φ
i) = ∂n−1
−−
iΛ
a ∂Ea
∂Φi −2iΛa ∂Ja
∂Φi
,
D+(∂n
++Φi) = 0,
D+(D+∂n
++Φi) = 2i∂n+1
++ Φi,
D+(∂n
++Φ
i) = D+∂n
++Φ
i,
D+(D+∂n
++Φ
i) = 0,
D+(∂n
−−Λa) = ∂n
−−Ea(Φ),
D+(∂n
−−Λ
a) = −2∂n
−−Ja(Φ). (3.11) D+(∂n
−−Φi) = 0,
D+(∂n
−−Φ
i) = ∂n−1
−−
iΛ
a ∂Ea
∂Φi −2iΛa ∂Ja
∂Φi
, (3.11) D+(∂n
−−Λ
a) = −2∂n
−−Ja(Φ). (3.11) D+(∂n
−−Λ
a) = −2∂n
−−Ja(Φ). (3.11) From these formulas we can guess that polynomials of ∂n
−−Φi should be in the cohomology. However, we need some extra assumptions about Ea and Ja in order to move further. – 16 – 3.1
Quasihomogeneous case As we have already learned, it is interesting to consider the case when the theory has an
R-symmetry. In such a case, we expect to have a stress-energy tensor in the cohomology. It is not hard to check that the following transformation: θ+ →e−iϵθ+,
Φi →e−iϵαiΦi,
Λa →e−iϵeαaΛa
(3.12) ,
Λa →e−iϵeαaΛa
(3.12) (3.12) is a symmetry of the classical action if and only if the following quasihomogeneity conditions
are satisfied: JHEP03(2018)079 is a symmetry of the classical action if and only if the following quasihomogeneity conditions
are satisfied: eαaJa +
X
i
αiΦi ∂Ja
∂Φi = Ja,
−eαaEa +
X
i
αiΦi ∂Ea
∂Φi = Ea,
(3.13) i
−eαaEa +
X
i
αiΦi ∂Ea
∂Φi = Ea,
(3.13) (3.13) where αi and eαa are real numbers. It is a matter of a standard calculation to find the real
conserved current j±± for this R-symmetry. It is then straightforward to write a superfield
which has it as the lowest component. The answer is: R++ = −i
2
X
i
αi
Φi∂++Φ
i −Φ
i∂++Φi
+ 1
2
X
i
(1 −αi)D+ΦiD+Φ
i
R−−= −i
2
X
i
αi
Φi∂−−Φ
i −Φ
i∂−−Φi
−
X
a
eαaΛaΛ
a. R−−= −i
2
X
i
αi
Φi∂−−Φ
i −Φ
i∂−−Φi
−
X
a
eαaΛaΛ
a. (3.14) (3.14) It is not a coincidence that we called it R. In fact, one can check that the equations
of motion imply ∂−−R++ + ∂++R−−= 0. Therefore, higher components of R are also
conserved currents. This is the supercurrent multiplet discussed before provided we can find
another real superfield Y−−−−(which has possibly improved stress-energy tensor T−−−−
as its lowest component) satisfying the required constraints. As one can check from (2.11),
the condition4 on Y−−−−is: D+Y−−−−= i
2D+∂−−R−−. (3.15) (3.15) Note that D+Y−−−−= −i
2D+∂−−R−−is then satisfied automatically. This defines Y−−−−
uniquely up to an arbitrary function of x−, as Y−−−−→Y−−−−+ f(x−) preserves the
above constraints. A simple computation allows to find a real superfield such that it satisfies
these constraints: Y−−−−=
X
i
h
∂−−Φi∂−−Φ
i −αi
4 ∂2
−−(Φ
iΦi)
i
+
X
a
i
2Λa∂−−Λ
a −i
2∂−−ΛaΛ
a
. (3.16) 4The fact that this Y−−−−together with Y++++ = i
4[D+, D+]J++ and Y++−−= i
4[D+, D+]J−−form
a conserved superspace current then follows automatically. 3.1
Quasihomogeneous case 4The fact that this Y−−−−together with Y++++ = i
4[D+, D+]J++ and Y++−−= i
4[D+, D+]J−−form
a conserved superspace current then follows automatically. – 17 – Therefore, we actually have a supercurrent multiplet described by R++, R−−and Y−−−−,
which is, moreover, an R-multiplet in the terminology of [20], as reviewed in section 2.2. The
Q+-cohomology class represented by the twisted stress-energy tensor eT−−−−from (2.24)
promotes to the D+-cohomology class represented by the superfield: eY = Y−−−−−i
2∂−−J−−
=
X
i
h
∂−−Φi∂−−Φ
i −αi
2 ∂−−(Φi∂−−Φ
i)
i
+
X
a
i
2Λa∂−−Λ
a −i
2∂−−ΛaΛ
a + ieαa
2 ∂−−(ΛaΛ
a)
. (3.17) (3.17) JHEP03(2018)079 This is precisely the stress-energy tensor in the cohomology as found in [11]. At first sight,
one could think that this is the end of the story. However, there are some subtleties here,
which we will now discuss. First of all, how is this R-multiplet related to the more general supercurrent multiplet
which we found in (3.6)? The answer is simple. If we also define V−= i
2D+R−−,
(3.18) (3.18) then R++, V−and Y−−−−form a supercurrent multiplet related to S++, W−and T−−−−
by the improvement transformation: then R++, V−and Y−−−−form a supercurrent multiplet related to S++, W−and T−−−−
by the improvement transformation: R++ = S++ + [D+, D+]U,
V−= W−+ ∂−−D+U,
Y−−−−= T−−−−+ ∂2
−−U,
U = −
X
i
αi
4 ΦiΦ
i. (3.19) U = −
X
i
αi
4 ΦiΦ
i. (3.19) (3.19) Note that the superfield U cannot be represented as a real part of some local chiral su-
perfield. Therefore this is an example of the improvement transformation allowed for the
general supercurrent multiplet but not allowed for the R-multiplet. As we will see mo-
mentarily, there might exist several R-multiplets which are not equivalent to each other as
R-mulitplets (cannot be related to each other by the R-multiplet improvements), but they
all are related to the same supercurrent multiplet S++, W−, T−−−−by the more general
improvement described above. So now we will discuss the possibility of having several inequivalent R-multiplets. Note
that the quasihomogeneity conditions (3.13) might have more than one solution. 3.1
Quasihomogeneous case (3.27) (3.27) – 19 – For the D+-closed element eY−−−−= Y−−−−−i
2J−−, we have: For the D+-closed element eY−−−−= Y−−−−−i
2J−−, we have: r the D+-closed element eY−−−−= Y−−−−−i
2J−−, we have: eY−−−−→eY−−−−−i
2∂−−(I−−+ ieI−−) = eY−−−−−i
2∂−−F−−. (3.28) (3.28) So the cohomology class [ eY−−−−] gets shifted by −i
2[∂−−F−−]. Let us summarize. We have the family of R-current multiplets generated by shifts
using the superspace current I±±. In the cohomology this corresponds to having an extra
left-moving U(1) current [F−−] generating an ambiguity of the stress-energy tensor in the
cohomology, as we can do shifts of the cohomology class [ eY−−−−] by [∂−−F−−]. JHEP03(2018)079 But the conformal theory to which our LG model flows in the IR supposedly should
have a unique stress-energy tensor, which thus gives a preferred stress-energy tensor for
the chiral algebra in the Q+-cohomology. One can ask a natural question: which of the
R-current multiplets above corresponds to the true stress-energy tensor of the theory in the
IR? The answer is simple: the correct stress-energy tensor is the one, for which the U(1)
current [F−−] is a primary operator in the cohomology, at least when it is possible to make
it primary (we will discuss this point later). It is clear that this corresponds to extrem-
izing the central charge of the corresponding Virasiro algebra (see the next subsection). To turn this statement into a criteria for picking the unique solution (αi, eαa) of (3.13),
we need to understand first how to compute the operator product expansions (OPE) in
the cohomology. 3.1
Quasihomogeneous case This
corresponds to having an extra flavor U(1) symmetry, which can then mix with the R-
symmetry to give another solution of (3.13) (in terms of current, this means to replace the
R-symmetry current jR by jR + jF , where jF is a Flavor symmetry current). The flavor symmetry does not rotate the thetas, so it acts just as: Φi →e−iϵqiΦi,
Λa →e−iϵeqaΛa. (3.20) Φi →e−iϵqiΦi,
Λa →e−iϵeqaΛa. (3.20) – 18 – The condition that this is a symmetry of the classical action is: The condition that this is a symmetry of the classical action is: eqaJa +
X
i
qiΦi ∂Ja
∂Φi = 0,
−eqaEa +
X
i
qiΦi ∂Ea
∂Φi = 0. (3.21) (3.21) We can see now that if {αi, eαa} is some solution of (3.13) and {qi, eqa} is some solution
of (3.21), then {αi+qi, eαa+eqa} is another solution of (3.13). This is actually the ambiguity
of the supercurrent multiplet which we were discussing before. In case we have extra
superspace currents, the basic supercurrent mutiplet S++, W−, T−−−−can be shifted. Let
us belabor this point somewhat further. JHEP03(2018)079 One can compute the current corresponding to the flavor symmetry (3.20) and find
the real superfield which contains it as the lowest component: I−−= −
X
a
eqaΛaΛ
a −i
2
X
i
qi
Φi∂−−Φ
i −Φ
i∂−−Φi
,
I++ = −1
2
X
i
qiD+ΦiD+Φ
i + i
2
X
i
qi
Φ
i∂++Φi −Φi∂++Φ
i
. (3.22) (3.22) On shell these are conserved at the level of superfields: On shell these are conserved at the level of superfields: ∂++I−−+ ∂−−I++ = 0. (3.23) (3.23) One can do a small computation to check that the following superfield: F−−= −
X
a
eqaΛaΛ
a −i
X
i
qiΦi∂−−Φ
i
(3.24) (3.24) is chiral on-shell, i.e. it satisfies D+F−−= 0 provided the equations of motion hold. In par-
ticular, it means that this F−−gives rise to the left-moving U(1) current in the cohomology. But it is also true that: Re F−−= I−−. (3.25) (3.25) Therefore, there exists a local expression for the superfield eI−−: eI−−= Im F−−= −1
2
X
i
qi∂−−(ΦiΦ
i). (3.26) (3.26) So, according to the general discussion from the section 2.2, we can shift the R-multiplet
using this I±±. Recall that the shift is: R++ →R++ + I++,
R−−→R−−+ I−−,
Y−−−−→Y−−−−+ 1
2∂−−eI−−. 3.1.1
The OPE in the cohomology The component action of the model that we study is: (3.29) S = SD + SF ,
(3.29) where the D-term action is: where the D-term action is: SD = 1
π
Z
d2x
−∂−−φ
i∂++φi −i
2ψ
i
+∂−−ψi
+ −iλ
a∂++λa −1
2GaG
a
+ i
2∂iEa(φ)λ
aψi
+ −i
2∂iE
a(φ)ψ
i
+λa + 1
2Ea(φ)E
a(φ)
,
(3.30) (3.30) and the F-term is: and the F-term is: SF = 1
π
Z
d2x
GaJa(φ) + G
aJa(φ) −iλaψi
+∂iJa(φ) −iλ
aψ
i
+∂iJa(φ)
. (3.31) (3.31) All couplings come from the E and J-type superpotentials. Note that φ is dimensionless in
2d, (and fermions are of dimension 1/2), therefore both Ea and Ja should have dimension 1. We will include an explicit coupling µ of dimension 1 in the theory, and replace Ea →µEa
and Ja →µJa in the above action, thinking of Ea(φ) and Ja(φ) as dimensionless functions
of dimensionless fields φi now. In section 2.2.3 we saw that in order to compute the OPE of the cohomology classes
we can turn offall dimensionful couplings in the theory. In particular, we can tune µ to – 20 – zero. This will remove all interactions from the above action. Thus to compute the OPE
of the cohomology classes, it is enough to consider the free theory: zero. This will remove all interactions from the above action. Thus to compute the OPE
of the cohomology classes, it is enough to consider the free theory: S0 = 1
π
Z
d2x
−∂−−φ
i∂++φi −i
2ψ
i
+∂−−ψi
+ −iλ
a∂++λa −1
2GaG
a
. (3.32) (3.32) Its correlators can be conveniently combined into superfield correlators: Its correlators can be conveniently combined into superfield correlators: D
Φ
i(x, θ′)Φj(y, θ)
E
= δij log
r−−r++
,
D
Λ
a(x, θ′)Λb(y, θ)
E
= δab
i
r−−,
(3.33) (3.33) JHEP03(2018)079 where r−−= x−−−y−−,
r++ = x++ −y++ + iθ+θ
+ + iθ′+θ
′+ + 2iθ
′+θ+. (3.34) (3.34) Now we want to compute the OPE of eY from (3.17) with itself. eY represented a candidate
stress-energy tensor in the cohomology and was given by: Now we want to compute the OPE of eY from (3.17) with itself. 3.1.1
The OPE in the cohomology (3.40) (3.40) In a generic situation, this equation allows to pick a unique solution (αi, eαa) and write a
correct stress-energy tensor. If the action admits f independent U(1) flavor symmetries
described by charges (qn
i , eqn
a), n = 1 . . . f, we should write the equation (3.40) for each
of them. Again, generically, one can expect this to give a condition to pick the unique
stress-energy tensor in the cohomology. In a generic situation, this equation allows to pick a unique solution (αi, eαa) and write a
correct stress-energy tensor. If the action admits f independent U(1) flavor symmetries
described by charges (qn
i , eqn
a), n = 1 . . . f, we should write the equation (3.40) for each
of them. Again, generically, one can expect this to give a condition to pick the unique
stress-energy tensor in the cohomology. JHEP03(2018)079 However, non-generic situations are possible, when this equation might either not fix
the stress-energy tensor completely, or might have no solutions at all. We will explore such
examples for the N = (2, 2) theories later: in such cases, indices a and i take the same
set of values, we have eαa = αi=a, and flavor symmetries (which should differ from the
N = (2, 2) R-symmetries) have eqa = qi=a. Therefore the equation (3.40) reduces to just
P
i qi = 0, which either holds identically and therefore imposes no constraints on αi, or
does not hold at all. In a former situation, the ambiguity of choosing the unique stress-
energy tensor is not removed and is just present in the IR. In a latter situation, there is
no solution to (3.40), which means that [F−−] cannot be made primary by choosing the
proper stress-energy tensor. There is an unwanted central term in the eYF−−OPE, which
cannot be removed and signals that there is an obstruction for the IR compact CFT to
exist. We will see in examples that there is a flat direction in the potential. This is usually
interpreted as a lack of normalizable vacuum. One can also note that if we decide to study the gauge theory obtained by gauging
the flavor symmetry with charges (qi, eqa), then the above equations become related to
anomalies. 3.1.1
The OPE in the cohomology Namely, the central term in the F−−F−−OPE becomes just the gauge anomaly
(so it is the t’Hooft anomaly in the LG model context): we need P
i q2
i −P
a eq2
a = 0 for the
gauge theory to exist [5]. Then equation (3.40) becomes the condition for the R-symmetry
defined by charges (αi, eαa) to be non-anomalous [11]. 3.1.1
The OPE in the cohomology eY represented a candidate
stress-energy tensor in the cohomology and was given by: eY =
X
i
h
1 −αi
2
∂−−Φi∂−−Φ
i −αi
2 Φi∂2
−−Φ
ii
+
X
a
i
2(1 + eαa)Λa∂−−Λ
a −i
2(1 −eαa)∂−−ΛaΛ
a
. (3.35) eY =
X
i
h
1 −αi
2
∂−−Φi∂−−Φ
i −αi
2 Φi∂2
−−Φ
ii +
X
a
i
2(1 + eαa)Λa∂−−Λ
a −i
2(1 −eαa)∂−−ΛaΛ
a
. (3.35) (3.35) Using the OPE above, we find that: Using the OPE above, we find that: eY(x) eY(y) ∼
c/2
(x−−−y−−)4 +
2 eY(y)
(x−−−y−−)2 +
∂−−eY(y)
x−−−y−−+ {Q+, . . . },
(3.36) (3.36) where the notation {Q+, . . . } for the unimportant term is slightly inaccurate: what we
actually mean is that the term that we drop becomes Q+-exact after we put θ+ = θ
+ = 0,
but as a shorthand we will denote it as {Q+, . . . }. The central term is: c =
X
i
(2 −6αi + 3α2
i ) +
X
a
(1 −3eα2
a). (3.37) (3.37) This matches the result of [11] and shows that we indeed have the stress-energy tensor in
the cohomology. Before we found that in case there is a U(1) flavor symmetry, there is another D+-
closed superfield F−−, which gives rise to the left-moving U(1) current in the cohomology. Recall that: F−−= −
X
a
eqaΛaΛ
a −i
X
i
qiΦi∂−−Φ
i. (3.38) (3.38) We can similarly compute its OPE: We can similarly compute its OPE: F−−(x)F−−(y) ∼
P
i q2
i −P
a eq2
a
(x−−−y−−)2 + {Q+, . . . }. (3.39) (3.39) This current creates ambiguity, as we explained before: we can replace eY by eY +
λ∂−−F−−for any λ ∈R and get another stress-energy tensor in the cohomology. The – 21 – unique one is picked by requiring that the [F−−] cohomology class be primary with respect
to the correct stress-energy tensor, whenever it is possible to impose such a condition. Equivalently, since shifting by the current shifts the central charge, one can ask that the
value of the central charge (3.37) be extremal with respect to the shifts (αi, eαa) →(αi +
λqi, eαa + λeqa). Any of these two criteria of course give the same equation:5 X
i
qi(1 −αi) +
X
a
eqaeαa = 0. 5While (3.37) is actually a left-moving central charge cL,
this is also equivalent to the cR-
extremization [23] since cL −cR is fixed by the gravitational anomaly. 3.1.2
Classical and quantum chiral algebra When we were discussing the OPE in the cohomology, we argued that, as a consequence
of conformal invariance, there should be no dimensionful couplings present in the OPE. We can generalize that further to say that the chiral algebra should not depend on any
dimensionful couplings at all. Any algebraic relations that involve dimensionful coupling
constants would violate the combination of scale and Lorentz invariance. One of the basic facts about theories we study is that they are free in the UV. In fact,
this provides an alternative argument for why the singular part of the OPE is independent – 22 – of couplings. Short-distance singularities of operators are simply governed by the free
theory, even before passing to the Q+ cohomology (however, we find the argument based
on Lorentz and scale invariance in the cohomology to be more transparent in our case). Independence of chiral algebras on dimensionful couplings implies a useful property,
which can be thought of as a sort of non-renormalization theorem. The exact quantum
chiral algebra in our theories is “almost determined” by the classical chiral algebra (and
might be called “almost tree level exact”, although this name could be misleading). All we
need to do to find the quantum counterpart is renormalize composite operators. Composite
operators can be thought of as several fundamental fields brought into one point, and in
the process we should subtract short-distance singularities. It might well happen (and
will happen in concrete examples later) that even though the classical operator is in Q+
cohomology, the infinite piece you have to subtract is not annihilated by Q+. In this
way, renormalization of composite operators representing classical cohomology classes can
remove part of the classical cohomology. The claim is that what you obtain using this
procedure is the exact answer. JHEP03(2018)079 To understand why this is true, we will think of an exact quantum theory as a set of
local operators, which satisfy OPE relations and operator equations of motion. As we said,
short-distance singularities are governed by the free theory, so singular part of the OPE
does not care about interactions and operator equations of motion. Non-singular part of
the OPE can be thought of as a definition of composite operators, and this is the point
where we should be careful, as already noted before. The remaining thing we need to care
about are operator equations of motion. 6In fact, the right hand side of (3.2) is already proportional to µ, so additional terms would be multiplied
by higher powers of µ. 3.1.2
Classical and quantum chiral algebra If we stare at classical equations (3.2), we can understand that they do not have any
short-distance singularities and can be made into operator equations. The question one
might ask is whether they receive any corrections at the quantum level. If there were such
corrections, they would be a result of interactions and would depend on the dimensionful
coupling6 µ. If this could change the answer for chiral algebra, it would mean that the
algebra depends on a dimensionful constant µ. We know that this is impossible on general
grounds, so we expect that quantum corrections to operator equations of motion are not
important for the chiral algebra computation. In fact, thinking slightly more general, the situation might be even simpler. Suppose we
have some renormalizable field theory, and we define it in the path integral approach. This
means that we choose our favorite regularization to make path integral finite-dimensional,
define the action and the measure in this regularization and add counterterms, if needed. Or, alternatively, think in terms of bare fields and couplings, without any counterterms. The standard way to derive equations of motion which hold under correlators, i.e., operator
equations of motion, is through integration by parts. For renormalizable field theories
defined in this way, these equations of motion hold exactly when written in terms bare
fields. If we write them in terms of physical fields and counterterms, then counterterms
of course contribute to equations of motion, but their role is to renormalize composite – 23 – operators that appear in equations of motion. This becomes very clear in the example of
the λφ4 theory. The equation of motion of the λφ4 with counterterms is: (□+ m2)φ = λφ3 + δmφ + δφ□φ + δλφ3,
(3.41) (3.41) and by some simple manipulations with diagrams, one can see that these three terms on
the right are precisely what one needs to define a composite operator λφ3. The mass
counterterm δmφ removes singularity coming from the self-contraction in φ3, while the
other two remove singularities coming from contractions between φ3 and one insertion of
the interaction vertex λφ4/4. It is quite obvious that this continues to higher orders of
perturbation theory, simply because the theory is renormalizable and has only these three
counterterms. JHEP03(2018)079 It is not completely clear how general this argument is and whether it holds for gauge
theories, but it definitely works for our LG models. 3.1.2
Classical and quantum chiral algebra Moreover, it is possible to show that
our models do not need any counterterms at all. So our conclusion is that equations (3.2) hold exactly once we properly define compos-
ite operators appearing there. This supports our claim that to compute quantum chiral
algebra, we need to find the classical one and then check which part of it survives after the
renormalization of composite operators. All these statements are true in perturbation theory. They might not hold if non-
perturbative corrections become relevant. For example, instantons might lift cohomology
classes [15], and this has to be studied separately. In our case we assume that the worldsheet
and the target are topologically trivial, so non-perturbative corrections are not expected. 3.1.3
Non-abelian global symmetries (3.44) (3.44) If we write [tα, tβ] = ifγ
αβtγ, then the OPE of these currents is given by: If we write [tα, tβ] = ifγ
αβtγ, then the OPE of these currents is given by: Jα(x)Jβ(y) ∼tr(tαtβ) −tr(τατβ)
(x−−−y−−)2
+
fγ
αβJγ(y)
x−−−y−−+ {Q+, . . . }. (3.45) (3.45) We have tr(tαtβ) = 2xΦδαβ and tr(τατβ) = 2xΛδαβ, where xΦ and xΛ are Dynkin indices
of the representations RΦ and RΛ respectively. Therefore, in the cohomology we find a
current algebra of G at the level r = 2(xΦ −xΛ). We have tr(tαtβ) = 2xΦδαβ and tr(τατβ) = 2xΛδαβ, where xΦ and xΛ are Dynkin indices
of the representations RΦ and RΛ respectively. Therefore, in the cohomology we find a
current algebra of G at the level r = 2(xΦ −xΛ). 3.1.3
Non-abelian global symmetries In addition to U(1) global symmetries, the action may also have non-abelian linearly
realized global symmetries that commute with SUSY. They generally are of the form
Φi →Ai
jΦj, Λa →Ba
b Λb. The kinetic part of the action implies that A ∈U(NΦ) and
B ∈U(NΛ), where NΦ is the number of chiral superfields Φi, i = 1 . . . NΦ, and NΛ is the
number of Fermi superfields Λa, a = 1 . . . NΛ. It is clear that by a unitary transformation Φi →U i
jΦj, Λa →V a
b Λb one can always
bring A and B into the diagonal form, and in such a basis they will describe just the U(1)
global symmetry. Therefore, in order to have something new compared to the previous
discussion, we assume that the action has some U(1) global symmetries and, on top of
that, also has some non-abelian symmetries. Altogether, they close to a subgroup G ⊂
U(NΦ) × U(NΛ). The free theory has the full U(NΦ) × U(NΛ) symmetry, which is then
broken to the subgroup G by the E and J superpotentials. Embedding G ⊂U(NΦ) × U(NΛ) defines an (NΦ + NΛ)-dimensional representation of
G on superfields of our model. This representation is reducible and can be decomposed
as a direct sum of an NΦ-dimensional representation RΦ on chiral superfields and an NΛ-
dimensional representation RΛ on Fermi superfields. Let the Hermitian generators of this
subgroup in the representation RΦ be called tα, α = 1 . . . |G|, and in the representation RΛ – 24 – — τα, α = 1 . . . |G|. The infinitesimal transformation is: — τα, α = 1 . . . |G|. The infinitesimal transformation is: Φi →Φi + iϵα(tα)i
jΦj,
Λa →Λa + iϵα(τα)a
bΛb. (3.42) (3.42) The condition on J and E for this to be a symmetry is: The condition on J and E for this to be a symmetry is: (tα)i
jΦj∂iJa(Φ) + (τα)b
aJb(Φ) = 0,
(tα)i
jΦj∂iEa(Φ) −(τα)a
bEb(Φ) = 0. (3.43) (3.43) JHEP03(2018)079 It is straightforward to repeat what we had done for abelian symmetries and to find the
corresponding element in the D+-cohomology: Jα = (τα)a
bΛbΛ
a + i(tα)i
jΦj∂−−Φ
i. 4
N = (2, 2) models If in a general N = (0, 2) LG model as described before we put Ea = 0, take a to be the
same sort of index as i, i.e., just put NΛ = NΦ (recall that everything is topologically trivial
in our discussion) and take Ja(Φ) = ∂W(Φ)
∂Φi=a for some holomorphic superpotential W(Φ), we
get a general N = (2, 2) LG model. In such a case (0, 2) superfields are promoted to (2, 2)
chiral superfields: Φi = Φi + i
√
2θ−Λi −iθ−θ
−∂−−Φi. (4.1) (4.1) With N = (2, 2) supersymmetry, we can go further in the discussion of general prop-
erties of the chiral algebra in the Q+-cohomology. First of all, let us get rid of the trivially
reducible case. Suppose that we can organize superfields Φi into two nonempty sets:
{Φ1, Φ2, . . . , Φs}, {Φs+1, Φs+2, . . . , ΦNΦ}, so that the superpotential can be written as
a sum: With N = (2, 2) supersymmetry, we can go further in the discussion of general prop-
erties of the chiral algebra in the Q+-cohomology. First of all, let us get rid of the trivially
reducible case. Suppose that we can organize superfields Φi into two nonempty sets:
{Φ1, Φ2, . . . , Φs}, {Φs+1, Φs+2, . . . , ΦNΦ}, so that the superpotential can be written as
a sum: W(Φ) = W (1)(Φ1, Φ2, . . . , Φs) + W (2)(Φs+1, Φs+2, . . . , ΦNΦ),
(4.2) (4.2) This superpotential just describes 2 separate LG models which do not interact with each
other. The space of observables in such a model is just the graded-symmetric tensor product
of the spaces for each of the two models, and the supercharge is the sum Q+ = Q
(1)
+ +Q
(2)
+ ,
where each term in the sum acts on the corresponding factor in the graded-symmetric
tensor product. It is a simple algebraic exercise to prove that the cohomology of such a
Q+ is just the graded-symmetric tensor product of the cohomologies of Q
(1)
+ and Q
(2)
+ . – 25 – Therefore, without any loss of generality, it is enough to study superpotentials which
cannot be decomposed as in (4.2), and can never be brought into such a decomposable
form by a holomorphic change of coordinates on the target. 4
N = (2, 2) models We will assume this from now
on. Note that it was shown in [8] that with such an assumption, no accidents happen in
the IR, which also simplifies life a lot. Since we put Ea = 0, quasihomogeneity conditions (3.13) now always have at least
one solution, eαa = 1, ∀a, αi = 0, ∀i. Therefore, according to our previous discussion, there
is always a stress-energy tensor in the cohomology. It is interesting, however, to study the
case when W(Φ) is quasi-homogeneous itself: JHEP03(2018)079 X
i
βiΦi ∂W
∂Φi = W(Φ). (4.3) (4.3) After all, as was noted in [24], this is the case most relevant for studying the IR fixed point
of the LG model. With this property, if we take αi = βi and eαa = βi=a, we get another
solution of (3.13). In other words, there exists a U(1) flavor symmetry corresponding to
the solution qi = βi, eqa = βi=a −1 of (3.21). If there is only one such flavor symmetry, we can see that the equation (3.40) picks
αi = βi and eαa = βi=a as defining the correct stress-energy tensor. Indeed, these values
satisfy (3.40), while another solution, eαa = 1, αi = 0, inserted in (3.40), gives P
i qi +
P
a eqa = P
i(2βi −1), which is generically non-zero. The last sum being zero corresponds
to various degenerate cases, for example if superpotential is just a quadratic polynomial
(which means that all fields are massive, the IR theory is trivial and the chiral algebra
should be trivial too). We will not concentrate on such cases. On the other hand, there can be more flavor symmetries in the model: Φi →e−iγiϵΦi,
(4.4) (4.4) if one can find such a system of charges γi that: if one can find such a system of charges γi that: if one can find such a system of charges γi that: X
i
γiΦi ∂W
∂Φi = 0. (4.5) (4.5) This gives a solution qi = γi, eqa = γi=a of (3.21). Note that both the solution qi = βi,
eqa = βi=a −1 and the solution qi = γi, eqa = γi=a describe flavor symmetries from the
N = (0, 2) point of view, since they just satisfy (3.21). 4
N = (2, 2) models However, from the N = (2, 2)
point of view, only the latter one is a flavor symmetry, while the former one becomes the
left-handed R-symmetry of the N = (2, 2) SUSY, which is seen from the fact that Φ’s and
Λ’s charges differ by one. The action of the LG model in the (2, 2) superspace is: S = 1
4π
Z
d2xd4θ Φ
iΦi + 1
4π
Z
d2xd2θ W(Φ) + 1
4π
Z
d2xd2θ W(Φ)
(4.6) (4.6) The superspace equations of motion are simply: The superspace equations of motion are simply: D+D−Φ
i = ∂W
∂Φi . (4.7) D+D−Φ
i = ∂W
∂Φi . (4.7) – 26 – – 26 – As was first noted in [10], we can find an element in the D+-cohomology represented by
the (2, 2) superfield: J =
X
i
1 −βi
2
D−ΦiD−Φ
i −iβiΦi∂−−Φ
i
,
(4.8) (4.8) which can then be expanded in components with respect to θ−and θ
−: the lowest com-
ponent is the left-handed R-current, the top component is the stress-energy tensor (which,
using our earlier N = (0, 2) terminology, corresponds to the solution αi = βi, eαa = βi=a
of (3.13)), and the fermionic components are the two left-handed supersymmetries. There-
fore, this J generates a left-moving N = 2 superconformal algebra in the D+-cohomology. If there exist additional U(1) flavor symmetries characterized by weights γi satisfy-
ing (4.5), then there is another D+-cohomology class represented by: JHEP03(2018)079 If there exist additional U(1) flavor symmetries characterized by weights γi satisfy
ing (4.5), then there is another D+-cohomology class represented by: Ψ = 1
2
X
i
γiΦiD−Φ
i,
(4.9) (4.9) 7The argument is as follows. Presence of the central term in (4.17) implies through the operator-state
correspondence that there is a state |ψ⟩in the IR CFT such that G−
+1/2|ψ⟩= κ|0⟩+ Q+|φ⟩, where G−
+1/2 is
one of the superconformal generators, |0⟩is the vacuum state and |φ⟩is some state. Taking the dimension-
zero component of this equality, we can assume that ψ has dimensions (1/2, 0), so that G−
+1/2|ψ⟩has
dimension zero. Since in a unitary theory there are no operators of negative dimension, Q+|φ⟩should not
be there: G−
+1/2|ψ⟩= κ|0⟩. Invariance of the vacuum implies G+
−1/2G−
+1/2|ψ⟩= 0. Since in a unitary
theory (G−
+1/2)† = G+
−1/2, by multiplying with ⟨ψ|, the last equality implies G−
+1/2|ψ⟩= 0, which gives a
contradiction unless κ = 0. so the derivative: (4.15) θ−
12 = θ−
1 −θ−
2 ,
θ
−
12 = θ
−
1 −θ
−
2 , θ12 = θ1 −θ2 ,
θ12 = θ1 −θ2 ,
r12 = x−−
1
−x−−
2
+ iθ
−
1 θ−
2 −iθ
−
2 θ−
1 ,
(4.14) r12 = x−−
1
−x−−
2
+ iθ
−
1 θ−
2 −iθ
−
2 θ−
1 ,
(4.14) (4.14) and the central charge is: c = 3
X
i
(1 −2βi). (4.15) (4.15) Equation (4.13) encodes the N = 2 superconformal algebra with the central charge c (this
equation, but in slightly different conventions, was present in [10]). Of course we could
have obtained the same value of the central charge using the more general equation (3.37),
which holds for more general N = (0, 2) LG models. One would have to put αi = βi,
eαa = βi=a there. JHEP03(2018)079 Notice that the central charge (4.15) is linear in βi. This means that if we have
U(1) flavor symmetries such that (4.5) holds, we can no longer get rid of the ambiguity
βi →βi + λγi by simply asking the central charge to take the extremal value. This is
related to the fact that the OPE of the cohomology class represented by (4.9) with itself
is regular: Ψ(x1, θ1)Ψ(x2, θ2) ∼{Q+, . . . }. (4.16) (4.16) that the OPE of [Ψ] with [J ] is the same as with [J +λD−Ψ], ∀λ ∈R. The J Ψ OPE is: J (x1, θ1)Ψ(x2, θ2) ∼κ θ−
12
(r12)2 −θ−
12θ
−
12
(r12)2 Ψ(x2, θ2) −iθ−
12
r12
D−Ψ(x2, θ2)
−2θ−
12θ
−
12
r12
∂−−Ψ(x2, θ2) −
i
r12
Ψ(x2, θ2) + {Q+, . . . },
(4.17) (4.17) where κ = P
i γi. Compare this with what one expects for the OPE of J with some
superconformal primary superfield P: where κ = P
i γi. Compare this with what one expects for the OPE of J with some
superconformal primary superfield P: J (x1, θ1)P(x2, θ2) ∼−2θ−
12θ
−
12
(r12)2 ∆P(x2, θ2) −iθ−
12
r12
D−P(x2, θ2)
−iθ
−
12
r12
D−P(x2, θ2) −2θ−
12θ
−
12
r12
∂−−P(x2, θ2)
−
i
r12
qP(x2, θ2) + {Q+, . . . },
(4.18) (4.18) where ∆is the conformal dimension of P and q is its R-charge. so the derivative: so the derivative: so the derivative: D−Ψ =
X
i
γi
1
2D−ΦiD−Φ
i + iΦi∂−−Φ
i
(4.10) (4.10) generates ambiguity, because we can replace J →J + λD−Ψ, ∀λ ∈R. Of course, this
is still the same ambiguity of the N = (0, 2) stress-tensor multiplet related to U(1) flavor
symmetries that we were discussing before. The only difference is that by now we have dealt
with the U(1) global symmetry which is the left-handed R-symmetry from the N = (2, 2)
point of view (it was described by the charges qi = βi, eqa = βi=a −1), and what we are left
with in (4.9) corresponds to the actual N = (2, 2) flavor symmetry. Similar to what we
had for a more general N = (0, 2) case, we could have analyzed this ambiguity using the
N = (2, 2) supersurrent multiplet, especially since its structure is described in details in
the appendix C of [20]. However, we chose not to do this, as it would not give us anything
essentially new compared to what we have already understood. Previous discussion of the OPE in the cohomology being determined by the free prop-
agators of course still holds. The free propagator of chiral superfields is: D
Φ
i(x1, θ1)Φj(x2, θ2)
E
= δij log
R−−
12 R++
12
,
(4.11) (4.11) where where R−−
12 = x−−
1
−x−−
2
+ iθ−
1 θ
−
1 + iθ−
2 θ
−
2 + 2iθ
−
1 θ−
2 ,
R++
12 = x++
1
−x++
2
+ iθ+
1 θ
+
1 + iθ+
2 θ
+
2 + 2iθ
+
1 θ+
2 . (4.12) (4.12) We can compute the OPEs: We can compute the OPEs: J (x1, θ1)J (x2, θ2) ∼−
c
3(r12)2 −2θ−
12θ
−
12
(r12)2 J (x2, θ2) −iθ−
12
r12
D−J (x2, θ2)
−iθ
−
12
r12
D−J (x2, θ2) −2θ−
12θ
−
12
r12
∂−−J (x2, θ2) + {Q+, . . . },
(4.13) – 27 – – 27 – where θ−
12 = θ−
1 −θ−
2 ,
θ
−
12 = θ
−
1 −θ
−
2 ,
r12 = x−−
1
−x−−
2
+ iθ
−
1 θ−
2 −iθ
−
2 θ−
1 ,
(4.14)
c = 3
X
i
(1 −2βi). so the derivative: What we see is that for
non-zero values of κ, Ψ is non-primary, and moreover it is not a descendant of any primary,
as can be seen from unitarity and global superconformal invariance of the vacuum of the
IR theory.7 Therefore, the non-zero κ becomes an obstruction for the IR CFT to exist. where ∆is the conformal dimension of P and q is its R-charge. What we see is that for
non-zero values of κ, Ψ is non-primary, and moreover it is not a descendant of any primary,
as can be seen from unitarity and global superconformal invariance of the vacuum of the
IR theory.7 Therefore, the non-zero κ becomes an obstruction for the IR CFT to exist. 7The argument is as follows. Presence of the central term in (4.17) implies through the operator-state
correspondence that there is a state |ψ⟩in the IR CFT such that G−
+1/2|ψ⟩= κ|0⟩+ Q+|φ⟩, where G−
+1/2 is
one of the superconformal generators, |0⟩is the vacuum state and |φ⟩is some state. Taking the dimension-
zero component of this equality, we can assume that ψ has dimensions (1/2, 0), so that G−
+1/2|ψ⟩has
dimension zero. Since in a unitary theory there are no operators of negative dimension, Q+|φ⟩should not
be there: G−
+1/2|ψ⟩= κ|0⟩. Invariance of the vacuum implies G+
−1/2G−
+1/2|ψ⟩= 0. Since in a unitary
theory (G−
+1/2)† = G+
−1/2, by multiplying with ⟨ψ|, the last equality implies G−
+1/2|ψ⟩= 0, which gives a
contradiction unless κ = 0. – 28 – This happens for example in a model with two superfields X and Y and superpotential
W = XY 2. Notice that since the ΨΨ OPE is regular, so is the D−ΨD−Ψ OPE. The absence of
central term in it means, as we have mentioned in section 3.1.1, that the corresponding
flavor symmetry can be gauged without encountering gauge anomalies. Possible non-zero
value of κ then becomes the anomaly for the right-handed R-symmetry. This would be
relevant if we were studying gauge theories. The theory can also have non-abelian flavor symmetries, which lead, as we have argued
before, to the current algebra in the cohomology. In our discussion of general (0, 2) theories,
the level of this current algebra was given by the difference of Dynkin indices: r = 2xΦ−2xΛ. The first term here corresponded to the way flavor symmetry acted on Φ’s, and the second
— on Λ’s. 5
Examples In this section we will consider a few examples of applications of our machinery to the
N = (2, 2) LG models, where we can say something about the chiral algebra and therefore
draw some conclusions about the theory to which the model flows in the IR. so the derivative: In the (2, 2)-supersymmetric case, the flavor symmetry acts in the same way on
Φ’s and Λ’s, as they are just components of the (2, 2) chiral superfields Φi. So xΦ = xΛ. We conclude that the current algebra in the cohomology corresponding to some flavor
symmetry of the N = (2, 2) supersymmetric LG model always has level zero. JHEP03(2018)079 5.1
Degenerate examples Consider the theory of two chiral superfields X and Y with superpotential W = XY 2. (5.1) (5.1) This theory has a non-trivial flavor symmetry. A possible charge assignment is: γX = 2,
γY = −1, so that This theory has a non-trivial flavor symmetry. A possible charge assignment is: γX = 2,
γY = −1, so that γY = −1, so that γXX ∂W
∂X + γY Y ∂W
∂Y = 0. (5.2) (5.2) As we know from the equation (4.9), there is an extra operator Ψ in the cohomology as a
result of this flavor symmetry. Since γX + γY = 1 ̸= 0, the OPE (4.17) tells us that this
operator is not primary. Moreover, as we explained in the Footnote 7, an operator satisfy-
ing (4.17) cannot be made primary in a unitary CFT with invariant vacuum. Therefore,
its existence indicates that the RG flow does not end at any compact CFT: the deep IR
theory does not have a normalizable vacuum. In fact, the superpotential has a flat direction
Y = 0, and we can conclude that the low-energy mode describing propagation along this
flat direction renders vacuum non-normalizable. One can get a compact conformal fixed
point if we add a perturbation ϵX2n+1 to the superpotential. This actually corresponds
to having the D series of minimal models at the IR fixed point, with the exact choice of
the model depending on n, even for small ϵ. By sending ϵ →0, the compact IR fixed
point will most likely go to infinity, signaling that the degenerate theory W = XY 2 will – 29 – behave differently. It would be great to have a better understanding of such non-compact
IR theories. This flavor symmetry could be gauged, however, as we have noted before, this would
make right-handed R-symmetry anomalous because of γX + γY ̸= 0. By considering a slightly different superpotential, namely: By considering a slightly different superpotential, namely: W = X2Y 2,
(5.3) (5.3) we get again a theory with flavor symmetry, but the charges now can be chosen as γX = 1,
γY = −1, so that γX + γY = 0. Therefore, the bad central term does not appear in (4.17),
and the theory should have a better IR limit, even though the superpotential still has flat
directions. 5.2
N = 2 minimal models A series of N = (2, 2) LG models are known to flow in the IR to the N = (2, 2) minimal
models. These superconformal theories are relatively simple. The central charge is given
by [25–29]:
3k A series of N = (2, 2) LG models are known to flow in the IR to the N = (2, 2) minimal
models. These superconformal theories are relatively simple. The central charge is given
by [25–29]: c =
3k
k + 2, k ≥1,
(5.4) (5.4) and there is a known spectrum of possible superconformal primaries. The A-D-E classifi-
cation of modular-invariant theories is known [30–33], and the corresponding LG superpo-
tentials have been identified before. So, we can try to compute the chiral algebra both for
the LG model and for the minimal model which is supposed to arise in the IR, therefore
providing more evidence for this relation, as well as demonstrating the power of chiral
algebras. 5.1
Degenerate examples For example, if we gauge this flavor symmetry, the flat directions are removed
and we still get a theory with the right-handed R-symmetry. We are not going to study
this example any further. JHEP03(2018)079 5.2.1
The Ak+1 series For a given k, the diagonal Ak+1 minimal model is the simplest one. Its set of primaries
has a subset of k + 1 fields which are chiral primary with respect to ]
SVir. Let us call them
Os, s = 0, . . . k, where O0 = 1 is the identity operator and Os has left-right conformal
dimensions (h, h) = (
s
2(k+2),
s
2(k+2)) and left-right U(1) charges (q, q) = (
s
k+2,
s
k+2). As we
see, they all are chiral primaries with respect to both SVir and ]
SVir. Therefore, together
with the N = 2 currents, they generate the chiral algebra of the theory, as well as the
anti-chiral algebra abtained analogously by taking the cohomology of Q−. We expect to get the same result from the LG model description. It is obtained by
considering only one chiral superfield Φ with the superpotential: W(Φ) = Φk+2
k + 2. (5.5) (5.5) The equations of motion are D+D−Φ −Φk+1 = 0,
D−D+Φ −Φ
k+1 = 0. (5.6) (5.6) – 30 – – 30 – Differentiating these equations and multiplying them by arbitrary polynomials of Φ, Φ and
their derivatives, we get a differential ideal I. The algebra bF0 consists of arbitrary poly-
nomials of variables ∂n
+∂m
−Dk
+Dp
−Φ and ∂n
+∂m
−D
k
+D
p
−Φ for non-negative integers n, m, k, p,
modulo the ideal I: Differentiating these equations and multiplying them by arbitrary polynomials of Φ, Φ and
their derivatives, we get a differential ideal I. The algebra bF0 consists of arbitrary poly-
nomials of variables ∂n
+∂m
−Dk
+Dp
−Φ and ∂n
+∂m
−D
k
+D
p
−Φ for non-negative integers n, m, k, p,
modulo the ideal I: bF0 = C[. . . , ∂n
+∂m
−Dk
+Dp
−Φ, ∂n
+∂m
−D
k
+D
p
−Φ, . . . ]/I. (5.7) (5.7) It is not hard to find another set of generators, which will generate
bF0 as a super-
commutative polynomial algebra itself. We already explained it in the context of general
N = (0, 2) LG models. Namely, we can take: It is not hard to find another set of generators, which will generate
bF0 as a super-
commutative polynomial algebra itself. We already explained it in the context of general
N = (0, 2) LG models. 5.2.1
The Ak+1 series Namely, we can take: G = {∂n
−−Φ, ∂n
++Φ, D−∂n
−−Φ, D+∂n
++Φ, ∂n
−−Φ, ∂n
++Φ, D−∂n
−−Φ, D+∂n
++Φ, n ≥0}. (5.8) JHEP03(2018)079 All other derivatives of elementary superfields Φ and Φ can be expressed, using equations
of motion, as polynomials of these generators, and moreover, there are no further algebraic
relations between these generators. So we have: All other derivatives of elementary superfields Φ and Φ can be expressed, using equations
of motion, as polynomials of these generators, and moreover, there are no further algebraic
relations between these generators. So we have: bF0 ≃C[G]. (5.9) (5.9) We will first compute the classical cohomology of D+ acting on this space. After that we
will check which part of it survives at the quantum level, when we take care to subtract
singular parts from composite operators. It is clear that the cohomology classes can only
be destroyed by this subtraction. Indeed, suppose we define: We will first compute the classical cohomology of D+ acting on this space. After that we
will check which part of it survives at the quantum level, when we take care to subtract
singular parts from composite operators. It is clear that the cohomology classes can only
be destroyed by this subtraction. Indeed, suppose we define: : AB : (z) = lim
ϵ→0 (A(z + ϵ)B(z) −(singular in ϵ)) . (5.10) (5.10) If AB was classically in the cohomology but the singular part is not D+-closed, the operator
: AB : is no longer in the cohomology. If AB was not in the cohomology even classically,
then neither is : AB :, which is quite obvious. Finally, if AB was classically D+-exact,
then there is no need to consider : AB :. Even if the singular part represented some non-
trivial quantum cohomology class, we would find it by starting with some other classical
cohomology class anyways. So, we will look for the classical cohomology first, and then
check which part of it survives subtraction of singularities.8 If AB was classically in the cohomology but the singular part is not D+-closed, the operator
: AB : is no longer in the cohomology. If AB was not in the cohomology even classically,
then neither is : AB :, which is quite obvious. Finally, if AB was classically D+-exact,
then there is no need to consider : AB :. 5.2.1
The Ak+1 series Even if the singular part represented some non-
trivial quantum cohomology class, we would find it by starting with some other classical
cohomology class anyways. So, we will look for the classical cohomology first, and then
check which part of it survives subtraction of singularities.8 To find how D+ acts on bF0 in terms of the generators, we act with D+ on the generators
from the set G and, using the equations of motion, express the result in terms of these
generators again. To explicitly describe D+, it is convenient to write it as a sum: D+ = d0 + d1,
(5.11) (5.11) D+ = d0 + d1, where d0 acts as follows: d0 :
∂n
−−Φ 7→0,
∂n
++Φ 7→0,
D−∂n
−−Φ 7→0,
∂n+1
−−Φ 7→0,
D+∂n
++Φ 7→0,
D−∂n
−−Φ 7→0,
D+∂n
++Φ 7→2i∂n+1
++ Φ,
∂n
++Φ 7→D+∂n
++Φ,
(5.12) d0 :
∂n
−−Φ 7→0,
∂n
++Φ 7→0,
D−∂n
−−Φ 7→0,
∂n+1
−−Φ 7→0,
D+∂n
++Φ 7→0,
D−∂n
−−Φ 7→0,
D+∂n
++Φ 7→2i∂n+1
++ Φ,
∂n
++Φ 7→D+∂n
++Φ,
(5.12) (5.12) 8In fact, computation of the classical cohomology is a hard combinatorial problem, while we are really
only interested in quantum cohomology. So we will not determine the classical cohomology completely, only
partly. As we will see, there is an N = 2 super-Virasoro algebra in cohomology, so our approach will be
to look for those classical cohomology classes which have a chance to be superconformal primaries at the
quantum level. – 31 – and d1 acts as: and d1 acts as: d1 :
∂n
−−Φ 7→0,
∂n
++Φ 7→0,
D−∂n
−−Φ 7→0,
D+∂n
++Φ 7→0,
∂n
++Φ 7→0,
D+∂n
++Φ 7→0,
D−∂n
−−Φ 7→∂n
−−(Φk+1),
∂n+1
−−Φ 7→i
2D−∂n
−−(Φk+1). (5.13 (5.13) This explicitly describes how D+ acts on the generators, and then extends to the full
algebra bF0 by linearity and Leibniz rule. Notice that d0 is just the D+ in the theory
with zero superpotential, while d1 includes corrections due to the superpotential. This
splitting of D+ is motivated by a perturbative computation of the D+-cohommology, i.e.,
the spectral sequence, which we are about to perform. JHEP03(2018)079 Let us introduce a filtration degree on bF0 by saying that for generators: ∀x ∈G, fdeg(x) = 1,
(5.14) (5.14) which then extends multiplicatively on the whole bF0. 5.2.1
The Ak+1 series We then define: bF(p)
0
= {S ∈bF0 : fdeg(S) ≥p},
(5.15) bF(p)
0
= {S ∈bF0 : fdeg(S) ≥p}, (5.15) which gives a filtration: bF0 ∼= bF(0)
0
⊃bF(1)
0
⊃bF(2)
0
⊃. . . (5.16) (5.16) Our differential D+ obviously preserves this filtration. In particular, d0 does not change
the filtration degree, while d1 increases it by k, if k > 0. This allows us to apply spectral
sequences to compute the cohomology of D+. But before that we will mention a trivial
technical lemma we will need later. Lemma 1. Let V be a Z2-graded vector space and S(V ) = ⊕k≥0Sk(V ) be the graded-
symmetric algebra of V . If there is a degree-1 differential d : V →V , i.e., d2 = 0,
then by the Leibniz rule it extends to a differential acting on the graded-symmetric algebra
d : S(V ) →S(V ), and moreover, its cohomology is: H(S(V ), d) = S (H(V, d)) . (5.17) (5.17) Now, having this lemma, we will proceed to compute the cohomology of D+. Now, having this lemma, we will proceed to compute the cohomology of D+. First let us consider the trivial case k = 0. Then both d0 and d1 do not change the
filtration degree. We can define a vector space spanned by the elements of G: V = Span(G). Since D+ = d0 + d1 does not change the filtration degree, it acts as a linear operator on
this V . Next we notice that bF0 ≃S(V ), so by the lemma H( bF0, D+) = S
H(V, D+)
. To compute the cohomology of D+ acting as a linear operator on V , we notice that all
elements of G are either not D+-closed or are D+-exact as a consequence of the equation of
motion D+D−Φ = Φ. So the cohomology is trivial for k = 0 (stress-energy supercurrent
J becomes D+-exact for k = 0 ad well). This could be expected because the k = 0 model
is massive, and therefore the IR theory it flows to is empty. – 32 – > 0. Then at the zeroth order of spectral sequence we have: Now, suppose k > 0. 5.2.1
The Ak+1 series Then at the zeroth order of spectral sequence we have: Ep
0 = bF(p)
0 / bF(p+1)
0
,
E0 ≡Gr( bF0) ∼=
M
p≥0
Ep
0,
(5.18) (5.18) where Gr( bF0) is the graded space associated with the filtered space bF0, and the differential
acting on it is just d0, which preserves grading. We note that E1
0 ≃Span(G), the vector
space spanned by the generators from G (which all have degree 1). Since d0 preserves
grading and, as one can easily see, E0 ≃S(E1
0), we just apply lemma and get H(E0, d0) =
S
H(E1
0, d0)
. By inspecting equations (5.12), we easily find the cohomology of d0 acting
on E1
0. The answer is H(E1
0, d0) = Span(S0), where the set S0 is: JHEP03(2018)079 S0 = {∂n
−−Φ, D−∂n
−−Φ, ∂n+1
−−Φ, D−∂n
−−Φ, n ≥0}. (5.19) (5.19) Therefore, we find the first term of the spectral sequence: Therefore, we find the first term of the spectral sequence: E1 = H(E0, d0) ≃C[S0]. (5.20) (5.20) Now, if k = 1, then for the first step of spectral sequence, d1 becomes the differential
acting on E1. If k > 1, then the differential acting on E1 is just zero, and E2 = H(E1, 0) ≃
E1. Next, if k > 2, we find that E3 ≃E1, and so on. This procedure goes on until we get
to the k-th term of spectral sequence: Ek ≃E1. As we know from spectral sequences, the
differential acting on Ek should be the degree-k part of D+, i.e., d1. So for the next term
we have: Ek+1 ≃H(E1, d1). (5.21) (5.21) Since there are no components of D+ of degree higher than k, the spectral sequence collapses
here and we conclude that: Since there are no components of D+ of degree higher than k, the spectral sequence collapses
here and we conclude that: H( bF0, D+) ≃H(E1, d1) ≃H(C[S0], d1). (5.22) (5.22) So all we need to do now is compute the cohomology of d1 acting on C[S0]. The way d1
acts on the elements of S0 is: So all we need to do now is compute the cohomology of d1 acting on C[S0]. The way d1
acts on the elements of S0 is: ∂n
−−Φ 7→0,
D−∂n
−−Φ 7→0,
∂n+1
−−Φ 7→i
2D−∂n
−−(Φk+1),
D−∂n
−−Φ 7→∂n
−−(Φk+1). 5.2.1
The Ak+1 series (5.23) d1 : (5.23) Even though we have considerably simplified the original problem, the direct computation
of the d1 cohomology is still too nasty. We can simplify it more by recalling that we already
have a stress-energy supercurrent in the cohomology, and therefore it is enough to look for
its superconformal primaries only. Our superpotential is of a quasi-homogeneous class,
with β =
1
k+2, so the stress-energy supercurrent is: J =
k + 1
2(k + 2)D−ΦD−Φ −
i
k + 2Φ∂−−Φ
(5.24) (5.24) and the corresponding central charge is c =
3k
k+2. Now suppose we found some polyno-
mial P ∈C[S0] which represents a D+-cohomology class. We have the following techni-
cal lemma: – 33 – – 33 – Lemma 2. Every d1-cohomology class [P] which is a superconformal primary with respect
to J , can be represented as a polynomial of Φ, D−Φ, D−Φ and ∂−−Φ, that is P ∈
C[Φ, D−Φ, D−Φ, ∂−−Φ]. Lemma 2. Every d1-cohomology class [P] which is a superconformal primary with respect
to J , can be represented as a polynomial of Φ, D−Φ, D−Φ and ∂−−Φ, that is P ∈
C[Φ, D−Φ, D−Φ, ∂−−Φ]. Lemma 2. Every d1-cohomology class [P] which is a superconformal primary with respect
to J , can be represented as a polynomial of Φ, D−Φ, D−Φ and ∂−−Φ, that is P ∈
C[Φ, D−Φ, D−Φ, ∂−−Φ]. The idea is that having higher derivatives of Φ and Φ in the expression for P will result
in higher poles in the J (x, θ)P(0, 0) OPE, which should not be there if [P] is primary. Elegant proof of this statement is not available at the moment, but calculations seem to
show that it is true, so we leave it as a conjecture. The operators Φ, Φ2, . . . Φk are all in the cohomology and are primaries — we will
write their OPE’s with J later. Φk+1 is exact and so is not in the cohomology, so any
polynomial of Φ is just a linear combination of 1, Φ, Φ2, . . . , Φk in the cohomology. Since
D−(P(Φ)) = P ′(Φ)D−Φ and (D−Φ)2 = 0, any polynomial of Φ and D−Φ is A(Φ) +
D−B(Φ), where the second term is a descendant. Let us figure out now if there are any
other primaries in the cohomology. 5.2.1
The Ak+1 series We try to construct d1-closed (or equivalently, D+-
closed) polynomials from Φ, D−Φ, D−Φ and ∂−−Φ, which are not just polynomials of Φ
and D−Φ. A simple computation shows that the most general such combination with even
statistics is: JHEP03(2018)079 E =
∞
X
n=0
Pn(Φ)(∂−−Φ)n
(n + 1)(k + 1)
2
D−ΦD−Φ −iΦ∂−−Φ
,
(5.25) (5.25) where Pn are arbitrary polynomials, while the most general odd closed element is: O =
∞
X
n=1
Cn(Φ)(∂−−Φ)nD−Φ,
(5.26) (5.26) where again Cn are arbitrary polynomials. where again Cn are arbitrary polynomials. To slightly simplify computations, we notice that since the operator d1 increases the
introduced above filtration degree fdeg by k, one can grade the cohomology by this degree,
and it is enough to assume that E has a given fixed degree (i.e., it is a homogeneous
polynomial). Next, we notice that we could introduce another grading — by the number of
derivatives in the expression. If we assign the bosonic derivative ∂−−a “derivative degree”
1 and the fermionic derivatives D−and D−a “derivative degree” 1/2, we can see that
the operator d1 actually lowers the “derivative degree” by 1/2. Therefore, again, we can
grade the cohomology by this degree, and it is enough to study the cohomology within the
sector with a given “derivative degree”. Fixing values of these two degrees — the filtration
degree and the “derivative degree” — we see that it is enough, without loss of generality,
to consider: Es,n = Φs(∂−−Φ)n
(n + 1)(k + 1)
2
D−ΦD−Φ −iΦ∂−−Φ
,
Os,n = Φs(∂−−Φ)nD−Φ. (5.27) (5.27) where s and n are non-negative integers. A simple calculation gives: D−Es,n = −i [s + 1 + (n + 1)(k + 1)] Os,n+1. (5.28) (5.28) – 34 – This suggests that any odd element of the above form Os,n+1 that we could have possibly
found in the cohomology would always by a descendant of some even element. This is
also true for Os,0 = ΦsD−Φ =
1
s+1D−Φs+1. Therefore, it is enough to study the expres-
sion Es,n given above. Can it represent a nontrivial cohomology class, and can it be a
superconformal primary? Observables Es,n and their lifting. Notice that for s ≥k: bservables Es,n and their lifting. Notice that for s ≥k: Observables Es,n and their lifting. 5.2.1
The Ak+1 series Careful computation of [Q+, e1], followed by taking the
ϵ →0 limit, shows that: [Q+, e1] = −(k + 1)
(k + 1)φk∂−−ψ−−1
2ψ−∂−−φk
−i(k + 2)[Q+, ∂2
−−φ]. (5.37) (5.37) JHEP03(2018)079 So indeed, e1 is not in quantum cohomology. We know that classical observables should
be lifted from cohomology in pairs. Therefore, the combination we got on the right, r1 =
(k +1)φk∂−−ψ−−1
2ψ−∂−−φk, should be some classical cohomology class which disappears
together with e1. And indeed, it is in the classical cohomology, as it is easy to check. Before, we found classical cohomology classes which had a chance of being superconformal
primaries, and this r1 was not among them, which suggests that it should be a descendant. Another computation shows that it is indeed a descendant. The lowest component of J is: j = J | =
k + 1
2(k + 2)ψ−ψ−−
i
k + 2φ∂−−φ,
(5.38) (5.38) it is a U(1) current in the N = 2 super-Virasoro. A computation shows that: j−1(φkψ−) =: jφkψ−:=
i
k + 2
(k + 1)φk∂−−ψ−−ψ−∂−−φk
+ [Q+, . . . ]. (5.39) (5.39) So this new operator, r1 = (k + 1)φk∂−−ψ−−1
2ψ−∂−−φk, is actually a superconformal
descendant of φkψ−. One can ask a similar question: what is this φkψ−? Clearly, it is in
the classical cohomology. But in fact, φkψ−=
1
k+1[Q−, φk+1], and recall that we have a
relation φk+1 = 0 in the classical cohomology. Therefore φkψ−also vanishes in the classical
cohomology. So we have discovered the following: classically, we have cohomology classes e1
and r1, but quantum-mechanically, we have [Q+, e1] = r1. And this r1 is a superconformal
descendant of φkψ−, which is actually zero in the classical cohomology. So this new operator, r1 = (k + 1)φk∂−−ψ−−1
2ψ−∂−−φk, is actually a superconformal
descendant of φkψ−. One can ask a similar question: what is this φkψ−? Clearly, it is in
the classical cohomology. But in fact, φkψ−=
1
k+1[Q−, φk+1], and recall that we have a
relation φk+1 = 0 in the classical cohomology. Therefore φkψ−also vanishes in the classical
cohomology. So we have discovered the following: classically, we have cohomology classes e1
and r1, but quantum-mechanically, we have [Q+, e1] = r1. And this r1 is a superconformal
descendant of φkψ−, which is actually zero in the classical cohomology. 5.2.1
The Ak+1 series Notice that for s ≥k: d1
h
Φs−k(∂−−Φ)n+1D−Φ
i
= iEs,n,
(5.29) (5.29) JHEP03(2018)079 so Es,n is exact for s ≥k. On the other hand, for s < k, Es,n is obviously not exact,
because, as we can see from the equation (5.13), the image of d1 always contains the field
Φ at least k + 1 times, while Es,n contains it s + 1 times. So we conclude that Es,n for
s < k indeed represents a non-trivial classical cohomology class. Classical observables Es,n satisfy the following multiplication rule: Es,nEt,m = −iEs+t+1,n+m+1. (5.30) (5.30) They can be combined with the observables Φs, for which we have: ΦsEt,n = Es+t,n. (5.31) (5.31) We see that Φ and Es,n generate a closed sector in the classical cohomology. As we will find
soon, these are not all classical cohomology classes, there exist more. But all observables
that have a chance of being superconformal primaries in the quantum cohomology are
within this sector. The stress-energy supercurrent J that we identified before is of course among these
observables:
1 J =
1
k + 2E0,0. (5.32) (5.32) In particular: J Es,n = −
i
k + 2Es+1,n+1. (5.33) (5.33) This equation implies that the only observables which have a chance of being superconfor-
mal primaries at the quantum level are Es,0 and E0,n. But because of: This equation implies that the only observables which have a chance of being superconfor-
mal primaries at the quantum level are Es,0 and E0,n. But because of: Es,0 = (k + 2)J Φs,
(5.34) (5.34) the former are simply descendants of Φs. So we only have E0,n left. Can E0,n represent cohomology classes in quantum theory? It turns out that only for
n = 0. The reason is that for n > 0, the infinite piece that one has to subtract in order to
define the composite operator E0,n is not Q+-closed. Consider the simplest operator E0,1. We call its lowest component e1: e1 = (k + 1)∂−−φψ−ψ−−iφ(∂−−φ)2. (5.35) (5.35) – 35 – – 35 – This is a composite operator whose precise definition requires subtraction of singularities: e1(x) = lim
ϵ→0
(k + 1)∂−−φ(x)ψ−(x)ψ−(x −ϵ) −i(∂−−φ(x))2φ(x −ϵ) −2ki
ϵ−−∂−−φ(x)
. (5.36)
(5.36) We see that the piece that we subtract is not Q+-closed, which already suggests that e1(x)
is probably not in the cohomology. 5.2.1
The Ak+1 series This might look confusing — how is it possible that a superconformal descendant of
zero is not zero? The resolution of this apparent paradox is that, actually, super-Virasoro
algebra does not act in the classical cohomology. It only acts in the quantum cohomology
by the OPE with the stress-energy supercurrent J , while there is no notion of OPE in
the classical cohomology. Therefore, there is no contradiction between the facts that φkψ−
vanishes in the classical cohomology, while its superconformal descendant r1 does not vanish
classically. The fact that latter is a descendant of the former is borrowed from the chiral
algebra in the quantum cohomology. And in the quantum cohomology, because of this
relation, both of them indeed have to vanish. This is quite satisfactory, because it also
explains why r1 should be lifted from the classical cohomology — because it vanishes in
quantum chiral algebra! In fact, by taking all possible superconformal descendants of the relation φk = 0,
we will get a lot of (probably, infinitely many) operators which vanish in the quantum – 36 – cohomology but represent non-vanishing classical cohomology classes. They all should be
lifted from the cohomology through the mechanism which we have just described. Also, it is not hard to convince oneself that not only E0,1, but all operators E0,n,
n > 0 get lifted from the cohomology at quantum level for the same reasons. Clearly,
there is some interesting (or at least non-trivial) mathematical structure in how classical
cohomology classes get paired and lifted from the cohomology. It is quite possible that
our observables Es,n and superconformal descendants of φk+1 are not the only classical
cohomology classes involved in this. However, we are not going to study this question here. We are only interested in the quantum cohomology here, so the conclusion we need
now is that the only primary operators in the cohomology are 1, Φ, Φ2, . . . , Φk. They,
together with the stress-energy supercurrent J , generate the full chiral algebra in the
Q+-cohomology. One can find that: JHEP03(2018)079 J (x1, θ1)Φs(x2, θ2) ∼−
2θ−
12θ
−
12
(r12)2 hs + iθ−
12
r12
D−+ 2θ−
12θ
−
12
r12
∂−−+
i
r12
qs
! Φs,
(5.40) (5.40) where hs = qs
2 =
s
2(k+2). We see that dimensions and charges match exactly our expecta-
tions for the Ak+1 minimal model. 5.2.2
D and E series of minimal models However, we have an extra primary operator of dimension 1: which are just the relations of the chiral ring, so we get the familiar result (explained in
section 2.3) that operators from the chiral ring of the N = (2, 2) theory are primaries of
the chiral algebra. However, we have an extra primary operator of dimension 1: JHEP03(2018)079 P = JV −JU,
(5.46) (5.46) which is not part of the chiral ring. The existence of this extra primary current in the
cohomology was already noticed in [34], where the author also conjectured that every
D2n+2 model has, in addition to the generators of the chiral ring, a single dimension-n
primary in the cohomology. which is not part of the chiral ring. The existence of this extra primary current in the
cohomology was already noticed in [34], where the author also conjectured that every
D2n+2 model has, in addition to the generators of the chiral ring, a single dimension-n
primary in the cohomology. We are not going to study n > 1 cases here. The only thing we want to mention is that
the spectral sequence approach we used for the Ak+1 models can be clearly generalized to
the D2n+2 case. For n > 1, the operator D+ will split as a sum of three terms: D+ = d0 + d1 + d2,
(5.47) (5.47) where d0 corresponds to the zero superpotential, d1 takes into account the effect of XY 2
term in the superpotential, and d2 encodes the effect of X2n+1 interaction. It should be
possible, though more technical than in the Ak+1 case, to compute the cohomology using
this splitting and check the conjecture made in [34]. where d0 corresponds to the zero superpotential, d1 takes into account the effect of XY 2
term in the superpotential, and d2 encodes the effect of X2n+1 interaction. It should be
possible, though more technical than in the Ak+1 case, to compute the cohomology using
this splitting and check the conjecture made in [34]. Finally, a small remark about the E series. The models E6 and E8 correspond to
superpotentials X3 + Y 4 and X3 + Y 5. Therefore, their chiral algebras are immediately
identified as those of A2 ⊗A3 and A2 ⊗A4 respectively. Therefore, they will also contain
extra primary operators, in addition to the chiral ring elements. 5.2.2
D and E series of minimal models We will not go into much details about the chiral algebras of D and E series of minimal
models. Instead we will just look at some of their features, leaving a more detailed study
for the future. The LG models which are expected to flow to D2n+2 minimal models in the IR are
described by the superpotential: W = XY 2 + X2n+1
2n + 1. (5.41) (5.41) Consider the n = 1 theory. It has W = XY 2 + X3
3 . If we make a change of variables V = X + Y
√
2
,
U = X −Y
√
2
,
(5.42) (5.42) We will get an LG model with W =
√
2
3 V 3 +
√
2
3 U 3. This is just a pair of non-interacting
A2 models. Thus the theory in the IR is expected to be just A2 ⊗A2, with the chiral
algebra being a tensor product as well. Recall from the previous subsection that, for the
W ∝V 3, the chiral algebra has only two primaries: the identity 1 and V , and there is
also a stress-energy supercurrent JV . Similarly for the second one: we have 1 and U as
primaries, and we have JU. By taking the tensor product of these two, we can identify
primaries in the chiral algebra of A2 ⊗A2 as: 1, V, U, V U, JV −JU. (5.43) (5.43) – 37 – Going back to X amd Y , the first four are simply: Going back to X amd Y , the first four are simply: 1, X, Y, X2 −Y 2. (5.44) (5.44) Moreover, since in the cohomology V 2 = U 2 = 0, these X and Y satisfy relations in the
chiral algebra: Moreover, since in the cohomology V 2 = U 2 = 0, these X and Y satisfy relations in the
chiral algebra: X2 + Y 2 = 0,
XY = 0,
(5.45) XY = 0,
(5.45) (5.45) which are just the relations of the chiral ring, so we get the familiar result (explained in
section 2.3) that operators from the chiral ring of the N = (2, 2) theory are primaries of
the chiral algebra. 6
Discussions and further directions We have only scratched the surface of the subject, demonstrating some general properties of
chiral algebras of N = (0, 2) theories and giving several simple examples. The most general
property was the RG invariance of the answer, which makes chiral algebras interesting
objects to study in the context of dualities. One obvious extension of this work would be to get a better description of chiral
algebras of N = (2, 2) LG models with quasi-homogeneous (or even general) polynomial
superpotentials. Our treatment allowed us to find answers in some cases, but it would
be much nicer to have a more general result, which would associate chiral algebra to any
polynomial superpotential. It would also be useful to find some classes of N = (0, 2) models
in which the chiral algebra could be described completely. JHEP03(2018)079 But the most interesting and immediate extension is, of course, the application of
chiral algebras to gauge theories. If the LG model has some flavor symmetry, one can
gauge it by coupling to gauge multiplets. One can argue that perturbatively, the way this
gauging is implemented in the chiral algebra is as follows. If G is the gauge group, one
should first take the G-invariant subalgebra of the ungauged chiral algebra, then tensor
multiply it by the “small” bc-system of dimension (1, 0) (where “small” means that zero
mode of c is excluded from the algebra). The ungauged chiral algebra has a current in it
which corresponds to the flavor symmetry we want to gauge. Using this current and the
bc-ghosts, one can construct a BRST operator. The condition of its nilpotency is precisely
the condition that there is no gauge anomaly, i.e., that the symmetry we want to gauge
really can be gauged. Then we have to compute the cohomology of this BRST operator. The answer is the gauged chiral algebra. (This procedure is identical to the gauging of
chiral algebras of 4D N = 2 theories from [35].) This procedure seems to hold in perturbation theory. One way to argue it is by
writing equations of motion of the gauge theory and, similar to what we did in this paper,
computing the cohomology of D+ using perturbation theory (or spectral sequence) in gauge
coupling. This approach is somewhat ugly, but it allows to argue that the answer is as we
claimed above. 5.2.2
D and E series of minimal models The E7 model has: W = X3 + XY 3,
(5.48) (5.48) therefore it has to be studied separately. In this case again we will have: D+ = d0 + d1 + d2,
(5.49) (5.49) where d0 is a D+ operator in the theory of two free chiral superfields without any super-
potential, d1 takes into account the X3 term and d2 takes care of XY 3. It is clear that at
the second step of the spectral sequence computation, when we consider the cohomology
of d1, we will essentially get the cohomology of the A2 model multiplied by the free theory
described by the chiral superfield Y . Computing the cohomology of d2 at the next step
then becomes much simpler, since we already know the answer for A2. – 38 – 6
Discussions and further directions Another, more conceptual proof would be to define the gauge theory using
the BRST formalism and the holomorphic gauge v++ = 0. This would give the action: S = S0 + {QB, Ψ} = S0 +
Z
d2x lAvA
++ +
Z
d2x bAD++cA,
(6.1) (6.1) where lA is the auxiliary field implementing gauge vA
++ = 0, and we added Faddeev-Popov
ghosts. One can extend supersymmetry to act trivially on ghosts. Then the supercharge Q+
and the BRST charge QB anticommute: {Q+, QB} = 0, and we really have two commuting
complexes. The theory is defined as the cohomology of QB, and within that cohomology
we want to find the chiral algebra in the cohomology of Q+. Since the complexes commute,
we could first find the cohomology of Q+, and then compute the cohomology of QB. It is
quite nice to discover that the gauging procedure we explained above arises in this way. However, some technical details still have to be clarified. A question of utmost importance is to understand how the gauging procedure should
be modified to account for non-perturbative effects, such as instantons. – 39 – Another extension, which is also important for gauge theories, is to study models
without R-symmetry. We can easily find gauge theories with anomalous R-symmetry. In case they are constructed by gauging some LG models that have (right-handed) R-
symmetry, it becomes natural to ask what happens to their chiral algebra during gauging
that manifests the breaking of R-symmetry. Acknowledgments The author would like to thank E. Witten for helpful discussions, as well as B. Le Floch,
I. Melnikov and E. Sharpe for useful comments on the draft. JHEP03(2018)079 Open Access. This article is distributed under the terms of the Creative Commons
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https://openalex.org/W2083606760
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https://researchonline.lshtm.ac.uk/id/eprint/7839/1/1475-2875-7-6.pdf
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English
| null |
Feasibility and acceptability of artemisinin-based combination therapy for the home management of malaria in four African sites
|
Malaria journal
| 2,008
|
cc-by
| 7,396
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Received: 22 October 2007
Accepted: 8 January 2008 Received: 22 October 2007
Accepted: 8 January 2008 Published: 8 January 2008 Malaria Journal 2008, 7:6
doi:10.1186/1475-2875-7-6 Malaria Journal 2008, 7:6
doi:10.1186/1475-2875-7-6 This article is available from: http://www.malariajournal.com/content/7/1/6 © 2008 Ajayi et al; licensee BioMed Central Ltd. j y
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. BioMed Central BioMed Central BioMed Central Malaria Journal Open Access Research Open Acc
Research
Feasibility and acceptability of artemisinin-based combination
therapy for the home management of malaria in four African sites
Ikeoluwapo O Ajayi*1, Edmund N Browne2, Bertha Garshong3,
Fred Bateganya4, Bidemi Yusuf1, Peter Agyei-Baffour2, Leticia Doamekpor3,
Andrew Balyeku4, Kaendi Munguti5, Simon Cousens6 and Franco Pagnoni7 p
Research
Feasibility and acceptability of artemisinin-based combination
therapy for the home management of malaria in four African sites
Ikeoluwapo O Ajayi*1, Edmund N Browne2, Bertha Garshong3,
Fred Bateganya4, Bidemi Yusuf1, Peter Agyei-Baffour2, Leticia Doamekpor3,
Andrew Balyeku4, Kaendi Munguti5, Simon Cousens6 and Franco Pagnoni7 Address: 1Malaria Research Laboratories, Institute of Medical Research and Training, College of Medicine, University of Ibadan, Nigeria,
2Department of Community Health, School of Medical Sciences, KNUST, Kumasi, Ghana, 3Health Research Unit, Ghana Health Service, Accra,
Ghana, 4Department of Sociology, Makerere University, Kampala, Uganda, 5Institute for Development Studies, College of Humanities and Social
Studies, University of Nairobi, Nairobi, Kenya, 6Infectious Diseases Epidemiology Unit, London School of Hygiene and Tropical Medicine,
London, UK and 7Implementation Research & Methods Unit, UNICEF/UNDP/World Bank/WHO Special Programme for Research and Training
in Tropical Diseases (TDR), Geneva, Switzerland Email: Ikeoluwapo O Ajayi* - ikeajayi2003@yahoo.com; Edmund N Browne - enlbrowne@yahoo.com;
Bertha Garshong - Bertha.Garshong@hru-ghs.org; Fred Bateganya - fredb@ss.mak.ac.ug; Bidemi Yusuf - bidemi_yusuf@yahoo.com; Pete Email: Ikeoluwapo O Ajayi* - ikeajayi2003@yahoo.com; Edmund N Browne - enlbrowne@yahoo.com;
Bertha Garshong - Bertha.Garshong@hru-ghs.org; Fred Bateganya - fredb@ss.mak.ac.ug; Bidemi Yusuf - bidemi_yusuf@yahoo.com; Peter Agyei-
Baffour - agyeibaffour@yahoo.co.uk; Leticia Doamekpor - leticia.Doamekpor@hru-ghs.org; Andrew Balyeku - abalyeku@gmail.com;
Kaendi Munguti - kaendim@iconnect.co.ke; Simon Cousens - Simon.Cousens@lshtm.ac.uk; Franco Pagnoni - pagnonif@who.int
* Corresponding author * Corresponding author Received: 22 October 2007
Accepted: 8 January 2008 Published: 8 January 2008
Malaria Journal 2008, 7:6
doi:10.1186/1475-2875-7-6 Abstract Background: The Home Management of Malaria (HMM) strategy was developed using chloroquine, a now obsolete drug, which
has been replaced by artemisinin-based combination therapy (ACT) in health facility settings. Incorporation of ACT in HMM
would greatly expand access to effective antimalarial therapy by the populations living in underserved areas in malaria endemic
countries. The feasibility and acceptability of incorporating ACT in HMM needs to be evaluated. Methods: A multi-country study was performed in four district-size sites in Ghana (two sites), Nigeria and Uganda, with
populations ranging between 38,000 and 60,000. Community medicine distributors (CMDs) were trained in each village to
dispense pre-packaged ACT to febrile children aged 6–59 months, after exclusion of danger signs. A community mobilization
campaign accompanied the programme. Artesunate-amodiaquine (AA) was used in Ghana and artemether-lumefantrine (AL) in
Nigeria and Uganda. Harmonized qualitative and quantitative data collection methods were used to evaluate CMD performance,
caregiver adherence and treatment coverage of febrile children with ACTs obtained from CMDs. Results: Some 20,000 fever episodes in young children were treated with ACT by CMDs across the four study sites. Cross-
sectional surveys identified 2,190 children with fever in the two preceding weeks, of whom 1,289 (59%) were reported to have
received ACT from a CMD. Coverage varied from 52% in Nigeria to 75% in Ho District, Ghana. Coverage rates did not appear
to vary greatly with the age of the child or with the educational level of the caregiver. A very high proportion of children were
reported to have received the first dose on the day of onset or the next day in all four sites (range 86–97%, average 90%). The
proportion of children correctly treated in terms of dose and duration was also high (range 74–97%, average 85%). Overall, the
proportion of febrile children who received prompt treatment and the correct dose for the assigned duration of treatment
ranged from 71% to 87% (average 77%). Almost all caregivers perceived ACT to be effective, and no severe adverse events were
reported. Conclusion: ACTs can be successfully integrated into the HMM strategy. Page 1 of 9
(page number not for citation purposes) http://www.malariajournal.com/content/7/1/6 Malaria Journal 2008, 7:6 Study design
Th
d The study was conducted in three phases. The first phase
involved advocacy, community mobilization, selection of
CMDs and establishment of key baseline indicators. Infor-
mation, Education and Communication (IEC) materials
were developed and research staff recruited and trained. The intervention phase involved the training of CMDs
and distribution of ACT. A year after commencement of
ACT distribution, a household survey, interviewing eligi-
ble caregivers about child fevers in the past two weeks, was
performed to evaluate the intervention. So far, 18 African countries have adopted HMM as part of
their malaria control programme [4]. Due to unacceptable
levels of resistance to chloroquine by Plasmodium falci-
parum, 44 countries in Africa have adopted artemisinin-
based combination therapy (ACT) as the first line treat-
ment for uncomplicated malaria [5]. However, in most
cases, the deployment of ACT is currently limited to
health facilities, with large scale implementation of HMM
using ACT delayed by concerns about the use of ACT at the
community level [6-8]. Background and in Ho district, an urban and a rural site were studied. In Nigeria, two districts Badeku and Ojoku/Ajia in Ona-
ara local government were studied. The sites in Ejisu-Jua-
ben, Ghana; Ona-ara Local Government, Nigeria; and
Buguri and Iganga districts, Uganda were all rural areas. Ghana and Nigeria are located in West Africa, while
Uganda is in East Africa. Table 1 describes the location
and population characteristics of the different study sites,
details of the community-based medicine distributors
(CMDs) deployed, their training and supervision proc-
esses, and type, dosing schedule and cost to the end user
of the antimalarial drug used. g
Home Management of Malaria (HMM) for children with
uncomplicated malaria in high transmission areas in
Africa is an integral part of malaria case management
within the overall Roll Back Malaria (RBM) strategy [1]. If
the Abuja target of 60% of uncomplicated malaria epi-
sodes receiving effective treatment within 24 hours [2]
and the Millennium Development Targets 5, 8 and 17
(reducing childhood mortality, halting the increase in
malaria incidence and providing access to affordable
essential drugs in developing countries) [3] are to be met,
there is an urgent need to increase access to effective
malaria treatment at the community level, especially in
underserved rural areas. Data collection methods A pilot study carried out in Ghana in 2004 [9] provided
preliminary evidence for the feasibility and acceptability
of the use of artemether-lumefantrine (AL) in the HMM
context. However, this study was essentially a qualitative
assessment of community acceptability. To provide addi-
tional evidence to support the use of ACT at the commu-
nity level, the UNICEF-UNDP-WORLD BANK-WHO
Special Programme for Research and Training in Tropical
Diseases (TDR) funded four studies in sites in Ghana,
Nigeria and Uganda, representing different health system
and epidemiological settings. This paper reports on the
feasibility, acceptability and utilization of ACT provided
at the community level. Qualitative and quantitative data collection methods were
used. Data collection tools for all the sites were harmo-
nized prior to the intervention with some country specific
modifications. The tools included the survey question-
naire and focus group discussion (FGD) and key inform-
ant interview (KII) guides, which were pre-tested and
translated into the local languages. The format of CMDs'
registers was also harmonized. The household survey
focused on the health-seeking behaviour of caregivers of
children with fever in the preceding two weeks, with an
emphasis on timeliness of treatment, compliance with the
prescribed treatment course, and perceptions of effective-
ness and drug related adverse events. The FGD and KII
guides focused on health-seeking behaviours, beliefs
about the aetiology of malaria, treatment practices and
community perceptions of the intervention. Sample size A major concern with using ACT at the community level
is the potential for poor adherence to the treatment sched-
ule by both caregivers and community medicine distribu-
tors (CMDs). This could facilitate the development of
parasite resistance to these expensive but currently highly
efficacious drugs. Other concerns relate to acceptability by
the community, the incidence of adverse events, cost and
ability to provide adequate storage conditions to ensure
drug stability in the community. It was decided that to yield useful results in terms of feasi-
bility, the study should cover populations with a mini-
mum population of around 40,000 – the size of a typical
district. The sample size for the household survey was cal-
culated using country-specific fever prevalence rates to
provide a precision of ± 5% for the estimates of coverage
of ACT treatment through CMDs, assuming a design effect
of two. The minimum number of households to be inter-
viewed ranged from 700 to 768. Study sites and population This multi-country study was performed in Ghana (two
sites), Nigeria and Uganda all in sub-Saharan Africa. In
Ghana two districts, Ejisu-Juaben and Ho were studied Page 2 of 9
(page number not for citation purposes) http://www.malariajournal.com/content/7/1/6 Malaria Journal 2008, 7:6 Table 1: Description of study sites and intervention implementation in four sites evaluating home management of malaria with
artemisinin combination therapy
Ejisu – Juaben District, Ghana
Ho District, Ghana
Badeku and Ojoku/Ajia
Districts, Nigeria
Bugiri and Iganga Districts,
Uganda
Population
characteristics
35 rural communities
Population c. 38,000
Perennial malaria
transmission
49 urban & rural communities
Population c. 42,000
Perennial malaria
transmission
40 rural communities
Population 43,000
Perennial, hyperendemic
malaria transmission
56 rural communities
Population c. 60,000
Perennial, hyperendemic
transmission
Community medicine
distributors
54 (51 male) trusted
members of community
(farmers, teachers, drug
sellers), chosen by the
community
76 (48 male) trusted
members of community
(farmers, teachers, drug
sellers), chosen by the
community
60 (4 male) including drug
sellers, health workers and
mothers chosen by the
communities
118 (62 male) pre-existing
cadre of CMDs (chosen by
the community)
5 days of training
2 days of training
2 days of training
2 days of training
Provided with bicycles, boots
& $3.50 monthly
Provided with T-shirts,
watches, raincoats, torches &
US$8 quarterly
Received commission of
20–30 US cents per pack
distributed. Provided with T-
shirts, transport
reimbursement, Festivity gift,
and certificates. Transport refund of USD
1.16 per meeting. T-shirts,
baseball caps, certificates
Bimonthly supervision by
research staff; Monthly
supervision by community
Health Officers
Monthly supervision by health
staff and quarterly meetings
Monthly supervision by
research staff
Monthly supervision by health
staff
CMDs were not obliged to follow up caregivers. However,
some of them followed up and some gave drugs under direct
observation. No active follow-up of
caregivers by CMDs. Program ran within existing
home management system. Study sites and population CMDs actively followed up
treated children to establish
treatment outcome
Drugs and dosing
schedule
Artesunate+Amodiaquine (ASAQ) once daily for 3 days
2 types of blister pack, for children < 1 year and children 1
year and above
Artemether-lumefantrine (Coartem®) twice daily for 3 days
2 types of blister pack, for children < 3 years and children 3
years and above
10 US cents for <1 year
20 US cents for 1 year and above
20 US cents for < 3 years
30 US cents for 3 years and
above Charged for first 6
months only, thereafter free
Free Table 1: Description of study sites and intervention implementation in four sites evaluating home manag
artemisinin combination therapy Bimonthly supervision by
research staff; Monthly
supervision by community
Health Officers
Monthly supervision by health
staff and quarterly meetings
CMDs were not obliged to follow up caregivers. However,
some of them followed up and some gave drugs under direct
observation. No active follow-up of
caregivers by CMDs. Program ran within existing
home management system. CMDs actively followed up
treated children to establish
treatment outcome
Artemether-lumefantrine (Coartem®) twice daily for 3 days
2 types of blister pack, for children < 3 years and children 3
years and above
20 US cents for < 3 years
30 US cents for 3 years and
above Charged for first 6
months only, thereafter free
Free Artesunate+Amodiaquine (ASAQ) once daily for 3 days
2 types of blister pack, for children < 1 year and children 1
year and above
10 US cents for <1 year
20 US cents for 1 year and above Community medicine distributors participation. Quarterly monetary allowances varied
between USD 4.5 (Uganda), USD 5 (Ejisu-Juaben) and
USD 8 (Ho). In Nigeria, the CMDs received a commission
of between USD 0.20 and USD 0.30 for each pack of AL
dispensed in addition to periodic gifts and reimburse-
ment of transportation fares. Community medicine distributors were trusted members
of the community who were chosen by the community
from a range of backgrounds, including farmers, chemi-
cal/medicine sellers, teachers, traders, community health
workers, artisans, 'mother trainers', and opinion leaders
(Table 1). In Ghana and Nigeria nurses and community
health officers at first-level formal health facilities were
counted as CMDs. 'Mother trainers' were lay mothers who
were selected from within the communities and trained to
distribute drugs. Drugs and dosing
schedule Ethics Ethical approval for conduct of this study was obtained
from the WHO Ethics Review Committee and at national
level from the appropriate Ethical Review Boards. Informed consent was obtained from community heads,
household heads and the caregivers who participated in
the study. Supervision Monthly supervision was undertaken in all sites, by health
staff or by the research team (Table 1). In each site, super-
vision included checking drug stocks, their storage condi-
tions, and the CMDs' registers. The research team
provided fuel and travel allowances to supervisors. In
Nigeria, unscheduled inspections were periodically
undertaken. Utilization of CMDs Based on the records maintained by the CMDs, some
20,000 fever episodes in young children were treated with
ACTs by CMDs across the four study sites (Table 2). The
total number of episodes treated varied considerably
between sites as did the average number of episodes
treated per CMD (from 17 per CMD in Nigeria to 93 in
Uganda). The higher number of episodes treated in
Uganda may be explained by the fact that home-based
management of fevers has been implemented there since
2001. A higher attrition rate of CMDs in Nigeria com-
pared to other study sites may also have contributed to the
lower figures recorded there. Slightly more girls (4,928)
than boys (4,595) were treated across the three sites in
which this information was recorded. In this study, the
accurate population denominators were not available. However, taking into account the width of the age groups,
utilization appeared to decline somewhat with age (from
3,571 children in the 6-month age band from 6–11
months to 6,627 children in the two year age band from
36–59 months). Utilization was generally prompt with at
least half of all children presenting on the day of onset or Drugs used in the study Two different ACTs were used in accordance with the
national drug policies of the participating countries; AL
was used in Uganda and Nigeria, and artesunate-amodi-
aquine (AS/AQ) was used in Ghana. Selection criteria for CMDs included being a permanent
resident (at least one year), trusted and respected by the
community, able to keep simple records, and a willing-
ness to serve. Uganda had a functioning pre-existing cadre
of CMDs who were co-opted into the study. The number
of CMDs per community depended on the community's
population size. An average of two CMDs per community
was used across the four sites (one CMD per 600 popula-
tion). For the AS/AQ combination, blister packs containing co-
packed artesunate and amodiaquine tablets of 50 and 153
mgs respectively were used. For children aged 12 to 59
months the recommended dose of AS/AQ was one tablet
of each drug once per day. However, for children of six to
11 months of age, tablets had to be broken in half and
repackaged to comply with the recommended AS/AQ
dose in that age group. Although CMDs were not paid a salary, various motiva-
tion mechanisms were used. Some of these included rain
coats, bicycles, boots, watches, T-shirts and certificates of In Nigeria and Uganda, a fixed combination of 20 mg of
artemether and 120 mg of lumefantrine (AL) in two types
of blister pack, one for children below three years of age Page 3 of 9
(page number not for citation purposes) http://www.malariajournal.com/content/7/1/6 http://www.malariajournal.com/content/7/1/6 Malaria Journal 2008, 7:6 notes, the responses to questions asked to explore issues
of interest were grouped together, coded, categorized and
analysed according to emerging themes. The findings were
interpreted and reported in the form of narratives. and another for children three years and above, were used. The recommended dose for children aged six to 35
months was one tablet twice daily for three days, and for
children aged 36 to 59 months two tablets twice daily for
three days. Caregivers were advised to administer the drug
after meals, preferably fatty food. Drug supply
I
ll h
i In all the sites, drugs were provided through existing pub-
lic health structures, at health district or sub-district level. In general, the distribution points for drugs were the local
health facilities. In Uganda, CMDs replenished their stock
at monthly meetings at health facility level. In Nigeria and
Ghana, CMDs replenished their stocks from the health
facilities as the need arose. In some instances drugs were
delivered by the research team or by health staff during
supervisory visits. A young woman in Nigeria said: within 24 hours of onset in all four sites. In the three sites
which recorded promptness as "same day" or "next day"
73–90% of children presented on the day of onset or the
next day. 'CMDs are always available. It is good we have two in our
community, when one is out the village the other one
attends to caregivers'. Data analysis EPI info version 6.02, SPSS version 11.0 and STATA ver-
sion 9.2 were used by the four sites to enter and analyse
data. A set of indicators and quantitative analyses to be
performed were agreed in discussion with research staff
from each site. Each research team then undertook the
analysis of their own data using standard statistical meth-
ods. The FGD and KII information was transcribed and
content analysis performed. From the transcripts and field Page 4 of 9
(page number not for citation purposes)
Table 2: Utilization of CMDs for treatment of children with fever in 4 sites (source: CMD registers)
Ejisu – Juaben
District, Ghana
Ho District,
Ghana
Badeku and Ojoku/Ajia
Districts, Nigeria
Bugiri and Iganga
Districts, Uganda
Totals
Number of CMDs
54
76
60
118
308
Total number febrile episodes in children less
than 5 treated with ACTs by CMDs
4522
3958
1044
11039
20563
Number (%) treated by age
6–11 months
789 (17%)
474 (12%)
99 (9%)
2209 (20%)
3571 (17%)
12–23 months
1531 (34%)
1157 (29%)
186 (18%)
2571 (23%)
5445 (26%)
24–35 months
1082 (24%)
1054 (27%)
236 (23%)
2548 (23%)
4920 (24%)
36–59 months
1120 (25%)
1273 (32%)
523 (50%)
3711 (34%)
6627 (32%)
Number (%) treated by sex
Female
2458 (54%)
1977 (50%)
493 (47%)
NA
4928 (52%)
Male
2064 (46%)
1980 (50%)
551 (53%)
NA
4595 (48%)
Number (%) treated by promptness of utilization
Same day
2636 (58%)
2125 (54%)
479 (49%)
5445 (49%) within
24 hours
14124 (69%)
Next day
1886 (42%)
1313 (33%)
240 (24%)
Later than next day
0 (0%)
520 (13%)
263 (27%)
5594 (51%)
6377 (31%) Table 2: Utilization of CMDs for treatment of children with fever in 4 sites (source: CMD registers) http://www.malariajournal.com/content/7/1/6 Malaria Journal 2008, 7:6 http://www.malariajournal.com/content/7/1/6 Caregiver adherence From the survey data, a very high proportion of children
were reported to have been treated promptly in all four
sites (Table 5). The proportion of febrile children who
received prompt treatment and received the correct dose
for the assigned duration of treatment ranged from 71%
in Uganda to 87% in Ghana (P < 0.001; Table 5). Adher-
ence by caregivers was linked to better treatment outcome. A woman participating in a FGD in Ejisu-Juaben district
said: Treatment coverage of febrile episodes by CMDs In two of the four sites there was some turnover of CMDs
but the attrition rate in these sites was relatively low. The
need for incentives for CMDs, however, was often men-
tioned as an issue. An opinion leader in Ho, Ghana, said: Data from the community-based cross-sectional surveys
were used to estimate the proportion of febrile children
receiving ACT from CMDs. A total of 2,190 children with
fever in the two weeks preceding the survey were identi-
fied of whom 1,289 (59%) were reported to have received
ACT from a CMD (Table 3). Coverage varied from 52% in
Nigeria to 75% in Ho District, Ghana (P < 0.001). Cover-
age rates did not appear to vary greatly with age, except
perhaps in Ejisu-Juaben District, Ghana, where coverage
appeared lower in the six to 11 months age group than in
older age groups (Table 3). Overall, across the four sites,
treatment coverage did not appear to vary strongly with
the educational level of the caregiver. There is this saying that "the one who feeds a child will
surely put her hands in her mouth"; our volunteers should
be appreciated so that they can enjoy the work they are
doing. For this, the community and I will think about it and
compensate them by giving some allowance because some-
times they have to go and call them from wherever they are. CMD performance Based on the information recorded by the CMDs in their
registers, a very high proportion of children received the
correct dose of ACTs (97% or greater in all sites; Table 4). From the survey data, CMDs were reported to have
explained the dosing schedule on a very high proportion
of occasions (>90% in all sites), but performed less well
with respect to explaining danger signs or possible adverse
events (Table 4). This was particularly pronounced in
Ejisu-Juaben District in Ghana. Availability of CMDs was
reported to be good, with 85% or more of caretakers
reporting that they found the CMD at the first time of vis-
iting. "When I went in for the drug he gave me instructions on
how to give the drug to the child. I was told to give it to him
continuously for three days – two tablets (white & yellow)
each day. I did that and afterwards the child was well
again. Happiness was then restored in my home". CMD performance Page 5 of 9
(page number not for citation purposes)
Table 3: Treatment coverage of febrile children aged 6–59 months with ACTs obtained from CMDs (source: household survey)
Ejisu – Juaben
District, Ghana
Ho District,
Ghana
Badeku and Ojoku/Ajia
Districts, Nigeria
Bugiri and Iganga
Districts, Uganda
Totals
Total number of febrile children identified
428
124
551
1087
2190
Number (%) treated with ACTs from a CMD
289 (68%)
93 (75%)
288 (52%)
619 (57%)
1289 (59%)
95% CI
61–74%
63–87%
44–61%
51–63%
Number treated (% coverage) by age in months
6–11
20 (45%)
13 (76%)
17 (53%)
16 (52%)
66(53%)
12–23
89 (74%)
21 (81%)
49 (49%)
179 (56%)
338 (60%)
24–35
64 (73%)
25 (76%)
69 (52%)
142 (56%)
300 (59%)
36–59
116 (66%)
28 (72%)
153 (54%)
282 (58%)
579 (59%)
Number treated (% coverage) by sex
Female
137 (65%)
46 (72%)
154 (54%)
NA
337 (60%)
Male
152 (70%)
47 (78%)
134 (50%)
NA
333 (61%)
Number treated (%) coverage by educational level of caregiver
None
71 (75%)
20 (77%)
80 (47%)
192 (54%)
363 (56%)
Primary
74 (68%)
28 (80%)
155 (56%)
315 (56%)
572 (58%)
Secondary
144 (64%)
45 (76%)
53 (52%)
112 (65%)
354 (63%)
Number treated (% coverage) by marital status of caregiver
Married
219 (67%)
87 (75%)
256 (54%)
571 (56%)
914 (47%)
Not married
70 (69%)
6 (75%)
30 (41%)
48 (63%)
127 (49%) e children aged 6–59 months with ACTs obtained from CMDs (source: household survey) Table 3: Treatment coverage of febrile children aged 6–59 months with ACTs obtained from CMDs (sou ment coverage of febrile children aged 6–59 months with ACTs obtained from CMDs (source: household sur http://www.malariajournal.com/content/7/1/6 Malaria Journal 2008, 7:6 Table 4: Measures of CMD performance in delivering ACTs in 4 sites (source: CMD registers + household survey)
Ejisu – Juaben
District, Ghana
Ho District,
Ghana
Badeku and Ojoku/Ajia
Districts, Nigeria
Bugiri and Iganga
Districts, Uganda
Totals
Correctness of prescription (from CMD register)
Number (%) of all children correctly dosed
4473 (99%)
3900 (98%)
1019 (98%)
10,671 (97%)
20063 (98%)
Number (%) of young children receiving an
over dose
30 (0.7%)
15 (3.2%)
11 (2%)
27 (0.4%)
83 (0.7%)
Number (%) of older children receiving an
under dose
8 (0.2%)
43 (1.2%)
12 (3%)
54 (1.5%)
117 (1%)
Number (%) of occasions on which CMD explained (from survey):
Treatment schedule
281 (97%)
93 (100%)
264 (92%)
569 (92%)
1207 (94%)
Danger signs
53 (18%)
83 (89%)
240 (83%)
462 (75%)
838 (65%)
Possible adverse events
44 (15%)
86 (93%)
241 (84%)
N/A
371 (55%)
Availability of CMDs (from survey)
Number (%) of mothers who did not find
the CMD at the first attempt
36 (12%)
5 (5%)
36 (12.5%)
86 (14%)
163 (12.5%)
Storage of ACTs (from supervision records)
Number (%) of supervisory visits at which
drugs were stored appropriately
523 (99%)
720 (100%)
960 (100%)
420 (100%)
2623 (99.8%)
Attrition of CMDs
Number (%) of CMDs withdrawing from
role
0 (0%)
3 (4%)
6 (10%)
0 (0%)
9 (3%) Table 4: Measures of CMD performance in delivering ACTs in 4 sites (source: CMD registers + household survey) mance in delivering ACTs in 4 sites (source: CMD registers + household survey) A local council chairperson of Bwalula village in Uganda
explained: child's condition becomes worse. If you are lucky to get to
the hospital early, after treatment you walk the same dis-
tance back or referred to the big hospital. The unlucky ones
either die before they get to the clinic or die at the clinic; but
ever since the introduction of this program, we have seen a
change. For a year now, since the introduction of the new
drug, no child has died and we have not sent any of them
to the hospital". child's condition becomes worse. If you are lucky to get to
the hospital early, after treatment you walk the same dis-
tance back or referred to the big hospital. The unlucky ones
either die before they get to the clinic or die at the clinic; but
ever since the introduction of this program, we have seen a
change. For a year now, since the introduction of the new
drug, no child has died and we have not sent any of them
to the hospital". From 1–2 weeks during Homapak, it now takes about three
months before my child gets sick. It is better than CQ when
you complete the dose. "As for the drug, it works like magic!" "As for the drug, it works like magic!" Safety and perceived effectiveness of ACTs The proportion of children for whom an adverse event
was reported to the CMD was low (circa 1%) in both sites
in which this information was available (Table 6). At the
survey the proportion of children reported to have experi-
enced an adverse event following treatment with ACT was
somewhat higher (4–8%) but none of these adverse
events were reported to be serious. In all sites almost all
caretakers perceived ACT to be effective. In Ho, Ghana, a
community member argued that: Discussion There are two major findings from this study. First, it
showed, at a larger scale than previously [9], that making
ACT available at the community level through trained
CMDs results in a high degree of adherence by sensitized
caregivers. Second, that community-based strategy can
deliver high coverage of febrile episodes in children with
prompt and adequate treatment. Importantly, these find- "The change is that, previously, when children fall sick, we
take some time to look for money and walk all the distance
to go to the hospital. Sometimes before we get there the Table 5: Adherence of caregivers to treatment schedule
Ejisu – Juaben
District, Ghana
Ho District,
Ghana
Badeku and Ojoku/Ajia
Districts, Nigeria
Bugiri and Iganga
Districts, Uganda
Totals
Number of episodes treated with ACTs from a
CMD
289
93
288
619
1289
Number (%) of children correctly treated (dose
and duration)
281 (97%)
69 (74%)
256 (89%)
490 (79%)
1096 (85%)
Number (%) of children treated promptly
(receiving first dose on the same or next day)
259 (90%)
89 (96%)
278 (97%)
531 (86%)
1157 (90%)
Number (%) of children treated promptly AND
correctly
252 (87%)
69 (74%)
231 (80%)
438 (71%)
990 (77%) Table 5: Adherence of caregivers to treatment schedule http://www.malariajournal.com/content/7/1/6 Malaria Journal 2008, 7:6 Table 6: Safety and perceived effectiveness of treatment of ACTs obtained from CMDs
Ejisu – Juaben
District, Ghana
Ho District,
Ghana
Badeku and Ojoku/Ajia
Districts, Nigeria
Bugiri and Iganga
Districts, Uganda
Totals
CMD registers
Number of episodes treated with ACTs from a
CMD (CMD registers)
4522
3958
1044
11039
20563
Number (%) of children reporting an adverse event
to a CMD
63 (1%)
10 (<1%)
NA
NA
Number (%) children reported recovered
NA
NA
NA
NA
Survey data
Number of episodes treated with ACTs from a
CMD (surveys)
289
93
288
619
1289
Number (%) of children reporting an adverse event
at survey
24 (8%)
7 (8%)
10 (4%)
34 (6%)
75 (6%)
Number (%) children reported recovered
NA
NA
284 (99%)
NA
284 (99%)
Number of caregivers (%) who perceived treatment
to be effective
286 (99%)
93 (100%)
280 (97%)
NA
659 (98%) Table 6: Safety and perceived effectiveness of treatment of ACTs obtained from CMDs ings are consistent in four different sites in sub-Saharan
Africa, both in West and East Africa. These studies were designed for the CMDs to provide
treatment on the basis of a clinical diagnosis. Discussion The possibil-
ity of moving away from a symptom based treatment to
more complex therapeutic protocols based on parasito-
logical confirmation is currently being investigated. To
bring parasitological diagnosis to the community level
will require reliable diagnostic tools, and evidence of fea-
sibility and acceptability by the community. There was high utilization of CMDs. In an estimated pop-
ulation of children aged 6 to 59 months across the sites of
27,450, the CMDs treated 20,563 episodes of fever during
the year of implementation (0.75 episodes of fever per
child on average)). This contrasts with the figure of
0.12–0.34 episodes per year reported from African health
facilities in available literature [10]. Furthermore, the
majority of caregivers reported that CMDs were available
at the first time of asking, and very little attrition of CMDs
was reported. This demonstrates the potential of CMDs to
increase access to care in underserved rural areas in Africa,
where government health facilities are often under-uti-
lized [11,12]. While every effort was made to ensure the
implementation of the study as close as possible to real
life conditions, its short duration and the research setting
are likely to have contributed to the low attrition rate of
CMDs observed in this study. Indeed, the need to provide
CMDs with incentives was repeatedly mentioned in inter-
views and FGDs, and financial motivation is known to be
a key factor determining long term performance of CMDs
[13]. It is clear that this need must be addressed to ensure
the long-term sustainability of CMDs performance. Adherence to the full treatment course has been high-
lighted as a matter of concern [14-16]. AL has to be taken
twice a day for three days (one or two tablets depending
on the age group), AS/AQ once daily for three days but
two different tablets (or half tablets) each time. Both of
these regimens are different from those for older antima-
larials like chloroquine or sulphadoxine-pyrimethamine
(SP). Nonetheless, reported adherence by caregivers to the
correct treatment schedule in terms of dose and duration
was high for both treatment regimes in all sites, ranging
from 79% in Uganda to 97% in Ejisu, Ghana (85% on
average across the sites). These findings are reassuring
with regard to the potential for development of resistance,
as exposure to sub-therapeutic drug levels is known to be
a major factor i Page 7 of 9
(page number not for citation purposes) n the selection of resistant parasites [17]. Concerns have been raised by several authors about the
potential misuse of ACT when deployed beyond the
health system [6,7], which could lead to wastage of
resources and the development of resistance to ACT. How-
ever, our findings indicate that there was appropriate pre-
scription in terms of dose by CMDs and correct use by
caregivers. In all study sites, 97% or more of the CMDs
prescribed the correct dose of ACTs, and almost all of
them explained the treatment schedule to the caregiver. Drug storage conditions were satisfactory in all sites. Furthermore, the majority of caregivers obtaining ACT
from a CMD reported that they treated their children
promptly (ranging from 86% to 97%, 90% on average
across the sites, Table 5). Data from CMDs' registers
(Table 2) support this finding, albeit with slightly lower
proportions of children recorded to be treated on the day
of onset or the next day in three sites. In Uganda, CMDs
reported treating only half of the episodes within 24
hours, a proportion which is still substantially higher than
previously reported by Nsungwa-Sabiiti [16]. The design Page 7 of 9
(page number not for citation purposes) Page 7 of 9
(page number not for citation purposes) http://www.malariajournal.com/content/7/1/6 http://www.malariajournal.com/content/7/1/6 http://www.malariajournal.com/content/7/1/6 Malaria Journal 2008, 7:6 of this study does not provide robust data on pharma-
covigilance. However, both CMDs and caregivers report a
very low incidence of adverse drug effects, none of which
were reported to be serious. While further studies are necessary to develop mecha-
nisms for ensuring long-term performance of CMDs and
to evaluate the effectiveness and safety of ACTs used
within the context of HMM programmes, the findings of
this study provide evidence to support scaling-up imple-
mentation of HMM with ACTs. Access to prompt and effective treatment is a cornerstone
of the current malaria control strategy [18]. Several strate-
gies have been developed to address the issue of access to
antimalarials in Africa, including home management of
malaria [1], improving the services of private medicine
sellers [19], a set of integrated interventions including
social marketing approaches [20] and improving the qual-
ity of health service performance [21]. n the selection of resistant parasites [17]. Our study provides
evidence that once the option of treating children with
effective antimalarials close to home is made available to
caregivers, a majority of them (ranging from 52% in
Nigeria to 75% in Ho, Ghana) make use of it, in most
cases in an appropriate and timely manner. This option
should be regarded as a complementary to, rather than
competing with, health facility delivery of care. As high-
lighted by Unger et al [21], interventions aiming at
improving use rates of general health services have also a
high potential of increasing malaria cure rates. Competing interests The author(s) declare that they have no competing inter-
ests. Authors' contributions All the authors except SC conceived the study; IA, ENB, BG
and FB were principal investigators for their respective
country's study site and together with FP, LD, BY and AB
participated in the research designed and supervised data
collection from the field. BY, PAB and SC performed most
of the quantitative data analysis while KM analysed the
qualitative data. FP, the WHO/TDR HMM research pro-
gram manager monitored the four sites. All authors con-
tributed to the draft. All authors read and approved the
final manuscript. Acknowledgements Overall effectiveness of drugs distributed at the commu-
nity level is known to be influenced by several factors
[16,22,23], not all of them addressed in this study. How-
ever, the high level of correct prescription by CMDs, the
promptness of treatment seeking and adherence to the
treatment schedule by caregivers, and the coverage (59%
on average across the sites) achieved in this study show
that some key factors determining the effectiveness of
antimalarial treatment can be successfully addressed. Thus, this approach has great potential to contribute to
reaching the Abuja target that "at least 60% of those suffer-
ing from malaria have prompt access to, and are able to cor-
rectly use, affordable and appropriate treatment within 24
hours of the onset of symptoms" [2]. The authors acknowledge the immense role of the community members,
opinion leaders, CMDs, the health workers, research assistants, field super-
visors and workers in Ejisu – Juaben and Ho district, Ghana, Ona-ara Local
Government, Nigeria and Bugiri and Iganga Districts, Uganda. We also acknowledge the valuable contribution of Dr. Josephine Namboze
(WHO/AFRO) and Wilson Were (WHO/GMP) who facilitated the proto-
col development workshops. This investigation received technical and
financial support from the UNICEF/UNDP/World Bank/WHO Special Pro-
gramme for Research and Training in Tropical Diseases. References 1. World Health Organization: The Roll Back Malaria Strategy for
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scientist can read your work free of charge
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Your research papers will be:
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Submit your manuscript here:
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Malaria policies versus practices, a reality check from Kinshasa, the capital of the Democratic Republic of Congo
|
BMC public health
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* Correspondence: mavoko@yahoo.com
1Département de Médecine Tropicale, Université de Kinshasa, B.P. 747, Kin XI,
Kinshasa, République Démocratique du Congo
2International Health Unit, Department of Epidemiology, University of
Antwerp, Campus Drie Eiken, Universiteitsplein 1, 2610 Kinshasa, République
Démocratique du Congo
Full list of author information is available at the end of the article RESEARCH ARTICLE Open Access Malaria policies versus practices, a reality check
from Kinshasa, the capital of the Democratic
Republic of Congo Hypolite Muhindo Mavoko1,2*, Gillon Ilombe1, Raquel Inocêncio da Luz2, Albert Kutekemeni3,
Jean-Pierre Van geertruyden2 and Pascal Lutumba1 Muhindo Mavoko et al. BMC Public Health (2015) 15:352
DOI 10.1186/s12889-015-1670-0 Study setting and design y
This was an observational study embedded in a two-
stage cluster randomized survey conducted in Kinshasa,
the capital city of the Democratic Republic of Congo
(DRC). This city is divided into 35 health zones among
them 12 were randomly selected: Bandalungwa, Binza
ozone, Bumbu, Kimbanseke, Kingabwa, Kokolo, Limete,
Makala, Maluku 1, Masina 1, Police and Selembao. In
each health zone, one public or private HC was ran-
domly selected among those reporting to the National
Health Information System. The former study consisted
of evaluating the accuracy of Optimal-IT®, as well as
Paracheck-Pf® malaria rapid diagnostic tests (RDTs) [5]. RDTs were performed and microscopy slides prepared at
recruitment. HC laboratory technicians had to read the
slides and were blinded to the RDTs results. The same
slides were read by expert microscopists for quality
control (QC) and they were blinded to previous results. The QC results constituted the gold standard to assess
the accuracy of RDTs and microscopy at PHC level. The sample size of 624 was calculated for the main
study based on the accuracy of Optimal-IT® RDT and
the same number was involved in these supplementary
analyses. In Sub Saharan Africa, which holds the gross of the
malaria burden, the proportion of people treated for
malaria with a confirmed diagnosis is low compared to
other regions of the world [4]. In addition, the accuracy
of malaria diagnosis at primary health care has been re-
ported to be poor in many areas [5-8]. This may explain
the fact that health practitioners tend to prescribe
antimalarial drugs regardless of laboratory results. Fur-
thermore, patients can purchase antimalarial drugs dir-
ectly on the informal market without any medical
prescription [9,10]. In 2010, only a few months after its publication, the
Congolese National Malaria Control Program (NMCP)
adopted
the
second
edition
of
the
WHO
malaria
treatment guidelines [2]. The major novelty was the
recommendation to give antimalarial treatment upon
parasitological evidence of malaria infection even in
children under the age of five years. The purpose
was to improve targeting of treatment and better
quality of care, in particular in an environment of in-
creased malaria control and or declining malaria en-
demicity [2]. Indeed non malaria cases would be
identified and may benefit from timely treatment
thereby reducing morbidity and mortality. Abstract BMC Public Health (2015) 15:352 Page 2 of 7 Methods Approximately half of the global population is exposed
to malaria [1]. Early parasitological diagnosis and effica-
cious treatment in all age groups are recommended by
the World Health Organization (WHO) as key strategies
to control malaria [2]. However, next to policies, avail-
ability of good quality drugs and parasite based diagnos-
tic is essential [3]. Other important factors are access to
health care services as well as the health seeking behav-
iour of the population. Policies are principally based on
high quality research evidence, but implementation re-
mains challenging on various levels because of some
field realities. Abstract Background: Artemisinin-based combination therapy (ACT) following a confirmed parasitological diagnosis is
recommended by the World Health Organization (WHO) and the Congolese National Malaria Control Program
(NMCP). However, commitment and competence of all stakeholders (patients, medical professionals, governments
and funders) is required to achieve effective case management and secure the “useful therapeutic life” of the
recommended drugs. The health seeking behaviour of patients and health care professionals’ practices for malaria
management were assessed. Methods: This was an observational study embedded in a two-stage cluster randomized survey conducted in one
health centre (HC) in each of the 12 selected health zones in Kinshasa city. All patients with clinical malaria diagnosis
were eligible. Their health seeking behaviour was recorded on a specific questionnaire, as well as the health care
practitioners’ practices. The last were not aware that their practices would be assessed. Results: Six hundred and twenty four patients were assessed, of whom 136 (21.8%) were under five years. Three
hundred and thirty five (55%) had taken medication prior to the current consultation (self -medication with
any product or visiting another HC) of whom 47(14%) took an antimalarial drug, and 56 (9%) were treated
presumptively. Among those, 53.6% received monotherapy either with quinine, artesunate, phytomedicines,
sulfadoxine-pyrimethamine or amodiaquine. On the other side, when clinicians were informed about laboratory
results, monotherapy was prescribed in 39.9% of the confirmed malaria cases. Only 285 patients (45.7%) were
managed in line with WHO and NMCP guidelines, of whom 120 (19.2%) were prescribed an ACT after positive
blood smear and 165 (26.4%) received no antimalarial after a negative result. Conclusion: This study shows the discrepancy between malaria policies and the reality on the field in Kinshasa,
regarding patients’ health seeking behaviour and health professionals’ practices. Consequently, the poor
compliance to the policies may contribute to the genesis and spread of antimalarial drug resistance and also
have a negative impact on the burden of the disease. Keywords: Malaria, Policies, Practices, Health seeking behaviour, Phytomedicines, Democratic Republic of Congo © 2015 Muhindo Mavoko 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. Muhindo Mavoko et al. Study setting and design In DRC,
confirmed uncomplicated malaria cases are recommended
to be treated either with artesunate+amodiaquine (ASAQ)
or artemether+lumefantrine (AL) as the first line,
and quinine+antibiotic having antimalarial activity as
second line [11]. Data were collected from May to June 2011. All the
patients attending the health facility and clinically diag-
nosed with malaria and to whom a blood smear (BS)
was requested, were eligible. Clinical diagnosis was
established by clinicians based on unspecific known mal-
aria symptoms like fever, history of fever, headache,
chills, weakness, muscle and joint pain and anorexia. Data was collected in 2 phases. First, a questionnaire
was filled during an interview. Socio-demographic data
were recorded and the patient/guardians’ health seeking
behaviour was assessed i.e. current illness, action taken
and health seeking practices prior to the current visit. In
a
second
phase,
the
medications
(antimalarial
and
others) prescribed by the clinicians before and after the
BS results as well as the laboratory results were also re-
corded. Clinicians were not informed on the data collec-
tion to avoid observer’s bias. Antimalarial drugs taken
before seeking care and/or prescribed at the PHC were
identified using the list of antimalarial drugs available on
the market in Kinshasa (e.g. Artemether+lumefantrine:
Coartem®, Coartesiane®, Lonart®, Luther®, etc.). Improving malaria case management requires the
involvement of all stakeholders and detection of the
operational bottle necks. Inspired by the Piot Model
[12], the health seeking behaviour of the patients and
practices of the health care providers were assessed
going from the first presumed malaria symptoms
until the delivery of care in formal health centres
(HC). The objective was to assess health seeking be-
haviour of patients and practices related to the man-
agement
of
uncomplicated
malaria
by
the
health
professional at primary health care level (PHC) com-
pared to the policies. Laboratory examination y
The collected BSs were stained with 10% Giemsa for
10 minutes. Thin BSs were fixed with methanol prior to
the staining. Microscopy reading was first performed by
the HC laboratory technicians and result was given to
clinicians, who were supposed to manage the cases
according to parasitological diagnosis and National Page 3 of 7 Muhindo Mavoko et al. BMC Public Health (2015) 15:352 age groups (p < 0.001) (Table 1). At PHC, 361 patients
(57.9%) tested positive for malaria, whereas only 123
(19.7%) were confirmed by the experts’ reading. guidelines and policies [11]. Slides were subsequently
transported to the parasitology unit, Kinshasa University
for QC. Prescribing practice
( The study was approved by the Committee for Medical
Ethics of the Antwerp University Hospital, Belgium
(approval reference: 14/36/236) and the Ethical Committee
of the School of Public Health, Kinshasa University, DRC
(approval reference: ESP/CE/082/10). Before inclusion,
written informed consent was obtained from all patients or
their legal guardians if minors. Prescribing practice
Only 285 cases (45.7%) were managed in line with WHO
guidelines as ACTs were prescribed to 120 positive
cases and nothing to 165 negative cases (Figure 1). ACTs consisted of ASAQ (100 cases, 30.0%), AL
(63 cases, 19.0%) and artesunate+sulfadoxine-pyrimeth-
amine (AS+SP) (7 cases, 2.1%). Monotherapy consisted of
quinine (88 cases, 26.7%), artesunate (31 cases, 9.4%), phy-
tomedicines (24 cases, 7.3%), sulfadoxine-pyrimethamine
(16 cases, 4.8%) and amodiaquine (1 case, 0.3%). Data analysis g
Among 373 patients who reported history of fever, 24
(6.4%) attended the HC the same day when fever started,
55 (14.8%) the following day, 154 (41.3%) after 2–3 days,
95 (25.5%) after 4–7 days, whereas 45 (10.1%) waited for
more than a week. Lowest proportion of malaria was
confirmed (both at PHC and by experts) among those
who came more than a week after the fever onset
(Table 2). At the same time, malaria was less confirmed
in the group that attended the HC directly (Table 2). Be-
fore the current consultation, 50 patients (8%) had taken
traditional treatment, 276 (44.2%) resorted to self-
treatment with modern drugs, 16 (2.8%) visited another
HC previously. Two hundred and eighty two (45.2%) did
not undertake any action prior to the current visit
(Figure 1). Before attending the HC, 100 patients (16%)
had taken an antimalarial drug, 288 (46.2%) took an-
other type of drug (antibiotics, antipyretic, etc.), and 235
(37.8%) took no drugs. Patients who had taken no anti-
malarial drugs more likely attended the HC during the
first 3 days of fever onset (p = 0.02). Data were double-entered and validated in Epi info ver-
sion 3.5.1 software and analysed using Stata version 11
(Stata Corp, Lakeway, College Station, Texas, USA). The
primary outcome was the proportion of patients to
whom a recommended antimalarial drug was prescribed
after a positive test and those for whom no prescription
was made after a negative test. Secondary outcomes in-
cluded description of patients’ health seeking behaviour
(time for consultation regarding the illness episode on-
set, medication taken before consultation) and malaria
testing outcome compared to patients’ health seeking
behaviour and clinicians’ practices. Descriptive statistics
were used to get frequencies and percentages. Cross tab-
ulations were performed to check relationship between
variables of interest using the Chi-square test of Pearson. Predictors of malaria confirmation were assessed by a
logistic regression, referring to experts’ microscopy read-
ing result. The cluster effect was checked and did not
influence the results. *out of 623; **out of 487. Baseline characteristics The lack of compliance with NMCP guidelines has
been reported elsewhere in sub-Saharan Africa [15-20]. Malaria control programs need to elaborate an efficient
strategy for the follow up of adherence to guidelines by
practitioners. In addition, a dialogue between policy
makers, researchers and program managers is needed to
discuss and address the gaps in the implementation of
malaria management policies [21]. The overtreatment reported in this survey could be
the result of unsatisfactory diagnostic accuracy at PHC
[5,22]. This may explain why clinicians seldom relied on
the BS results. Another explanation could be related to
the mistrust of policies evidence, as reported in other
studies [23,24]. In Tanzania, Reyburn et al. [25] found
that almost half of patients with a negative malaria test
were treated with an antimalarial drug. The responsibil-
ity of the overtreatment is to be shared with the patients
who can influence health practitioners’ practice [26,27]. In Ghana, participants to a focus group discussion
claimed that a test should result in a diagnosis, even fol-
lowing a negative result [28]. In this aspect, clinicians
may choose to prescribe antimalarial drugs, mostly
when laboratory diagnoses for other infections are
not available. most prescribed antimalarials in these cases. Clinicians
changed the prescription for 7 of them in light of the
BS results from SP to ACT. Prior treatment before
attending the HC did not influence prescription be-
havior (p = 0.09). Clinicians tended to prescribe more antimalarial drugs
to children younger than five, even when laboratory re-
sults were negative. Clear guidelines for management of
non-malaria febrile illnesses as well as appropriate tests
and drugs are urgently needed [29]. By the time this
survey was conducted, ASAQ and AL were the recom-
mended first line ACTs. It was noticed that clinicians
tended to prescribe more ASAQ when BS result was
positive. When prescription was done before BS or in
spite of negative BS result, either Quinine or AL was
preferred. Assuming that they were not aware about the
introduction of AL in the national guidelines some
months prior to this assessment, the hypothesis is that
they had preference for non-recommended antimalarial
drugs in absence of malaria confirmation. For 330 patients (53.0%), antimalarial prescription
was done based on BS results. In that case, ASAQ was
most frequently prescribed (30.3%), followed by quinine
monotherapy (26.7%) and AL (19.1%). Baseline characteristics Fifty six patients (9.0%) received an antimalarial pre-
scription while BS results were still unknown and this
was observed more frequently in children under five
years, but the difference was not significant (p = 0.54). Quinine (34.0%), AL (34.0%) and SP (10.7%) were the A minimum of 50 patients were enrolled from each HC,
with a total of 624. Of those, 136 were under five years
(21.8%). In general, history of fever was the most fre-
quent symptom (59.8%), and it was significantly more
reported in children under five years compared to other Table 1 Baseline characteristics of 624 patients in primary health care setting, Kinshasa, DRC, 2011
Variable
All age group
<5 years
≥5 year
p
(N = 624)
(N = 136)
(N = 488)
n (%)
n (%)
n (%)
Female (n, %)
362 (58.0)
62 (45.6)
300 (61.5)
0.001
History of fever
373 (59.8)
123 (90.4)
250 (51.2)
<0.001
Ownership mosquito net
333 (53.4)
76 (55.8)
257 (52.7)
0.51
Slept under mosquito net last night
224 (35.9)
56 (41.1)
168 (34.4)
0.15
Malaria self-treatment prior current consultation using any antimalarial drug
100 (16.0)*
18 (13.2)
82 (16.8)**
0.31
*
t
f 623 **
t
f 487 ics of 624 patients in primary health care setting, Kinshasa, DRC, 2011 Page 4 of 7 Muhindo Mavoko et al. BMC Public Health (2015) 15:352 Table 2 Diagnosis outcome compared to the patients’
health seeking practice in primary health care setting,
Kinshasa, DRC, 2011
Patients’ practice
Malaria cases
confirmed at
PHC
Malaria cases
confirmed by
experts
n (%)
n (%)
Days from fever onset to the current
consultation (n/ N)
0 (24/ 373)
17 (70.8)
7 (29.2)
1 (55/ 373)
30 (54.6)
15 (27.3)
2 to 3 (154/ 373)
102 (66.2)
50 (32.5)
4 to 7 (95/ 373)
61 (64.2)
21 (22.1)
>7 (45/ 373)
26 (57.8)
9 (20.0)
Care seeking bahaviour
Self-treatment with traditional
products (50/ 624)
33 (66.0)
12 (24.0)
Self-treatment with modern
products (276/ 624)
166 (60.1)
72 (26.1)
Visited another HC (16/ 624)
9 (56.3)
2 (12.5)
No action (282/ 624)
153 (54.3)
37 (13.1) Table 2 Diagnosis outcome compared to the patients’
health seeking practice in primary health care setting,
Kinshasa, DRC, 2011 drugs and the patient/ care giver’s compliance to the treat-
ment [13,14]. Nevertheless, this study did not explore those
aspects. Baseline characteristics Monotherapy was
prescribed to 39.8% of patients who tested positive for
malaria at PHC. Malaria was more likely confirmed by
experts’ reading when the patients had reported history
of fever (p < 0.001), undertook no treatment before
attending the HC (p < 0.001) and lacked mosquitonet
(p = 0.05). In the multivariate analysis, history of fever,
treatment seeking behaviour and not sleeping under
mosquitonet were significantly associated with malaria
confirmation (Table 3). Combination treatment using ACTs is the key strategy
to reduce the impact of failing monotherapies [30,31]. Unfortunately and against the national guidelines,
this survey highlighted a worrying proportion of pre-
scribing monotherapy, including artemisinin mono-
therapy (AMT), a practice that could increase treatment
failures and contribute to the spread of resistance. Despite
the WHO’s call to stop AMT production and marketing
to refrain the development of drug resistance [31], the
field reality is still different. However, a trend towards their
disappearance from the market in Kinshasa is notably ob-
served. In 2009 they constituted 10% and 41% of antimal-
arial drugs stocked in public/ Not-for-Profit and private *Malaria confirmed by experts. Acknowledgement We thank the study patients and the staff of the involved health centres. We
are also grateful to Vito Baraka for proofreading of the manuscript. This study
was funded by VLIR-UOS through project number ZRDC2008MP053, which
aimed to create a clinical research unit and the capacity building at the Tropical
Medicine Department, Kinshasa University. Apparently, none of the clinicians involved was aware,
6 months after the NMCP guidelines update, that quin-
ine should not be used as a monotherapy. This survey
did not focus on the knowledge nor the background of
practitioners and further research will be needed to as-
sess explanatory causes and identify the bottlenecks
where training or adapted measurements can be taken. The lack of this information is a limitation of the study,
but these findings may be reproducible in other settings. References Directives nationales de
traitement du paludisme en République Démocratique du Congo. Kinshasa:
Ministère de la Santé; 2010. 12. Mumba M, Visschedijk J, van Cleeff M, Hausman B. A Piot model to analyse
case management in malaria control programmes. Trop Med Int Health. 2003;8(6):544–51. 12. Mumba M, Visschedijk J, van Cleeff M, Hausman B. A Piot model to analyse
case management in malaria control programmes. Trop Med Int Health. 2003;8(6):544–51. Authors’ contributions HMM, PL and JPV designed the study protocol. HMM and GI supervised the
activities on the field. HMM performed the statistical analysis. RIL, HMM and
JPV drafted the manuscript. HMM, JPV, PL, GI, RIL and AK reviewed the paper
and all authors approved the final version. Competing interests Competing interests
The authors declare that they have no competing interests. It is worth mentioning the ongoing initiative in the coun-
try supported by the Global Fund that aims to supply RDTs
as well as recommended medications. The support is pres-
ently available in 60% of the health zones in DRC. At the
time this survey was conducted, two of the twelve HC in-
volved were supplied, although on an irregular basis. References 1. WHO. World Malaria Report 2011. Geneva: World Health Organization; 2011. 2. WHO. Guidelines for the treatment of malaria. Geneva: World Health
Organization; 2010. p
g
;
2. WHO. Guidelines for the treatment of malaria. Geneva: World Health
Organization; 2010. 3. Bate R, Coticelli P, Tren R, Attaran A. Antimalarial drug quality in the most
severely malarious parts of Africa – a six country study. PLoS One. 2008;3(5):2132. 3. Bate R, Coticelli P, Tren R, Attaran A. Antimalarial drug quality in the most
severely malarious parts of Africa – a six country study. PLoS One. 2008;3(5):2132. 4. WHO. World Malaria Report 2008. Geneva: World Health Organization; 2008. 5. Muhindo MH, Ilombe G, Meya R, Mitashi PM, Kutekemeni A, Gasigwa D,
et al. Accuracy of malaria rapid diagnosis test Optimal-IT® in Kinshasa, the
Democratic Republic of Congo. Malar J. 2012;11:224. 6. Mukadi P, Gillet P, Lukuka A, Atua B, Kahodi S, Lokombe J, et al. External
quality assessment of malaria microscopy in the Democratic Republic of the
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7. McKenzie FE, Sirichaisinthop J, Miller RS, Gasser RA, Wongsrichanalai C. Dependence of malaria detection and species diagnosis by microscopy on
parasite density. Am J Trop Med Hyg. 2003;69:372–6. 8. Zurovac D, Midia B, Ochola SA, English M, Snow RW. Microscopy and
outpatient malaria case management among older children and adults in
Kenya. Trop Med Int Health. 2006;11:432–40. 9. Luxemburger C, Nosten F, Kyle DE, Kiricharoen L, Chongsuphajaisiddhi T,
White NJ. Clinical features cannot predict a diagnosis of malaria or
differentiate the infecting species in children living in an area of low
transmission. Trans R Soc Trop Med Hyg. 1998;92:45–9. Approximately a third (35.9%) of those owning a mos-
quitonet slept under it the night before consultation. The protective impact on sleeping under mosquitonet
was shown by the multivariate analysis. Hence the cam-
pains are highly needed to sensitize the mass on the
benefits of using the mosquitonet. 10. Mbonye AK, Bygbjerg IC, Magnussen P. Prevention and treatment practices
and implications for Malaria control in Mukono District Uganda. J Biosoc Sci. 2008;40:283–96. 10. Mbonye AK, Bygbjerg IC, Magnussen P. Prevention and treatment practices
and implications for Malaria control in Mukono District Uganda. J Biosoc Sci. 2008;40:283–96. 11. Programme National de Lutte contre le Paludisme. Directives nationales de
traitement du paludisme en République Démocratique du Congo. Kinshasa:
Ministère de la Santé; 2010. 11. Programme National de Lutte contre le Paludisme. Author details
1 1Département de Médecine Tropicale, Université de Kinshasa, B.P. 747, Kin XI,
Kinshasa, République Démocratique du Congo. 2International Health Unit,
Department of Epidemiology, University of Antwerp, Campus Drie Eiken,
Universiteitsplein 1, 2610 Kinshasa, République Démocratique du Congo. 3Programme National de Lutte contre le Paludisme, Ministère de la Santé
Publique, Kinshasa, République Démocratique du Congo. Almost half of the patients (44.2%) undertook a treat-
ment prior to the current consultation and they tended
to be more infected with malaria. This means that either
the medication taken was not effective or the treatment
course was not appropriate. Most of them used antimal-
arial, antibiotics and antipyretics. Such inappropriate use
of antibiotics and antimalarials is dangerous as it may
contribute to the emergence and spread of drug resistant
bacteria and Plasmodium [29,33]. Self-treatment has
been reported to be common, mainly in rural areas of
Africa [9]. People should be encouraged to address to
the health facilities as soon as the clinical episode starts,
for proper diagnosis and treatment. In the urban settings
like Kinshasa, geographical access is not the main obs-
tacle but affordability of the health services can be the
main limiting factor. This could explain why few people
with history of fever attended the HC the same day. Ac-
cess to health may be increased by increasing the health
insurance coverage either through the Government or
the private stakeholders. Received: 14 June 2014 Accepted: 20 March 2015 Discussion Results of this survey highlight the alarming situation of
malaria management at PHC in the DRC because of
a huge discrepancy between policy and field reality. Almost half of the patients (45.7%) were managed in line
with NMCP guidelines. However, 7 patients (2.1%)
received a prescription for AS+SP which is not offi-
cially recommended in DRC but still in line with the
WHO recommendations. Besides, a proper prescription is
not enough to ensure accurate malaria management. The
steps forward are availability of quality-assured antimalarial Page 5 of 7 Muhindo Mavoko et al. BMC Public Health (2015) 15:352 Figure 1 Flow chart of the patients’ and clinicians’ practices in primary health care setting, Kinshasa, DRC, 2011. igure 1 Flow chart of the patients’ and clinicians’ practices in primary health care setting, Kinshasa, DRC, 2011. confirmed by experts’ reading based on practices of patients and medicals in primary health care
a, DRC, 2011 Table 3 Malaria confirmed by experts’ reading based on practices of patients and medicals in primary health care
setting, Kinshasa, DRC, 2011
Variable (n)
Blood smear positive (%)*
OR (95% CI)
p
Adj OR* (95% CI)
p
Age group
<5 years (136)
33 (24.3)
1.4 (0.9-2.3)
0.13
-
NS
≥5 years (488)
90 (18.4)
1
History of fever
Yes (373)
102 (27.4)
4.1 (2.5-7.1)
<0.001
3.6 (2.1-6.1)
<0.001
No (251)
21 (8.4)
1
1
Antimalarial treatment prior current consultation
Yes (100)
17 (17)
1
-
No (523)
106 (20)
0.8 (0.4-1.4)
0.45
NS
Treatment seeking practice
Yes (342)
86 (25.2)
2.2 (1.4-3.5)
<0.001
1.6 (1.0-2.5)
0.04
No (282)
37 (13.1)
1
Mosquitonet ownership
Yes (333)
56 (16.8)
1
0.05
-
NS
No (291)
67 (23.0)
1.4 (1.0-2.4)
Slept under mosquito net last night
Yes (224)
35 (15.6)
1
0.40
1
0.02
No (109)
21 (19.3)
1.3 (0.7-2.4)
1.7 (1.0-2.6)
Malaria treatment prescribed before blood smear result
Yes (56)
16 (28.6)
0.6 (0.3-1.2)
0.08
-
NS
No (568)
107 (18.9)
1
*Malaria confirmed by experts. Muhindo Mavoko et al. BMC Public Health (2015) 15:352 Page 6 of 7 Page 6 of 7 sector outlets respectively [13]. Four years later their avail-
ability decreased below 1% in both sectors [32]. Competing interests Conclusion Data of this study indicate that neither the health seek-
ing behaviour of the patients nor the treatment practices
of the health care providers were appropriate. This may
have a negative impact on the burden of the disease and
contribute to the genesis and spread of drug resistance. However, national malaria related health policies are in
line with WHO recommendations and regularly up-
dated. Health education, including health promotion and
training on fever management is urgently needed. 13. O’Connell KA, Gatakaa H, Poyer S, Njogu J, Evance I, Munroe E, et al. Got
ACTs? Availability, price, market share and provider knowledge of anti-malarial
medicines in public and private sector outlets in six malaria-endemic countries. Malar J. 2011;10:326. 13. O’Connell KA, Gatakaa H, Poyer S, Njogu J, Evance I, Munroe E, et al. Got
ACTs? Availability, price, market share and provider knowledge of anti-malarial
medicines in public and private sector outlets in six malaria-endemic countries. Malar J. 2011;10:326. 14. Banek K, Lalani M, Staedke SG, Chandramohan D. Adherence to artemisinin-
based combination therapy for the treatment of malaria: a systematic
review of the evidence. Malar J. 2014;13:7. 14. Banek K, Lalani M, Staedke SG, Chandramohan D. Adherence to artemisinin-
based combination therapy for the treatment of malaria: a systematic
review of the evidence. Malar J. 2014;13:7. 15. Sayang C, Gausseres M, Vernazza-Licht N, Malvy D, Bley D, Millet P. Treatment
of malaria from monotherapy to artemisinin-based combination therapy by
health professionals in rural health facilities in southern Cameroon. Malar J. 2009;8:174. 15. Sayang C, Gausseres M, Vernazza-Licht N, Malvy D, Bley D, Millet P. Treatment
of malaria from monotherapy to artemisinin-based combination therapy by
health professionals in rural health facilities in southern Cameroon. Malar J. 2009;8:174. Page 7 of 7 Page 7 of 7 Muhindo Mavoko et al. BMC Public Health (2015) 15:352 16. Krause G, Sauerborn R. Comprehensive community effectiveness of health
care. A study of malaria treatment in children and adults in rural Burkina
Faso. Ann Trop Paediatr. 2000;20:273–82. 17. Rowe AK, Ponce de León GF, Mihigo J, Santelli AC, Miller NP, Van-Dúnem P. Quality of malaria case management at outpatient health facilities in
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antimalarial drugs and evaluation of the attitude and practices for the
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treatment on prescribing patterns in Gezira state, Sudan. East Mediterr
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Pract. 2014;15(1):165. 23. Kramera AR, Dickinson KL, Anderson RM, Fowler VG, Miranda ML, Mutero
CM, et al. Using decision analysis to improve malaria control policy making. Health Policy. 2009;92:133–40. y
24. Amin AA, Zurovac D, Kangwana BB, Greenfield J, Otieno DN, Akhwale WS,
et al. The challenges of changing national malaria drug policy to
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enough: physician rationales for altering standard treatment guidelines
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test result is negative, they should be able to tell us what is wrong with us”:
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settings: a global review of evidence and practice. Geneva: World Health
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30. White NJ. Malaria – time to act. N Eng J Med. 2006;55:1956–7. 30. White NJ. Malaria – time to act. N Eng J Med. 2006;55:1956–7. 31. WHO. Guidelines for the Treatment of Malaria. Geneva: World Health
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ACTWatch en République Démocratique du Congo, 2013. http://www. actwatch.info/sites/default/files/content/publications/attachments/ACTwatch
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chemotherapy. Parassitologia. 1999;41:301–8. 33. White NJ. Delaying antimalarial drug resistance with combination
chemotherapy. Parassitologia. 1999;41:301–8. Submit your next manuscript to BioMed Central
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and take full advantage of:
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• Immediate publication on acceptance
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Submit your manuscript at
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https://openalex.org/W3102565266
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https://repository.ubn.ru.nl/bitstream/handle/2066/227649/1/227649.pdf
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English
| null |
Key role for lipids in cognitive symptoms of schizophrenia
|
Translational psychiatry
| 2,020
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cc-by
| 9,766
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2020, Article / Letter to editor (Translational Psychiatry, 10, 1, (2020), pp. 1-12, article 399)
Doi link to publisher: https://doi.org/10.1038/s41398-020-01084-x Version of the following full text: Publisher’s version
Downloaded from: https://hdl.handle.net/2066/227649
Download date: 2024-10-24 Version of the following full text: Publisher’s version
Downloaded from: https://hdl.handle.net/2066/227649
Download date: 2024-10-24 © The Author(s) 2020
OpenAccessThisarticleislicensedunderaCreativeCommonsAttribution4.0InternationalLicense,whichpermitsuse,sharing,adaptation,distributionandreproduction
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/. Abstract Schizophrenia (SZ) is a psychiatric disorder with a convoluted etiology that includes cognitive symptoms, which arise
from among others a dysfunctional dorsolateral prefrontal cortex (dlPFC). In our search for the molecular
underpinnings of the cognitive deficits in SZ, we here performed RNA sequencing of gray matter from the dlPFC of SZ
patients and controls. We found that the differentially expressed RNAs were enriched for mRNAs involved in the Liver
X Receptor/Retinoid X Receptor (LXR/RXR) lipid metabolism pathway. Components of the LXR/RXR pathway were
upregulated in gray matter but not in white matter of SZ dlPFC. Intriguingly, an analysis for shared genetic etiology,
using two SZ genome-wide association studies (GWASs) and GWAS data for 514 metabolites, revealed genetic overlap
between SZ and acylcarnitines, VLDL lipids, and fatty acid metabolites, which are all linked to the LXR/RXR signaling
pathway. Furthermore, analysis of structural T1-weighted magnetic resonance imaging in combination with cognitive
behavioral data showed that the lipid content of dlPFC gray matter is lower in SZ patients than in controls and
correlates with a tendency towards reduced accuracy in the dlPFC-dependent task-switching test. We conclude that
aberrations in LXR/RXR-regulated lipid metabolism lead to a decreased lipid content in SZ dlPFC that correlates with
reduced cognitive performance. A R T I C L E O p e n A c c e s s Key role for lipids in cognitive symptoms
of schizophrenia Dorien A. Maas
1,2,3, Marijn B. Martens4, Nikos Priovoulos5, Wieteke A. Zuure1, Judith R. Homberg3,
Brahim Nait-Oumesmar
2 and Gerard J. M. Martens1,4 Correspondence: Dorien A. Maas (d.a.maas@outlook.com)
1Faculty of Science, Centre for Neuroscience, Department of Molecular Animal
Physiology, Donders Institute for Brain, Cognition and Behavior, Radboud
University Nijmegen, Geert Grooteplein Zuid 26-28, 6525 GA Nijmegen, The
Netherlands
2Sorbonne Université, Paris Brain Institute – ICM, Inserm U1127, CNRS UMR
7225, Hôpital Pitié-Salpêtrière, Paris, France
Full list of author information is available at the end of the article Note: To cite this publication please use the final published version (if applicable). Maas et al. Translational Psychiatry (2020) 10:399
https://doi.org/10.1038/s41398-020-01084-x Translational Psychiatry Introduction negative and cognitive symptoms of SZ8. Cognitive symp-
toms and related changes in the prefrontal cortex (PFC) of
SZ patients are already present before disease onset9 and
contribute negatively to functional outcome10–13. Cognitive
deficits are found in individuals at high risk to develop SZ14
and family members of SZ patients15, albeit to a lower
degree. The various subregions of the PFC are involved in
deficits in specific cognitive domains16. For example,
although ventro-lateral PFC functioning remains largely
unaffected,
impaired
dorsolateral
(dl)PFC-dependent
processes are thought to underlie a range of cognitive
deficits in SZ17–19. In addition, dlPFC activation during
the performance of cognitive tasks is decreased in SZ
patients18,20,21. Schizophrenia (SZ) is a psychiatric disorder with a con-
voluted etiology and a lifetime prevalence of 0.84%. It is
thought that an interplay between genetic, epigenetic, and
environmental risk factors is involved in SZ etiology1. Symptoms of SZ include positive, negative, and cognitive
symptoms2. The positive symptoms comprise delusions
and hallucinations3, the negative symptoms are a loss of
typical affective functions2, and the most prominent cog-
nitive symptoms of SZ are deficits in attention4 and
executive functioning5–7. There are currently no effective
pharmacological
treatment
strategies
that
target
the Correspondence: Dorien A. Maas (d.a.maas@outlook.com)
1Faculty of Science, Centre for Neuroscience, Department of Molecular Animal
Physiology, Donders Institute for Brain, Cognition and Behavior, Radboud
University Nijmegen, Geert Grooteplein Zuid 26-28, 6525 GA Nijmegen, The
Netherlands Transcriptomic studies on the PFC of SZ patients have
increased our understanding of the molecular mechan-
isms
contributing
to
the
PFC-dependent
cognitive
impairment in SZ. The majority of transcriptomic studies
performed
on
SZ
dlPFC
(RNA
sequencing22–29
or © The Author(s) 2020 © The Author(s) 2020
OpenAccessThisarticleislicensedunderaCreativeCommonsAttribution4.0InternationalLicense,whichpermitsuse,sharing,adaptation,distributionandreproduction
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/. Maas et al. Translational Psychiatry (2020) 10:399 Page 2 of 12 microarray30–32 analyses) have been conducted on a mix
of gray and white matter. However, gray and white matter
display discrete gene expression patterns33, and therefore
investigating the transcriptome of a gray and white-matter
mix does not allow the detection of gene expression dif-
ferences that arise from and are specific to either gray or
white matter. One transcriptomic study has been per-
formed on SZ PFC gray matter, but did not specify the
PFC subregion that was used34. Yet, spatial differences in
gene expression patterns exist throughout the cortex35
and PFC subregions have distinct contributions to the
cognitive deficits in SZ16. Only two transcriptomic studies
published to date have analyzed solely the gray matter of
the SZ dlPFC subregion, with one study reporting dif-
ferences in the axon guidance pathway36 and the other
analyzing the expression of only the delta 4-desaturase,
sphingolipid 2 (DEGS2) gene37. mapped reads. Gene expression levels (fragments per
kilobase of transcript per million mapped reads) were
calculated using RSEM and NOIseq algorithms were then
used to determine genes differentially expressed in SZ
patients and controls. Significantly differentially expressed
genes (probability > 0.8) were used for analysis with the
Ingenuity Pathway Analysis software package (Qiagen). RNA sequencing and RNA-sequencing data analysis were
performed by researchers that were blinded for disease
state. RNA-sequencing data are freely available through
https://doi.org/10.6084/m9.figshare.12640460.v1 Samples and RNA sequencing Human post-mortem dlPFC brain tissue from four
chronic SZ patients and four control individuals was
obtained from the Dutch Brain Bank (Amsterdam, The
Netherlands). Sample size was based on tissue availability. Sections of 300 µm were obtained in a cryostat (Leica) at
−15 °C and two to three punches were collected from
different places in the gray matter and in the white matter
using a 2.00 mm punch needle (Harris). Punches were
frozen on dry ice and stored at −80 °C until RNA isolation
using RNeasy lipid tissue mini kit (74804 Qiagen). Isolated
RNA was sent for quality control, RNA sequencing, and
bioinformatics data analysis to BGI Genomics. Agilent
2100 Bio Analyzer was used to determine RNA quality
and RNA integrity numbers of all RNA samples were 6.7
or
higher. RNA
sequencing
was
performed
using
BGISEQ-500 platform generating 6.71 Gb bases per
sample. Using hierarchical indexing for spliced alignment
of transcripts or HISAT, clean reads were mapped to the
reference genome UCSC HG38 with an average of 92.06% Quantitative real-time PCR For quantitative real-time PCR (qPCR) analysis, per
sample 350 μg RNA was treated with DNase I (Fermentas)
and cDNA was synthesized using the Revert Aid H-minus
first-strand cDNA synthesis kit (Thermo Scientific). cDNA was subsequently diluted 1 : 20 in MilliQ H2O and
stored at −20 °C until qPCR analysis. qPCR samples
contained 2.0 μL diluted cDNA, 0.8 μL 5 μM forward
primer, 0.8 μL 5 μM reverse primer, 5 μL SybrGreen mix
(Roche), and 1.8 μL MilliQ H2O. qPCR was performed
with a Rotor Gene 6000 Series (Corbett Life Sciences)
using a three-step paradigm with a fixed gain of 8. Fifty
cycling steps of 95, 60, and 72 °C were applied and
fluorescence was acquired after each cycling step. Primers
were designed with NCBI Primer-Blast and synthesized by
Sigma (for primer pair sequences, see Supplementary
Table S1). Melting temperature was used to check whe-
ther a single PCR product was generated and the take off
and amplification values of the housekeeping genes (Ppia
and Gapdh) were used to determine the normalization
factor with GeNorm38 after which normalized mRNA
expression levels were calculated. qPCR data were ana-
lyzed using Levene’s test for equality of variances and two-
tailed independent samples T-tests in SPSS Statistics 21. Individual data points and means were plotted using
Graphpad Prism 4. Researchers were blinded for disease
state during qPCR analysis. In the current study, we sequenced the transcriptome of
the gray matter of dlPFC in SZ and controls. As we found
that the differentially expressed genes were enriched in
Liver
X
Receptor/Retinoid
X
Receptor
(LXR/RXR)-
mediated lipid metabolism genes, we next investigated
whether SZ has a genetic link with lipid metabolism. We
indeed identified shared genetic etiology between SZ and
among other acylcarnitines, very-low-density lipoprotein
(VLDL)
lipids,
and
fatty
acid
metabolites. Finally,
exploratory analyses of structural magnetic resonance
imaging (MRI) data were in accordance with a lower lipid
content of the dlPFC gray matter in SZ patients as com-
pared to controls and correlated with reduced cognitive
performance. Thus, distortions in lipid homeostasis play a
key role in the cognitive symptoms of SZ. Materials and methods
Samples and RNA sequencing Samples and RNA sequencing Shared genetic etiology Using
PRSice,
single-nucleotide
polymorphisms
(SNPs) were clumped to remove linkage disequilibrium
(LD) with an LD threshold of 0.1, a distance threshold of
250 kb, and the 1000 Genomes Project data as genotype
reference47. A range of SNP significance thresholds was
used (pT < 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5) to calculate
shared genetic etiology and the p-values obtained using
these thresholds were corrected with Bonferroni multiple
comparisons correction for the number of metabolites
tested. dlPFC was extracted from the atlas and projected to each
individual scan. The dlPFC regions of interest were cor-
rected at the individual level with a gray-matter mask
made with FSL-FAst and the output was visually verified. The average MP-RAGE signal in the dlPFC gray matter of
SZ patients and controls was examined. Two linear
models were fitted including the average left and right
gray-matter dlPFC MP-RAGE signal as the dependent
variable and age, sex and group as the independent vari-
ables. A retrospective motion-estimate (Average Edge
Strength) was also calculated with the homonymous
Matlab toolbox51 and entered as an independent variable. The analyses were repeated for data acquired at both 3T
scanners (Trio, Siemens Healthineers). We then utilized a
linear model to analyze the correlation between dlPFC
gray-matter MP-RAGE signal and accuracy in the task-
switching test in SZ patients accounting for age and
motion. For details on the task-switching test, see ref. 48. Individual data points and means were plotted using
Graphpad Prism 4. Researchers were blinded for disease
state during data analysis. RNA sequencing reveals LXR/RXR activation as the top-
enriched canonical pathway in gray matter of SZ dlPFC RNA sequencing reveals LXR/RXR activation as the top-
enriched canonical pathway in gray matter of SZ dlPFC The Consortium for Neuropsychiatric Phenomics made
available an MRI dataset including 125 healthy individuals
(median age = 28 years old, 53% female) and 50 indivi-
duals (median age = 37.5 years old, 76% female) diag-
nosed with SZ or schizoaffective disorder. This dataset
includes a T1-weighted magnetization prepared–rapid
gradient echo (MP-RAGE) sequence (repetition time =
1.9 s,
echo
time = 2.26 ms,
field-of-view = 250 mm,
matrix = 256 × 256, slice thickness = 1 mm, 176 slices), as
well as cognitive behavioral data from the task-switching
test. For details on the dataset, see ref. 48. For all MRI
analyses, open source code was used. The MP-RAGEs
were corrected for B0/B1 inhomogeneities using the N4
algorithm. A study-specific template of the MP-RAGE
scans was created in the common space between the scans
with an iterative diffeomorphic warp estimate using the
ANTS package49. The template was diffeomorphically
registered to the MarsAtlas50. A segmentation of the enriched canonical pathway in gray matter of SZ dlPFC
RNA sequencing was performed on gray matter from
dlPFC of four SZ patients and four controls (see Supple-
mentary Table S2 for subject and tissue characteristics). Gene expression density was similar for all samples
(Supplementary Fig. S1a) and differential expression
analysis showed 132 significantly upregulated genes and
5 significantly down-regulated genes in SZ dlPFC gray
matter (Supplementary Fig. S1b). Ingenuity pathway
analysis of the significantly differentially expressed genes
revealed that “LXR/RXR activation” was the most sig-
nificantly enriched canonical pathway in the dlPFC of SZ
patients (p = 3.89E-07 in Benjamini–Hochberg corrected
T-test; see Table 1 for the top five canonical pathways
with statistical values and molecules involved); the other
canonical pathways were at least 30 times less enriched. The LXR/RXR pathway regulates cholesterol homeostasis
in the brain. The increased abundance of transcripts that Table 1
Ingenuity pathway analysis of genes differentially expressed in SZ vs. control dlPFC gray matter. Canonical pathway
p-value (Benjamini–Hochberg corrected)
Genes
LXR/RXR activation
3.89E −07
AGT, APOC2, C4A/C4B, IL1RL1, S100A8, SERPINA1, TNFRSF11B
Complement system
1.15E −05
C1QA, C1QB, C1QC, C4A/C4B
Antigen presentation pathway
1.43E −05
HLA-DMA, HLA-DQB1, HLA-DRB3, HLA-DRB5
PD1-PD-L cancer immunotherapy pathway
5.45E −05
HLA-DMA, HLA-DQB1, HLA-DRB3, HLA-DRB5, TNFRSF11B
T-helper cell differentiation
1.71E −04
HLA-DMA, HLA-DQB1, HLA-DRB5, TNFRSF11B Table 1
Ingenuity pathway analysis of genes differentially expressed in SZ vs. control dlPFC gray matter. Shared genetic etiology Two SZ genome-wide association studies (GWASs) and
four metabolite GWAS datasets were used to calculate
shared genetic etiology between SZ and metabolite levels. We first calculated shared genetic etiology between 561
metabolites and SZ using previously published SZ GWAS
data that was obtained from 33,426 SZ patients from
European ancestry39. We then replicated the calculation
using a second SZ GWAS dataset, namely the GWAS data
from 36,989 SZ patients as provided by the Psychiatric
Genomics
Consortium40,
which
includes
the
same
patients from European descent, but also includes indi-
viduals with East-Asian ancestry. The metabolite GWAS
data were obtained from Rhee et al.41 including 268 Maas et al. Translational Psychiatry (2020) 10:399 Page 3 of 12 Page 3 of 12 metabolite GWASs, Draisma et al.42 including 129
metabolite GWASs, Kettunen et al.43 including 123
metabolite GWASs, and Ahola-Olli et al.44 including 41
cytokine GWASs, and included 2076, 7478, and 24,925
participants of European decent, and 2019 Finnish parti-
cipants, respectively. Shared genetic etiology was calcu-
lated using the freely available program PRSice version
1.2345 with PLINK version 1.9 and based on the method
of Johnson et al.46. Metabolite GWAS data were taken as
base samples and SZ GWAS data as the target sample,
and correlation results were weighted by the SZ group
size. Using
PRSice,
single-nucleotide
polymorphisms
(SNPs) were clumped to remove linkage disequilibrium
(LD) with an LD threshold of 0.1, a distance threshold of
250 kb, and the 1000 Genomes Project data as genotype
reference47. A range of SNP significance thresholds was
used (pT < 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5) to calculate
shared genetic etiology and the p-values obtained using
these thresholds were corrected with Bonferroni multiple
comparisons correction for the number of metabolites
tested. metabolite GWASs, Draisma et al.42 including 129
metabolite GWASs, Kettunen et al.43 including 123
metabolite GWASs, and Ahola-Olli et al.44 including 41
cytokine GWASs, and included 2076, 7478, and 24,925
participants of European decent, and 2019 Finnish parti-
cipants, respectively. Shared genetic etiology was calcu-
lated using the freely available program PRSice version
1.2345 with PLINK version 1.9 and based on the method
of Johnson et al.46. Metabolite GWAS data were taken as
base samples and SZ GWAS data as the target sample,
and correlation results were weighted by the SZ group
size. RNA sequencing reveals LXR/RXR activation as the top-
enriched canonical pathway in gray matter of SZ dlPFC control dlPFC gray and white matter. a Normalized mRNA expression of
angiotensinogen (Agt), apolipoprotein C2 (Apoc2), and complement 4b (C4b) in SZ vs. control dlPFC gray matter. These mRNAs are components of
the canonical pathway “LXR/RXR activation” in the Ingenuity pathway analysis. b Normalized mRNA expression of the LXR/RXR signaling cascade
components Liver X receptor β (Lxrβ), retinoid X receptor β (Rxrβ), ATP-binding cassette transporter A1 (Abca1), peripheral myelin protein 22 (Pmp22),
apolipoprotein E (ApoE), and sterol regulatory element-binding protein 1 (SREBP1) in SZ vs. control dlPFC gray matter. c, d Normalized mRNA
expression of the same genes as in a and b in the white matter of dlPFC. n = 4 samples per group, #p < 0.1, *p < 0.05 in independent samples T-test. are associated with activation of the LXR/RXR pathway
indicates a change in cholesterol metabolism in SZ dlPFC
gray matter. p = 0.885, df = 3.378). The LXRβ/RXRβ pathway acti-
vates the transcription factor sterol regulatory element-
binding proteins (e.g., SREBP1) and as such stimulates
cholesterol and oxysterol efflux from the cell via ATP-
binding cassette transporter A1 (Abca1), which is
regulated by peripheral myelin protein 22 (Pmp22)52–54. Upon efflux from the cell, cholesterol is packed in the
brain in high-density lipoprotein (HDL)-like particles
containing apolipoproteins, predominantly apolipopro-
tein E (ApoE)52. Abca1 and Pmp22, but not Srebp1 and
Apoe, mRNA expression were upregulated in SZ vs. control dlPFC gray matter (Fig. 1b; independent samples
T-test Srebp1 t = 1.047, p = 0.335, df = 6, Apoe t =
1.606, p = 0.206, df = 3.032, Abca1 t = 3.836, p = 0.023,
df = 3.538, Pmp22 t = 2.219, p = 0.068, df = 6), indicat-
ing increased cholesterol efflux in SZ dlPFC gray matter. Notably, in the dlPFC white matter, no changes in LXR/
RXR-related mRNA expression were found (Fig. 1c, d),
highlighting
the
importance
of
studying
mRNA g
y
Upregulation of the “LXR/RXR activation” canonical
pathway components angiotensinogen (Agt), apolipopro-
tein C2 (Apoc2), and complement 4b (C4b) in SZ vs. control dlPFC gray matter was confirmed by qPCR
(Fig. 1a; independent samples T-test t = 2.407, p = 0.053,
df = 6, t = 2.673, p = 0.056, df=3.986, t = 2.155, p =
0.083, df=3.059, respectively; see Supplementary Table S1
for primer sequences). We next investigated whether
other mRNAs related to LXR/RXR activation were also
differentially expressed in SZ dlPFC gray matter. RNA sequencing reveals LXR/RXR activation as the top-
enriched canonical pathway in gray matter of SZ dlPFC Canonical pathway
p-value (Benjamini–Hochberg corrected)
Genes
LXR/RXR activation
3.89E −07
AGT, APOC2, C4A/C4B, IL1RL1, S100A8, SERPINA1, TNFRSF11B
Complement system
1.15E −05
C1QA, C1QB, C1QC, C4A/C4B
Antigen presentation pathway
1.43E −05
HLA-DMA, HLA-DQB1, HLA-DRB3, HLA-DRB5
PD1-PD-L cancer immunotherapy pathway
5.45E −05
HLA-DMA, HLA-DQB1, HLA-DRB3, HLA-DRB5, TNFRSF11B
T-helper cell differentiation
1.71E −04
HLA-DMA, HLA-DQB1, HLA-DRB5, TNFRSF11B Table 1
Ingenuity pathway analysis of genes differentially expressed in SZ vs. control dlPFC gra Maas et al. Translational Psychiatry (2020) 10:399 Page 4 of 12 Fig. 1 Expression of LXR/RXR-related mRNAs in SZ vs. control dlPFC gray and white matter. a Normalized mRNA expression of
angiotensinogen (Agt), apolipoprotein C2 (Apoc2), and complement 4b (C4b) in SZ vs. control dlPFC gray matter. These mRNAs are components of
the canonical pathway “LXR/RXR activation” in the Ingenuity pathway analysis. b Normalized mRNA expression of the LXR/RXR signaling cascade
components Liver X receptor β (Lxrβ), retinoid X receptor β (Rxrβ), ATP-binding cassette transporter A1 (Abca1), peripheral myelin protein 22 (Pmp22),
apolipoprotein E (ApoE), and sterol regulatory element-binding protein 1 (SREBP1) in SZ vs. control dlPFC gray matter. c, d Normalized mRNA
expression of the same genes as in a and b in the white matter of dlPFC. n = 4 samples per group, #p < 0.1, *p < 0.05 in independent samples T-test. Fig. 1 Expression of LXR/RXR-related mRNAs in SZ vs. control dlPFC gray and white matter. a Normalized m gray and white matter. a Normalized mRNA expression of Fig. 1 Expression of LXR/RXR-related mRNAs in SZ vs. control dlPFC gray and white matter. a Normalized mRNA expression of
angiotensinogen (Agt), apolipoprotein C2 (Apoc2), and complement 4b (C4b) in SZ vs. control dlPFC gray matter. These mRNAs are components of
the canonical pathway “LXR/RXR activation” in the Ingenuity pathway analysis. b Normalized mRNA expression of the LXR/RXR signaling cascade
components Liver X receptor β (Lxrβ), retinoid X receptor β (Rxrβ), ATP-binding cassette transporter A1 (Abca1), peripheral myelin protein 22 (Pmp22),
apolipoprotein E (ApoE), and sterol regulatory element-binding protein 1 (SREBP1) in SZ vs. control dlPFC gray matter. c, d Normalized mRNA
expression of the same genes as in a and b in the white matter of dlPFC. n = 4 samples per group, #p < 0.1, *p < 0.05 in independent samples T-test. /RXR-related mRNAs in SZ vs. control dlPFC gray and white matter. a Normalized mRNA expression of Fig. 1 Expression of LXR/RXR-related mRNAs in SZ vs. RNA sequencing reveals LXR/RXR activation as the top-
enriched canonical pathway in gray matter of SZ dlPFC LXRβ is
the isoform of LXR that is expressed most abundantly in
the brain and forms heterodimers with RXRβ52. We found
an upregulation of Rxrβ, but no changes in the mRNA
expression of Lxrβ in the SZ dlPFC gray matter as com-
pared to controls (Fig. 1b; independent samples T-test
Rxrβ
t = 2.202,
p = 0.070,
df = 6,
Lxrβ
t = 0.156, Maas et al. Translational Psychiatry (2020) 10:399 Page 5 of 12 Page 5 of 12 free-water pool and the macromolecule pool. Following
the saturation, the macromolecular pool quickly relaxes
and subsequently accelerates the relaxation of the free-
water pool in a process termed magnetization transfer. We hypothesized that a difference in lipid content and
thus macromolecular pool would contribute to a change
in magnetization transfer. We tested this by comparing
the dlPFC gray-matter MP-RAGE signal between SZ
patients and controls. We found that the MP-RAGE sig-
nal was significantly decreased in the dlPFC gray matter of
SZ patients as compared to controls, both in the left and
right hemispheres, and accounting for age, sex, motion,
and scanning site (Fig. 2a and Supplementary Table S6;
linear model left dlPFC estimate = −26.025, t = −4.433,
p < 0.001, right dlPFC estimate = −25.249, t = −4.319,
p < 0.001; Supplementary Fig. S2). These results are in
accordance with a lower macromolecular content and
thus a lower lipid content of the SZ dlPFC gray matter. Notably, we found a correlation between the accuracy on
the dlPFC-dependent task-switching test and the MP-
RAGE signal in both the left and right dlPFC accounting
for age and motion (Fig. 2b; linear model left dlPFC
estimate = 4.286, t = 1.946, p = 0.0579, right dlPFC esti-
mate = 4.330, t = 1.969, p = 0.0551). These data are con-
sistent with a lower lipid content of the SZ dlPFC gray
matter that correlates with a reduced accuracy in the
dlPFC-dependent task-switching test and as such is in line
with an important role for a distorted lipid metabolism in
the cognitive deficits of SZ. expression patterns in the gray and white matter
separately. In addition to the LXR/RXR activation pathway, the
canonical pathway analysis revealed significant enrich-
ment of four immune-related pathways in SZ dlPFC gray
matter (Table 1), in line with the dysregulation of mRNA
expression of genes related to inflammation and the
immune system in SZ PFC25,38. Discussion SZ is a psychiatric disorder with an unknown etiology
and its cognitive deficits are associated with the dlPFC. Here we performed RNA sequencing of post-mortem
dlPFC gray matter of SZ patients and controls to gain
insight into the molecular mechanisms contributing to
the cognitive dysfunction in SZ. We found an enrichment
of differentially expressed genes in the LXR/RXR activa-
tion pathway and validated upregulation of components
of the LXR/RXR lipid metabolism pathway in SZ dlPFC
gray, but not white, matter. We further revealed shared
genetic etiology between SZ and a number of lipid-related
metabolites, confirming a genetic link between SZ and
lipid metabolism. Finally, the results obtained with the
MP-RAGE signals from structural MRI data are in
accordance with a decreased lipid content in the dlPFC
gray matter of SZ patients and correlated with reduced
performance in the task-switching cognitive test. Shared genetic etiology between SZ and lipid metabolism Shared genetic etiology between SZ and lipid metabolism S a ed ge et c et o ogy bet ee
S
a d
p d
etabo s
To further investigate the role of lipid metabolism in
SZ, we analyzed the shared genetic etiology between SZ
and 514 circulating metabolites, including amino acids,
nutrients, organic compounds, cytokines, growth factors,
and lipids. Following Bonferroni correction (Table 2 and
Supplementary Table S4), we found significant overlap
between genetic risk for SZ and 35 metabolites (Table 2
and Supplementary Table S4; p < 0.05) using the results
of the 2018 SZ GWAS study published by the Bipolar
and Schizophrenia working group of the Psychiatric
Genomics Consortium39. Using a second 2014 SZ
GWAS dataset provided by the Psychiatric Genomics
Consortium40, the genetic association between SZ risk
and 25 of the 35 metabolites was replicated, and 21
additional metabolites that share genetic etiology with
SZ were identified (Table 2 and Supplementary Table
S5). Strikingly, the 56 metabolites that share significant
genetic etiology with SZ are all related to lipids (except
for IP10 and IL16) and fall within three themes: acyl-
carnitines, VLDL lipids, and fatty acid metabolites. We
conclude that disruptions in lipid homeostasis are
genetically associated with SZ. The finding that two
immune-related metabolites IP10 and IL16 (Table 2)
share genetic etiology with SZ is in line with the invol-
vement of the immune system in the disorder. RNA sequencing reveals LXR/RXR activation as the top-
enriched canonical pathway in gray matter of SZ dlPFC Furthermore, the top
upstream regulator in the Ingenuity pathway analysis was
interferon-γ (Supplementary Table S3; p = 2.22E −16)
and this and other proinflammatory cytokines are asso-
ciated with SZ55–57. Lipid content of dlPFC gray matter is lower in SZ than in
controls and correlates with reduced accuracy in the task-
switching test We further investigated the effect of the disrupted lipid
homeostasis in SZ dlPFC gray matter using a publicly
available
dataset
from
the
Consortium
for
Neu-
ropsychiatric Phenomics. This dataset contains amongst
others structural MRI scans and performance in the task-
switching cognitive test of 50 SZ patients and 125 control
individuals48. From this dataset, we analyzed the T1-
weighted MP-RAGE signal. The macromolecular pool in
the brain consists mainly of lipids, as illustrated by the
typical gray–white matter contrast obtained in T1-
weighted MRI scans. The T1 inversion pulse saturates the Gray and white matter have a different cellular com-
position and function, and distinct transcriptomes33. Gray
matter of the cortex consists mainly of neurons and glial
cells, while white matter consists primarily of myelinated
axons. Previous RNA-sequencing studies on mixes of gray
and white matter from the dlPFC of SZ patients and Maas et al. Translational Psychiatry (2020) 10:399 Page 6 of 12 Table 2
Metabolites that share significant genetic etiology with SZ. Metabolite
Lowest significant p-value
treshold
Bonferroni- corrected
p-value
Lowest significant p-value
treshold
Bonferroni- corrected
p-value
SZ GWAS 201840
SZ GWAS 201448
C5.1.DC1
0.1
0.000246
0.05
0.015176
IP105
0.3
0.000997
0.05
0.048767
CH2.DB.ratio2
0.1
0.00153
0.1
0.014121
LPE16_0_LIPID4
0.001
0.001988
0.001
0.000768
XS.VLDL.TG3
0.2
0.002406
0.3
0.039835
C14.1.OH1
0.05
0.002534
0.05
0.008502
DB.in.FA2
0.1
0.003962
0.1
0.010729
XS.VLDL.P3
0.3
0.004685
0.2
0.008044
IDL.C3
0.05
0.006454
NA
NA
PC38_2_LIPID4
0.3
0.007062
0.2
0.000573
CH2.in.FA2
0.2
0.008944
0.1
0.006678
Bis.DB.ratio2
0.2
0.009453
0.05
0.018628
DHA2
0.4
0.010114
0.05
0.00935
SM.C26.04
0.1
0.010245
0.05
0.002634
XS.VLDL.L3
0.05
0.010881
0.2
7.49E-05
TAG54_6_LIPID4
0.001
0.016009
0.001
0.009793
SM.OH.C24.14
0.1
0.017865
0.05
0.006078
FAw34
0.05
0.01807
0.05
0.002175
fumarate_maleate_valerat_CMH
0.05
0.019523
0.05
0.001032
Ratio_PC3806_LPC2206_LIPID4
0.05
0.023256
NA
NA
IDL.FC3
0.4
0.024663
NA
NA
LPC20_3_LIPID4
0.3
0.026304
NA
NA
XS.VLDL.PL3
0.05
0.027834
0.2
0.00178
XL.VLDL.TG3
0.3
0.032077
0.3
0.001178
PC32_0_LIPID4
0.1
0.032928
0.1
0.000627
PC.ae.C44.34
0.1
0.034516
NA
NA
IDL.L3
0.1
0.034726
NA
NA
S.VLDL.C3
0.4
0.035649
0.4
0.047852
IDL.P3
0.1
0.036891
NA
NA
lysoPC.a.C20.44
0.3
0.039526
0.2
0.019734
Bis.FA.ratio2
0.1
0.040777
0.05
0.005426
S.VLDL.L3
0.5
0.0408
NA
NA
GROa
0.001
0.042828
NA
NA
MCP1
0.001
0.045366
0.001
0.001432
LPC22_6_LIPID4
0.1
0.048797
NA
NA
Cit
NA
NA
0 05
0 00031 Table 2
Metabolites that share significant genetic etiology with SZ. Page 7 of 12 Maas et al. Lipid content of dlPFC gray matter is lower in SZ than in
controls and correlates with reduced accuracy in the task-
switching test Translational Psychiatry (2020) 10:399 Table 2 continued
Metabolite
Lowest significant p-value
treshold
Bonferroni- corrected
p-value
Lowest significant p-value
treshold
Bonferroni- corrected
p-value
SZ GWAS 201840
SZ GWAS 201448
PC38_6_LIPID4
NA
NA
0.1
0.002651
CE20_5_LIPID3
NA
NA
0.2
0.002948
TAG56_6_LIPID4
NA
NA
0.2
0.005974
PC40_6_LIPID4
NA
NA
0.3
0.008719
PC.aa.C24.04
NA
NA
0.1
0.010428
TAG56_8_LIPID4
NA
NA
0.2
0.012203
IL165
NA
NA
0.05
0.01252
TAG58_10_LIPID4
NA
NA
0.3
0.014247
TAG56_6_LIPID4
NA
NA
0.3
0.020381
XXL.VLDL.PL3
NA
NA
0.3
0.027232
L.VLDL.P3
NA
NA
0.05
0.030895
XL.HDL.L3
NA
NA
0.5
0.032042
aconitate_CMH
NA
NA
0.1
0.032102
XL.VLDL.L3
NA
NA
0.4
0.03703
TAG58_11_LIPID4
NA
NA
0.3
0.037887
PC38_4_LIPID4
NA
NA
0.4
0.038142
LDL.D3
NA
NA
0.001
0.042824
FAw64
NA
NA
0.05
0.044601
ADP_CMH
NA
NA
0.4
0.049512
1A
l
i i hallucinations, but not between controls and SZ patients
without auditory hallucinations36. This highlights that
mRNA expression in dlPFC gray matter might differ
among subgroups of SZ patients. As in the present study
we did not compare subgroups of SZ patients, we may
have missed more subtle mRNA expression differences
between patients and controls. In addition, for unknown
reasons we did not find the previously reported decreased
mRNA expression of the SZ-associated DEGS2 gene in SZ
dlPFC gray matter37 nor the decreased expression of
sodium channel subunit SCN2A, the latter probably due
to the fact that we studied a different PFC subregion34. Thus, future transcriptomic studies should include SZ
patient subgroups and various PFC subregions. controls have shown among others altered abundance of
transcripts involved in glucocorticoid signaling29, pre-
synaptic function32, inflammation25, nuclear receptor
signaling23, synaptic vesicle recycling, transmitter release,
and cytoskeletal dynamics31. Our RNA-sequencing study
on only the dlPFC gray matter confirms the dysregulation
of inflammation-related genes in SZ. More importantly,
among the differentially expressed dlPFC gray-matter
genes the most enriched pathway involved LXR/RXR-
mediated cholesterol lipid homeostasis. LXR/RXR-related
genes were upregulated in the dlPFC gray matter of SZ
patients, but were unaltered in the dlPFC white matter. The previous transcriptomic studies on a mix of SZ dlPFC
gray and white matter have likely missed the enrichment
of this pathway because of the relatively high contribution
of the lipid content of the white matter. The LXR/RXR pathway is activated by binding of oxy-
sterols to LXR. Oxysterols are metabolites produced
during the breakdown of cholesterol and able to cross the
blood brain barrier. Lipid content of dlPFC gray matter is lower in SZ than in
controls and correlates with reduced accuracy in the task-
switching test In the brain, LXRβ forms hetero-
dimers with RXRβ and their activation leads to increased Interestingly, dlPFC gray-matter mRNA expression
differences in the axon guidance pathway are known to
exist between controls and SZ patients with auditory Maas et al. Translational Psychiatry (2020) 10:399 Page 8 of 12 Fig. 2 MP-RAGE signal in SZ and control dlPFC gray matter and correlation with task-switching accuracy. a Left: schematic representation of
the dlPFC gray matter in the left and right brain hemispheres. Right: average MP-RAGE signal from the left and right dlPFC gray matter in SZ versus
control (CON). **p < 0.001 in a linear model corrected for sex, age, motion, and scanning site. b Scatter plot of the accuracy in the task-switching test
and the MP-RAGE signal from the dlPFC gray matter in the left and right hemisphere of SZ patients. Fig. 2 MP-RAGE signal in SZ and control dlPFC gray matter and correlation with task-switching accuracy. a Left: schematic representation of
the dlPFC gray matter in the left and right brain hemispheres. Right: average MP-RAGE signal from the left and right dlPFC gray matter in SZ versus
control (CON). **p < 0.001 in a linear model corrected for sex, age, motion, and scanning site. b Scatter plot of the accuracy in the task-switching test
and the MP-RAGE signal from the dlPFC gray matter in the left and right hemisphere of SZ patients. HDL, and apolipoprotein levels64,65, and lower short-
chain acylcarnitine levels in the blood66. Moreover, in SZ
patients using antipsychotic medication the occurrence of
metabolic syndrome is increased and cholesterol levels are
correlated with cognitive impairment63,67, implicating a
role for peripheral lipid metabolism in brain functioning
and cognitive deficits in SZ. In the present study, we find
that SZ shares genetic etiology with a number of meta-
bolites, most of which were replicated using a second SZ
GWAS study. Among the metabolites that share genetic
etiology with SZ, we found an enrichment of acylcarni-
tines, VLDL lipids and fatty acid metabolites. A previous
polygenic risk score analysis has revealed that the severity
of cognitive deficits is linked to genetic variations in genes
involved in retinoid signaling68, a pathway that, similar to
the LXR/RXR pathway, is linked to lipid metabolism. Lipid content of dlPFC gray matter is lower in SZ than in
controls and correlates with reduced accuracy in the task-
switching test Therefore, our results together with this earlier finding
highlight a genetic contribution to the observed altera-
tions in lipid homeostasis in SZ that are thus likely not
solely caused by antipsychotic treatment. efflux of cholesterol via ABCA1- and PMP22-regulated
mechanisms into HDL-like particles containing apolipo-
protein, inhibition of cholesterol uptake by the cell and
stimulation of fatty acid synthesis52–54. We indeed found
moderate upregulation of Rxrβ, Apoc2, Abca1, and Pmp22
in SZ dlPFC gray matter as compared to controls. Inter-
estingly, LXR signaling is involved in the development of
ventral midbrain dopaminergic neurons52,58 and there is a
genetic association between PMP22 and SZ59. In vitro
studies have shown contradictory effects of antipsychotics
on LXR signaling in that one study has reported an
increased mRNA expression of Abca1 and Apoe60,
whereas a second study has shown that antipsychotics
reduce cholesterol synthesis and export from the endo-
plasmic reticulum, and do not induce LXR activation61. Nevertheless, a disturbance of LXR-mediated cholesterol
homeostasis appears to play a role in SZ etiology, but
further studies are necessary. A number of links exist between a distorted lipid
homeostasis and SZ. For example, a meta-analysis has
revealed that metabolic syndrome in SZ patients, a con-
dition in which cholesterol and triglyceride levels are
abnormal, is associated with a high degree of cognitive
impairment62. Metabolic syndrome also impairs cognition
in otherwise healthy individuals63. Blood triglyceride
levels are correlated with positive symptom severity and
blood HDL levels with global functioning of SZ patients64. Unmedicated SZ patients have lower total cholesterol, Notably, acylcarnitines, fatty acid production, choles-
terol efflux into HDL-like particles, and LXR/RXR acti-
vation share a common molecular pathway (Fig. 3). During fatty acid oxidation, unsaturated fatty acids
esterify with acyl-CoA to form acylcarnitine that is sub-
sequently
transported
into
the
mitochondrial
inner
membrane. Once inside the inner mitochondrial mem-
brane, acylcarnitines are subjected to β-oxidation, which Page 9 of 12 Maas et al. Translational Psychiatry (2020) 10:399 Fig. 3 Molecular pathways involving LXR/RXR signaling, acylcarnitines, VLDL lipids, and fatty acid metabolites. Dark green rectangles
represent genes upregulated in SZ dlPFC. Light green ovals represent lipid-related metabolites that share significant genetic etiology with SZ. Arrows
indicate the nature of molecular interactions (see legend in the figure for details). References54,68,84–94 have been used to construct the molecular
pathways. Fig. 3 Molecular pathways involving LXR/RXR signaling, acylcarnitines, VLDL lipids, and fatty acid metabolites. Lipid content of dlPFC gray matter is lower in SZ than in
controls and correlates with reduced accuracy in the task-
switching test Dark green rectangles
represent genes upregulated in SZ dlPFC. Light green ovals represent lipid-related metabolites that share significant genetic etiology with SZ. Arrows
indicate the nature of molecular interactions (see legend in the figure for details). References54,68,84–94 have been used to construct the molecular
pathways. involving fatty acid oxidation and cholesterol efflux, pro-
duction, and transport may well play a role in SZ. involving fatty acid oxidation and cholesterol efflux, pro-
duction, and transport may well play a role in SZ. Using a publicly available dataset48, we show that the
T1-weighted MP-RAGE signal is significantly decreased in
SZ dlPFC gray matter. The T1-weighted MP-RAGE signal
creates contrast between gray and white matter, which is
thought to be due to magnetization transfer effects, where produces acetyl-CoA that can either enter the citric acid
cycle, or is transported to the cytosol where it participates
in lipid biosynthesis (fatty acid and cholesterol synthesis). Cholesterol can be transported out of the cell via HDL-
like particles. Based on our transcriptomic study and the
shared genetic etiology between SZ and several lipid-
related metabolites, we conclude that lipid homeostasis Using a publicly available dataset48, we show that the
T1-weighted MP-RAGE signal is significantly decreased in
SZ dlPFC gray matter. The T1-weighted MP-RAGE signal
creates contrast between gray and white matter, which is
thought to be due to magnetization transfer effects, where Using a publicly available dataset48, we show that the
T1-weighted MP-RAGE signal is significantly decreased in
SZ dlPFC gray matter. The T1-weighted MP-RAGE signal
creates contrast between gray and white matter, which is
thought to be due to magnetization transfer effects, where Maas et al. Translational Psychiatry (2020) 10:399 Page 10 of 12 Page 10 of 12 increased lipid content results in increased signal. Our
results are therefore consistent with a decreased lipid
content in the gray matter of the dlPFC of SZ patients. This conclusion does not take into account the added
effects from differences in spin density or in the relaxation
rate of the free-water pool itself to the MP-RAGE signal. However, the relaxation rate of the free-water pool itself is
largely homogeneous across the brain69,70. Our finding of
a decreased lipid content in SZ dlPFC gray matter thus
warrants future validation with quantitative magnetiza-
tion transfer methods. Notably, the decreased MP-RAGE
signal in SZ dlPFC gray matter correlates with decreased
accuracy in the task-switching test in SZ patients. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations. References 1. Modai, S. & Shomron, N. Molecular risk factors for schizophrenia. Trends Mol. Med. 22, 242–253 (2016). 1. Modai, S. & Shomron, N. Molecular risk factors for schizophrenia. Trends Mol. Med. 22, 242–253 (2016). 2. Tandon, R., Nasrallah, H. A. & Keshavan, M. S. Schizophrenia, “just the facts”
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controls and correlates with reduced accuracy in the task-
switching test The
task-switching test examines executive functioning and
relies on the dlPFC71–73. In SZ patients, reduced accu-
racy74 and reaction time differences75 in this test have
been reported. We find that in SZ altered performance in
the task-switching test might arise from a decreased lipid
content in the dlPFC. Kapittelweg 29, 6525 EN Nijmegen, The Netherlands. 4NeuroDrug Research
Ltd, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands. 5Spinoza Centre for
Neuroimaging, Meibergdreef 75, Amsterdam-Zuidoost, 1105 BK Amsterdam,
The Netherlands Kapittelweg 29, 6525 EN Nijmegen, The Netherlands. 4NeuroDrug Research
Ltd, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands. 5Spinoza Centre for
Neuroimaging, Meibergdreef 75, Amsterdam-Zuidoost, 1105 BK Amsterdam,
The Netherlands Author contributions D.A.M. and G.J.M.M. designed the project and wrote the manuscript. D.A.M. performed data acquisition and analysis, and drafted the manuscript. W.A.Z. performed data acquisition of RNA sequencing and qPCR experiments. M.B.M. performed data analysis of shared genetic etiology, N.P. performed data
analysis of structural MRI data. J.R.H. and B.N.O. contributed to supervision of
the project. G.J.M.M. supervised the project. Supplementary Information accompanies this paper at (https://doi.org/
10.1038/s41398-020-01084-x). Supplementary Information accompanies this paper at (https://doi.org/
10.1038/s41398-020-01084-x). Received: 17 February 2020 Revised: 2 October 2020 Accepted: 26 October
2020 Received: 17 February 2020 Revised: 2 October 2020 Accepted: 26 October
2020 About half of the dry weight of the brain is attributable
to lipids and about 80% of brain lipids are part of myelin
sheaths. In SZ PFC, abnormalities in myelination are
evident and decreased PFC myelin content contributes to
disease
symptomatology76,77. Furthermore,
poly-
unsaturated fatty acid levels in the blood are correlated
with white-matter integrity in frontal regions of the SZ
brain78, whereas increased LDL levels are associated with
white-matter alterations79, and white matter, as well as
myelin abnormalities in the PFC contribute to cognitive
deficits in SZ76. Myelin lipids are produced by oligoden-
drocytes (OLs) and LXRβ-knockout mice show a hypo-
myelination phenotype, because cholesterol deficiency
inhibits OL differentiation and myelination80,81. During
brain development, LXRβ is also involved in the forma-
tion of OL precursor cells82 and exerts transcriptional
control over myelin-related genes83. Therefore, abnorm-
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Susceptibilidad de afección por enjambres de medusas (Pelagia noctiluca) en las playas del litoral occidental de la provincia de Málaga
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Investigaciones geográficas/Investigaciones geográficas
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Investigaciones Geográficas
Instituto Interuniversitario de Geografía
Universidad de Alicante
Nº 77, enero-junio de 2022, pp. 239-258.
ISSN: 0213 - 4691. eISSN: 1989 - 9890.
DOI: 10.14198/INGEO.18723
Cita bibliográfica: de la Fuente Roselló, A., Sortino Barrionuevo, J.F., Reyes Corredera, S. J., & Perles Roselló, M.J. (2022). Susceptibilidad de
afección por enjambres de medusas (Pelagia noctiluca) en las playas del litoral occidental de la provincia de Málaga. Investigaciones Geográficas,
(77), 239-258. https://doi.org/10.14198/INGEO.18723
Susceptibilidad de afección por enjambres de
medusas (Pelagia noctiluca) en las playas del
litoral occidental de la provincia de Málaga
Susceptibility of affection by jellyfish swarms (Pelagia noctiluca)
on the beaches of the western coast of the province of Malaga
Ana de la Fuente Roselló1*
Juan Francisco Sortino Barrionuevo2
Sergio Jesús Reyes Corredera3
María Jesús Perles Roselló4
Resumen
En el artículo se analiza y cartografía la susceptibilidad de afección por enjambres de medusas (Pelagia
noctiluca) en las playas del litoral occidental de la provincia de Málaga. Los indicadores de peligrosidad
de aparición de enjambres se elaboran a partir de un inventario de datos empíricos de avistamientos
de medusas, registrados a través de la aplicación InfoMedusa (datos diarios para los meses estivales del
periodo 2015-2020). Se proponen distintos indicadores de peligrosidad (frecuencia y severidad), y se
calculan parámetros específicos que corrijan el problema del número desigual de observaciones en las
playas. Los resultados cartográficos muestran que el patrón de distribución espacial no es aleatorio. Se
observa una mayor frecuencia de episodios en las playas más occidentales del tramo estudiado, y el análisis
preliminar de los factores condicionantes en cada playa permiten deducir una asociación de las playas con
más propensión a recibir enjambres con la amplitud del ángulo de apertura de la misma, así como con
la orientación al suroeste del tramo de costa en el que se sitúan las playas. Estas condiciones facilitan la
entrada de las corrientes de levante procedentes del mar de Alborán, origen predominante de los enjambres.
Palabras clave: Costa del Sol; Infomedusa; cartografía de susceptibilidad; playas.
Abstract
The article analyses and maps the hazard of jellyfish swarms (Pelagia Noctiluca) on the western coast
of Malaga province. Hazard indicators are based on empirical data from jellyfish sightings recorded
with ‘InfoMedusa’ (daily observations for the summer months of the 2015-2020 period). Various hazard
indicators are proposed (frequency and severity) and specific parameters are calculated to correct the
problem of unequal numbers of beach observations and its repercussions on the representativeness of
the results. The cartographic results show that the spatial distribution pattern is not random. A higher
frequency of episodes is observed on the westernmost beaches of the area under study and a preliminary
analysis of the conditioning factors in each beach allows us to associate the beaches with the greatest
propensity to receive swarms with the width of the opening angle of these beaches – as well as the
southwest orientation of the stretch of coast where the beaches are located. These conditions facilitate the
entry of eastern currents from the Alboran sea (the main origin of the swarms).
Keywords: Costa del Sol; Infomedusa; susceptibility mapping; beaches.
1 Departamento de Geografía, Universidad de Málaga, España. anadelafuente@uma.es.* Autora para correspondencia
2 Departamento de Geografía, Universidad de Málaga, España. francis.sortino@uma.es
3 Departamento de Geografía, Universidad de Málaga, España. sergioreyes@uma.es
4 Departamento de Geografía, Universidad de Málaga, España. mjperles@uma.es
Recepción: 14/01/2021.
Aceptación: 29/07/2021.
Publicación: 26/01/2022 (en prensa: 25/11/2021).
Este trabajo se publica bajo una licencia de Creative Commons Reconocimiento 4.0 Internacional.
© Ana de la Fuente Roselló, Juan Francisco Sortino Barrionuevo, Sergio Jesús Reyes Corredera, María Jesús Perles Roselló
de la Fuente Roselló, A., Sortino Barrionuevo, J.F., Reyes Corredera, S. J. y Perles Roselló, M.J.
1. Introducción
Las costas y playas son elementos de indiscutible importancia para el desarrollo económico del territorio en el que se localizan, constituyendo un pilar básico en actividades como el turismo de sol y playa,
cuyo atractivo resulta muy sensible a la degradación de estos espacios y a cualquier cambio que genere
incertidumbre en su uso (Enríquez y Bujosa, 2020). En las últimas décadas, probablemente motivado
por la influencia del ser humano en el funcionamiento del clima y sus consecuencias sobre de los mares
y océanos, se han incrementado de una forma alarmante la presencia de floraciones de medusas que acaban por llegar hasta la costa (Dong, 2019; Mills, 2001; Purcell, 2011). En el entorno mediterráneo, estos
problemas están especialmente motivados por la medusa clavel (Pelagia noctiluca), que supone la especie
potencialmente más dañina de esta área (Bellido el al., 2020). Se trata de una de las especies de medusas
más importantes en el Mediterráneo debido a su amplia distribución, abundancia, función ecológica y
también especialmente motivado por su interacción negativa con la población (Canepa, Fuentes, Sabatés,
Piraino y Boero, 2014), siendo además una de las especies más usuales en los enjambres de medusas en
la provincia de Málaga.
El incremento de la frecuencia de aparición de floraciones es analizado por distintos autores. Brotz,
Cheung, Keisner, Pakhomov y Pauly (2012) constatan cambios en las poblaciones de medusas y analizan
la tendencia al aumento de la presencia de éstas en las costas a partir del año 1950. Asimismo, Purcell,
Uye y Lo (2007) pronostica un progresivo incremento de poblaciones de medusas en áreas costeras
asociado a la presión antropogénica sobre el mar. Kogovsek, Bogunovic y Malej (2010) analizan las
ocurrencias históricas en el mar Adriático en los últimos 200 años combinadas con variables ambientales
(temperatura, salinidad, pH, clorofila, peso seco de zooplancton y descargas principales en los ríos),
concluyendo que la periodicidad de aparición en forma de plaga se ha acortado en las últimas décadas,
aumentando así la recurrencia de las floraciones de esta especie. Los archivos de la Station Zoologique, en
el Observatorio Oceanográfico Villefranche-sur-Mer, contienen registros de años con presencia de Pelagia
noctiluca y años sin visitas de ésta, indicando que en el periodo de 200 años que va desde 1785 a 1985,
se produjeron apariciones masivas de esta especie, con una periodicidad aproximada de doce años. En
el caso concreto de la Costa del Sol, el Aula del Mar observa un incremento de frecuencia y magnitud de
apariciones de medusas en el litoral malagueño, que parece indicar un cambio de tendencia. En la última
década, ha registrado proliferaciones en periodos cíclicos de 2-4 años, cuando con anterioridad los ciclos
esperados se estimaban en 10-20 años (Aula del Mar, 2021).
La llegada de enjambres de medusas a las playas de un determinado litoral en cantidad y frecuencia
extraordinaria constituye una anomalía con consecuencias negativas sobre la sociedad, que permite clasificarlo, según la Oficina de las Naciones Unidas para la Reducción del Riesgo de Desastres (UNISDR), como
un riesgo ambiental de tipo biológico. Estos enjambres acarrean una gran problemática para las costas
que lo sufren puesto que, aparte de la pérdida de biodiversidad que conllevan, traen consigo importantes
efectos negativos en sectores como la pesca (Sabatés et al., 2010) pero sobre todo en el sector turístico
(Ghermandi, Galil, Gowdy y Nunes, 2015; Nunes et al., 2015; Rubio y Gutiérrez, 2020).
El enfoque territorial de análisis del riesgo (Cutter, 1994; Calvo, 2001; Olcina, 2008; Perles y Mérida,
2010; Veyret, Beucher y Bonnard, 2005) destaca la importancia de la posición de los elementos causantes
y receptores del daño a la hora de evaluar el riesgo. Perles y Cantarero (2010), de forma más concreta,
insisten en la importancia de conocer el patrón espacial con el que se comporta el evento peligroso. Proponen articular el patrón espacial de peligrosidad en áreas causantes de la peligrosidad, líneas o superficies transmisoras de los flujos, y áreas receptoras de los impactos del proceso. Para que un peligro incida
sobre un área receptora tiene que existir algún vector de transferencia de materia y energía que los una,
y además, el área causante ha de estar situada en una posición topológica que permita esta transferencia.
Este enfoque es idóneo para analizar la peligrosidad de llegada de los enjambres de medusas a los distintos puntos del litoral.
Existen investigaciones que se centran en el análisis y predicción del comportamiento espacial y/o temporal de los enjambres, y en la búsqueda de factores condicionantes del proceso. En este sentido, Bellido
et al. (2020) indagan en la relación entre los índices atmosféricos y la incidencia de enjambres costeros y
encuentran una conexión entre años de incidencia alta de proliferación de enjambres de medusas y lluvias
copiosas durante invierno y primavera. (Bellido et al., 2020; Gutiérrez-Estrada et al., 2021). Kogovsek et
al. (2010) analizan las ocurrencias históricas en el mar Adriático en los últimos 200 años, combinadas con
variables ambientales (temperatura, salinidad, pH, clorofila, peso seco de zooplancton y descargas principales en los ríos), concluyendo que la periodicidad de aparición en forma de plaga se ha acortado en las
últimas décadas, aumentando así la recurrencia de las floraciones de esta especie. Asimismo, Goy, Morand
y Etienne (1988) manifiestan que, usando un modelo de pronóstico (variables climáticas, temperatura,
lluvia y presión atmosférica), se pueden predecir periodos con Pelagia noctiluca.
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Susceptibilidad de afección por enjambres de medusas (Pelagia noctiluca) en las playas del litoral occidental de la provincia de Málaga
Los estudios orientados a conocer y cartografiar el comportamiento espacial de la especie con un
sentido más aplicado, puede citarse el Proyecto Medusa, que se crea en 2007, por la Agencia Catalana
del Agua en colaboración con el Instituto de Ciencias del Mar de Barcelona (ICM-CSIC), con el objeto de
controlar la presencia de medusas y entender su funcionamiento en el espacio y en el tiempo a lo largo de
la costa catalana. Este proyecto ha realizado sondeos diarios en época estival en diferentes playas, donde se
mapean las mayores concentraciones de medusas varadas. Asimismo, se incorporan en las conclusiones de
su estudio la importancia de factores como la producción de medusas en alta mar, así como las estructuras
artificiales que propician las condiciones idóneas para su crecimiento y reproducción. También se incluyen
los factores que propician su debilitamiento, como son los vientos del sureste (Canepa et al., 2014).
Con respecto a los antecedentes más próximos al área de estudio, cabe señalar el análisis realizado
en este ámbito (Rubio y Gutiérrez, 2020) que, para analizar la distribución del fenómeno, utiliza como
fuente la recurrencia de búsquedas de información sobre medusas en internet, la publicación de noticias
relacionadas de los principales periódicos de Andalucía y encuestas a usuarios de playas en la Costa del
Sol. Con este material concluye un patrón de distribución en el que destacan los avistamientos estivales,
espacialmente concentrados entre la Costa del Sol Oriental (Málaga) y la Costa Tropical (Granada). La
escala de trabajo del citado informe, a diferencia del desarrollado en la presente investigación, no se
acerca de forma expresa al litoral, ni desciende al nivel de detalle de la playa, sino se desarrolla a escala
regional. Gutiérrez-Estrada et al. (2021) enfocan su estudio al desarrollo de un sistema predictivo de la
llegada de medusas a tierra en la Costa del Sol. A una escala más detallada, el trabajo de Olmedo (2019)
constituye un referente del comportamiento y peligrosidad de los enjambres de medusas en las playas del
sector oriental de la provincia de Málaga.
A nivel de la Costa del Sol (Málaga), un antecedente directo de este trabajo es el informe presentado
por el Aula del Mar basado en la aplicación móvil InfoMedusa (https://infomedusa.es). Esta aplicación fue
creada por la Diputación Provincial en colaboración con el Aula del Mar para el seguimiento de la aparición
masiva de Pelagia noctiluca en las distintas playas que componen este litoral. Suministra datos informativos
sobre el comportamiento, evolución, trayectoria e intensidad de la plaga, suponiendo por ello una importante base para la elaboración de modelos predictivos (Bellido, Souviron, Báez, Ferri-Yañez y Salas, 2018).
En el ámbito español existen otras plataformas de recopilación de datos tales como MedusApp, Grumering
o iMedJelly, con diferentes periodos temporales y escalas espaciales en el fenómeno de avistamiento de medusas, aunque ninguna de ellas tan abundante ni extensa en el área de estudio como InfoMedusa.
A partir de la problemática causada por la llegada de enjambres a la costa, en la realización de esta
investigación, se ha considerado de interés analizar cuáles son los patrones espaciales de comportamiento
de las medusas a nivel litoral. Se pretende identificar las playas más proclives a recibir medusas como
paso previo para la identificación de factores relacionados con esta distribución, y poder proponer, en
última instancia, medidas de mitigación idóneas para estas zonas.
De forma específica, se abordan los siguientes objetivos:
1) Explotar la base de datos proporcionada por el programa InfoMedusa, para generar un inventario
básico de avistamientos de medusas en las playas del litoral occidental de la provincia de Málaga,
para el periodo 2015-2020.
2) Analizar la frecuencia de aparición de medusas en cada playa de la zona de estudio (recurrencia
diaria en periodo estival durante seis años), así como su severidad. Ajustar los indicadores de peligrosidad a las particularidades y sesgos de la fuente de datos y del inventario.
3) Elaborar una cartografía de peligrosidad por llegada de enjambres en las playas de la Costa del Sol
occidental.
4) Comprobar que la distribución espacial de la peligrosidad de afección muestra un patrón aleatorio
o, por el contrario, permite identificar tendencias estructurales en la mayor susceptibilidad de
algunas playas.
5) Proponer hipótesis preliminares sobre los factores que pueden estar condicionando la mayor predisposición a recibir medusas manifestada en algunas playas.
2. Metodología
Para la elaboración de la cartografía de peligrosidad se han desarrollado las siguientes fases de trabajo:
1) Delimitación espacial y cartografía de las playas del litoral occidental de la provincia de Málaga.
Identificación de sus principales características fisiográficas y otros factores que puedan relacionarse como posibles factores condicionantes de la mayor o menor presencia de medusas.
2) Volcado y tratamiento inicial de depuración de la base de datos proporcionada por el programa
InfoMedusa. Tránsito de la información cualitativa (comentarios) hacia cuantitativa (dato numérico) y asignación del número de registros a cada playa.
Investigaciones Geográficas, nº 77, pp. 239-258.
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de la Fuente Roselló, A., Sortino Barrionuevo, J.F., Reyes Corredera, S. J. y Perles Roselló, M.J.
3) S elección para el estudio de las playas con un número de registros válidos que garanticen la fiabilidad de los datos.
4) Análisis de la frecuencia y severidad de aparición de medusas en cada playa de la zona de estudio
(recurrencia diaria durante los meses estivales durante un periodo de seis años).
5) Elaboración de la cartografía de susceptibilidad mediante el software de procesamiento geoespacial ArcGis.
6) Análisis de posibles factores explicativos del patrón espacial de peligrosidad observado.
2.1. Selección del área de estudio
El análisis de la frecuencia y severidad de la aparición de medusas se ha aplicado al conjunto de playas
del litoral occidental malagueño. Tras una primera fase de revisión y depuración de los datos en todas
las playas, se han seleccionado, para una segunda fase de estudio, las 12 playas que reúnen una totalidad
de registros suficiente como para ser consideradas fiables, además de una casuística variada en lo que se
refiere a factores que puedan resultar explicativos de una mayor o menor propensión a recibir enjambres.
(playas de Guadalhorce, Guadalmar, Los Álamos, La Carihuela, Las Gaviotas, Los Boliches, Dunas de
Artola, El Cable, La Rada, El Cristo, Playa Ancha y Sabinillas, recogidas en la Figura 1).
Se han seleccionado playas de extensión diversa, que se distribuyen a lo largo de unos 90 km del
litoral (Tabla 1). Se ha procurado que en la muestra exista una casuística variada en función de los criterios recogidos en la guía de playas del Ministerio para la Transición Ecológica y el Reto Demográfico
(MITECO). Se ha atendido principalmente a características como el carácter, clasificando las playas en
urbana, cuando esta se encuentra integrada en el entramado urbano, semiaislada, si tiene acceso a alguna
urbanización de baja densidad edificativa y aislada, cuando no existen urbanizaciones en el frente costero.
También se han tenido en cuenta las condiciones de baño (oleaje moderado y aguas tranquilas), el tipo y
composición de la arena el cual, aunque muy similar en la totalidad del tramo de costa analizado, puede
distinguir entre arena y grava, y si se localizan en zonas protegidas. Además, en la selección, se ha procurado que las playas abarquen la práctica totalidad de los términos municipales que componen el tramo
de costa. En la Figura 2 se muestran imágenes en las que se observa las mencionadas características. De
forma añadida, la alta dependencia de este sector del territorio respecto a la actividad turística justifica
sobradamente el interés del análisis en la zona.
Se ha determinado que la unidad de análisis y representación de la peligrosidad en esta investigación
sea la playa, frente a otras opciones utilizadas en otras investigaciones similares, como por ejemplo el
municipio. La playa es la delimitación fisiográfica mínima en las que las características morfológicas y
los procesos funcionales costeros se comportan de forma sistémica, y constituyen una unidad. Por este
motivo, la playa constituye la unidad especial idónea a la hora de interpretar la mayor susceptibilidad
a recibir medusas de algunos sectores del litoral, y las causas que pueden explicar esta mayor afección.
Figura 1. Playas seleccionadas como área de estudio
Elaboración propia
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Susceptibilidad de afección por enjambres de medusas (Pelagia noctiluca) en las playas del litoral occidental de la provincia de Málaga
Tabla 1. Características de las playas objeto de estudio
Playa
Término
municipal
Longitud
(metros)
Anchura
(metros)
Grado de
urbanización
Paseo
marítimo
Condiciones
de baño
Fachada
litoral
Tipo de
arena
Composición
Zona
protegida
Descripción
Guadalhorce
Málaga
950
60
Aislada
No
Oleaje
moderado
Dunas /
Humedal
Oscura
Arena
RENPA
Paraje
natural
Playa junto a la desembocadura del río Guadalhorce, con escasa
afluencia de público.
Guadalmar
Málaga
400
30
Semiurbana
No
Oleaje
moderado
Urbana /
Dunas
Oscura
Arena
No
Forma parte de la zona conocida como playa de San Julian, cerca
de la desembocadura del río Guadalhorce. Presenta un alto grado
de urbanización
La Carihuela
Torremolinos
2100
30
Urbana
Sí
Oleaje
moderado
Urbana
Oscura
Arena
No
Playa con un alto nivel de servicios, ambiente familiar y muy frecuentada.
Los Álamos
Torremolinos
1500
60
Semiurbana
No
Oleaje
moderado
Semiurbana
Dorada
Arena
No
Playa familiar. La más alejada del centro de Torremolinos.
Las Gaviotas
Fuengirola
1200
40
Urbana
Sí
Aguas
tranquilas
Urbana
Oscura
Arena
No
Buen nivel de servicios y alta ocupación.
Los Boliches
Fuengirola
1100
40
Urbana
Sí
Aguas
tranquilas
Urbana
Oscura
Arena
No
Playa muy popular, bien equipada, en el centro urbano de Fuengirola.
Dunas de
Artola
Marbella
1200
30
Aislada
No
Oleaje
moderado
Semiurbana
Dorada
Arena
RENPA
Parque
Natural
Playa situada en una zona protegida denominada Dunas de Artola.
El Cable
Marbella
400
40
Semiurbana
No
Oleaje
moderado
Dunas /
Semiurbana
Oscura
Arena
No
Playa formada por una franja de arena estrecha, con poca afluencia
de público.
El Cristo
Estepona
700
25 a 10
Semiurbana
No
Aguas
tranquilas
Semiurbana
/ Humedal
Oscura
Arena
Zona
protegida
de interés
ornitológico
Playa del casco urbano, aunque algo retirada del centro, junto al
Puerto Deportivo de Estepona. Forma una bonita cala, regenerada
recientemente, configurando una zona abrigada y resguardada de
los vientos.
La Rada
Estepona
2300
80 a 50
Urbana
Sí
Aguas
tranquilas
Urbana
Oscura
Arena
No
Playa situada frente al casco urbano, ha sido recientemente regenerada. Muy extensa y concurrida, cuenta con gran número de
servicios.
Urbana
Oscura
Grava
Zona
protegida
de interés
ornitológico
Aguas tranquilas y gran afluencia de público. Dotada con un buen
número de servicios.
Urbana
Oscura
Arena
No
Se extiende entre el Puerto Deportivo y el río Manilva. El frente
litoral está ocupado por el núcleo de San Luis de Sabinillas.
Playa Ancha
Casares
1300
50
Urbana
Sí
Aguas
tranquilas
Sabinillas
Manilva
1700
50
Urbana
Sí
Aguas
tranquilas
Fuente: MITECO, 2021. Elaboración propia
Investigaciones Geográficas, nº 77, pp. 239-258.
243
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Figura 2. Imágenes de playas seleccionadas
Guadalhorce
Guadalmar
Dunas de Artola
Fuente: Diario Sur (s.f.)
Fuente: Turismo y Planificación
Costa del Sol SLU (s.f.)
Fuente: Consejería de Agricultura, Ganadería,
Pesca y Desarrollo Sostenible (s.f.)
Los álamos
Las Gaviotas
Los Boliches
Fuente: Diario Sur (s.f.)
Fuente: Diputación de Málaga (s.f.)
Fuente: Narrogeographic (16/09/2011)
La Carihuela
El Cable
La Rada
Fuente: Aforo Costa del Sol (s.f.)
Fuente: Costalsur (s.f.)
Fuente: Diario Sur (s.f.)
El Cristo
Playa Ancha
Sabinillas
Fuente: Turismo y Planificación
Costa del Sol SLU (s.f.)
Fuente: Ayuntamiento de Casares (s.f.)
Fuente: Inspain (s.f.)
2.2. Características de la base de datos InfoMedusa y tratamiento y depuración de los datos
La investigación se fundamenta en los datos ofrecidos por la aplicación informática InfoMedusa
(https://infomedusa.es), activada en 2013 por parte de la Diputación Provincial en colaboración con
244
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Susceptibilidad de afección por enjambres de medusas (Pelagia noctiluca) en las playas del litoral occidental de la provincia de Málaga
el Aula del Mar. La aplicación tiene como objetivo informar sobre la actividad de las medusas en las
playas del litoral malagueño y es realizada gracias a la colaboración ciudadana, que participa mediante
la introducción de comentarios acerca de la presencia o no de medusas clasificadas por playas. La base
de datos asociada constituye una importante fuente a tratar y explotar, pero requiere una labor previa de
transformación de los registros, expresados en forma de comentarios generales sobre el estado de la mar,
en datos fehacientes y concretos sobre ausencia o presencia de enjambres.
Para la utilización de la base de datos en cuestión, ha sido necesario tomar una serie de decisiones
iniciales, y aplicar una serie de procesos de depuración:
1) Interpretación y filtrado de comentarios, con la eliminación de errores y clasificación de los comentarios relevantes o útiles para el estudio. Hay que señalar que los comentarios son expresados
de forma libre por los usuarios, por lo que existe un variado patrón de observaciones.
2) Delimitación del periodo de información en el que los datos poseen la suficiente calidad como
para poder ser utilizados para un propósito científico, que se concreta en la serie temporal que
transcurre desde el año 2015 al 2020 durante los meses de junio a septiembre.
3) Selección de las playas a analizar en el tramo de estudio seleccionado en razón de la totalidad de
registros de datos de cada playa. Se han eliminado del análisis las playas del tramo occidental de la
Costa del Sol con menos de 500 registros. Este valor de exclusión se ha obtenido tomando como
referencia el total de anotaciones del año 2018, el cual supone, con diferencia, el que posee un
mayor número total de comentarios. A partir del registro total de datos del año de referencia sobre
el total de playas de la costa occidental de Málaga presentes en la aplicación (170), se ha obtenido
el valor medio de 576, 4, que se ha redondeado a la baja para facilitar el análisis.
Total comentarios año 2018: 98.004.
Total playas: 170.
Promedio: 576, 49.
4) Diseño de matriz de volcado de datos diarios depurados por playas, que contiene la serie temporal
comentada, las playas objeto de análisis y las variables seleccionadas.
Para sistematizar los datos obtenidos de los comentarios de los usuarios de la aplicación, la información se ha clasificado en cuatro categorías (Tabla 2).
Tabla 2. Clasificación de los comentarios de InfoMedusa
Clasificación de medusas
Comentario
0
Comentario irrelevante
1
No presencia de medusas
2
Presencia de medusas dispersas
3
Presencia de enjambres
Elaboración propia
Se han consultado de forma directa y manual un total de 16.726 comentarios para las playas objeto
de este estudio. Tras los procesos de filtrado y depuración de la base de datos han resultado un total de
1.885 registros útiles para el estudio.
2.3. Análisis de la frecuencia y severidad de aparición de medusas. Indicadores calculados
Para obtener datos diarios de la presencia o ausencia de medusas en cada playa en un formato binario (sí/no), los dos grados de severidad diferenciados en el inventario (Presencia de medusas dispersas/
presencia de enjambres) han sido unificados en uno sólo, ya que ambos indican la presencia de medusas.
Se han calculado distintos parámetros indicadores de la frecuencia de medusas en la playa: por una parte,
se ha procedido a ponderar el número de registro por la superficie útil de la playa, entendiendo que la
superficie de la playa puede resultar un indicador aproximado de la afluencia o potencial de visitas de
cada una de ellas. El indicador obtenido se ha denominado Número de días con medusas ponderado por la
superficie de la playa. Asimismo, para evitar el problema de la diferente afluencia en función de la localización, se ha aplicado un coeficiente de minoración para las playas alejadas de los circuitos de acceso
urbano, multiplicando por 0,5 la superficie de la playa.
Paralelamente, para el análisis comparativo de las anotaciones diarias de presencia/ausencia de medusas entre playas, se han calculado datos porcentuales relativos al total de registros en cada playa (número
Investigaciones Geográficas, nº 77, pp. 239-258.
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de días con y sin medusas para cada playa en relación al total de registros de la misma). Se han obtenido
dos indicadores: Porcentaje de días con medusas respecto al total de registros de la playa y Porcentaje de días
sin medusas respecto al total de registros de la playa. A continuación, para clasificar en intervalos los distintos indicadores de peligrosidad calculados y asignar los correspondientes grados de gravedad, se han
utilizados dos criterios: intervalos de naturaleza estadística (media y desviación estándar) en el caso de
las variables Número de días con y sin medusas ponderado por la superficie de la playa, e intervalos regulares
para el indicador Porcentaje de días con y sin medusas. En este último caso, se ha optado por estandarizar
los intervalos para el conjunto de las playas a partir del valor máximo presente en la muestra, esto es, el
porcentaje de días con medusas máximo en las playas del área de estudio, a fin de observar con mayor
claridad las diferencias entre playas y afinar la comparación entre ellas. Las Tablas 3, 4 y 5 recogen los
intervalos asignados.
Tabla 3. Media y desviación estándar de las variables número de días con y sin medusas
ponderado por la superficie de la playa
Cálculos
Número de días con medusas
Número de días sin medusas
Ʃ superficie de la playa
248,54
366,91
20,71
30,58
σ
24,38
38,47
1/2 σ
12,19
19,24
Elaboración propia
En razón de los estadísticos obtenidos, se han definido tres intervalos de gravedad del peligro (Tabla 4).
Tabla 4. Días con medusas ponderado por la superficie de la playa. Intervalos de peligrosidad
Intervalos en función
de la superficie de la playa
Días con medusas
Días sin medusas
1
<9
<12
2
10-31
12-50
3
>31
>50
Elaboración propia
Tabla 5. Porcentaje de días con y sin medusas. Intervalos asignados
Intervalos en función
de datos relativos
Días con medusas
Días sin medusas
1
< 38
<52
2
38-47
52-61
3
>47
>61
Elaboración propia
La representación gráfica y cartográfica de estos indicadores y sus intervalos se ha realizado siguiendo
la semiótica de color del semáforo. La representación cartográfica se ha ejecutado mediante el programa
de procesamiento geoespacial ArcGis.
2.4. Asignación de intervalos de peligrosidad a los distintos indicadores
Como se ha citado anteriormente, el número de registros (comentarios en InfoMedusa, 2020) no es
homogéneo entre las distintas playas. Para discriminar las playas que son más y menos fiables para este
estudio en razón del número de anotaciones de usuarios disponible, se han calculado los valores medios
de comentarios para el conjunto del área de estudio y su desviación estándar (Tabla 7). A partir de estos
datos, se han considerado, por una parte, fiables aquellas playas que tienen un número de registros en
246
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Susceptibilidad de afección por enjambres de medusas (Pelagia noctiluca) en las playas del litoral occidental de la provincia de Málaga
torno a la media menos ½ desviación estándar y/o superiores a ella. Por otra parte, se consideran playas
poco fiables las que disponen de un número inferior a la media menos ½ desviación estándar (Tabla 6).
Tabla 6. Número de registros por playa. Media y desviación estándar
Cálculos
Registros de datos
Ʃ
594,90
49,58
σ
57,37
1/2 σ
28,68
Elaboración propia
Tabla 7. Intervalos de fiabilidad de la playa en función del número de registros
Intervalos de fiabilidad
de las playas
Días con medusas
Poco fiable
< 21
Fiable
21-78
Muy fiable
>78
Elaboración propia
En una fase posterior, para evaluar la fiabilidad de los datos ofrecidos por la playa, se ha utilizado una
estrategia añadida. Se ha procedido a constatar que, si una playa presenta un elevado número de días con
medusas, no coincida con la observación contraria, esto es, que tenga a la vez un número elevado de días
sin medusas, lógica que invalidaría la consistencia de la afirmación primera. Se ha considerado que, si la
playa ofrece un número elevado de días con medusas y a la vez un número elevado de días sin medusas,
la contradicción hace perder fiabilidad a ambas observaciones. A partir de este análisis, la fiabilidad de los
datos ofrecidos por cada playa se rige por el razonamiento que se muestra en la Tabla 8.
Tabla 8. Criterios aplicados para deducir la fiabilidad de los datos en cada playa
Playa fiable
Se considera que una playa es fiable cuándo tiene un más de 21 de registros
(dato ponderado por la superficie de la playa) y una diferencia entre los días con
y sin medusas mayor al 60%.
Playa fiable proclive a las medusas
Playa con un porcentaje de registro de días con medusas mayor al 60%.
Playa fiable no proclive a las medusas
Playa con un porcentaje de registro de días sin medusas mayor al 60%.
Playa no fiable
Se considera que una playa no es fiable cuándo tiene un número de registros por
debajo de la media y/o una escasa diferencia entre los días con medusas y los
días sin medusas (del 50 al 60%)
Elaboración propia
3. Resultados
Tras el proceso de consulta directa y manual de un total de 16.726 comentarios referentes a las playas
seleccionadas, los comentarios han sido depurados y filtrados en razón de su calidad y representatividad
estadística, ofreciendo como resultado un total de 1.885 registros útiles para el estudio, datos que centran
el análisis en un total de 12 playas del litoral occidental. Como puede observarse en la Figura 3, los resultados muestran que existe una notable disparidad en el número de comentarios útiles (nº de registros)
entre las distintas playas, que oscila entre 57 en Guadalhorce y 264 en La Carihuela.
Los valores de frecuencia expresados en valores absolutos son más elevados en las playas con más
registros, tanto para el indicador de días con medusas como para su contrario, días sin medusas (Figuras
4 y 5). Este hecho resta valor comparativo a los indicadores de peligrosidad expresados en valores absolutos, tales como el Número absoluto de días con medusas representado en la Figura 6, en la que, como
puede observarse, las playas Los Álamos, La Carihuela, Los Boliches y La Rada presentan los valores más
elevados de frecuencia de afección por medusas.
Investigaciones Geográficas, nº 77, pp. 239-258.
247
de la Fuente Roselló, A., Sortino Barrionuevo, J.F., Reyes Corredera, S. J. y Perles Roselló, M.J.
Figura 3. Número de días con registros de medusas
Fuente: InfoMedusa, 2020. Elaboración propia
Figura 4. Días con medusas por playas. Medias del periodo de análisis y datos absolutos
Fuente: InfoMedusa, 2020. Elaboración propia
Figura 5. Días sin medusas por playas. Medias del periodo de análisis y datos absolutos
Fuente: InfoMedusa, 2020. Elaboración propia
248
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Susceptibilidad de afección por enjambres de medusas (Pelagia noctiluca) en las playas del litoral occidental de la provincia de Málaga
Figura 6. Días con medusas por playas. Datos absolutos
Fuente: InfoMedusa, 2020. Elaboración propia
Al objeto de matizar el problema que introduce en la interpretación el hecho de que las playas tengan
un número de registro tan dispar, se ha calculado el indicador de frecuencia ponderado por la superficie
de la playa. En la Tabla 9 se muestra el resultado de la ponderación de los días con medusas con respecto
a la superficie total de la playa, así como los cálculos realizados para la obtención del valor medio y los
intervalos de gravedad establecidos con relación al mismo. Los valores superiores a la media aparecen
resaltados en la Tabla 9.
Tabla 9. Número de días con medusas ponderado por la superficie de la playa
Playa
Días con medusas
Guadalhorce
3,81
Guadalmar
23,49
Los Álamos
7,50
La Carihuela
4,52
Las Gaviotas
9,90
Los Boliches
81,00
Dunas de Artola
42,38
El Cable
9,31
La Rada
7,29
El Cristo
28,79
Playa Ancha
13,05
Sabinillas
9,51
Ʃ
248,54
σ
24,38
1/2 σ
12,19
20,71
Elaboración propia
Investigaciones Geográficas, nº 77, pp. 239-258.
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de la Fuente Roselló, A., Sortino Barrionuevo, J.F., Reyes Corredera, S. J. y Perles Roselló, M.J.
Como resultado de la aplicación de la ponderación por superficie se ha obtenido el resultado mostrado en la Figura 7.
Figura 7. Número de días con medusas ponderado por la superficie total de la playa
Fuente: InfoMedusa, 2020. Elaboración propia
Considerando la superficie de cada playa, los datos varían notablemente, destacando sobre todo la
playa de Los Boliches y, en menor medida, Dunas de Artola, como las que más porcentaje de medusas han
registrado en función de su superficie, siendo éstas las únicas playas que se sitúan en el intervalo 3, por
encima de la media considerada. El mapa resultante clasificado en intervalos se muestra en la Figura 8.
Figura 8 Intervalos de días con medusas ponderado por la superficie total de la playa
Fuente: InfoMedusa, 2020. Elaboración propia
Sin un patrón de continuidad, se pueden distinguir tres niveles de presencia de medusas: inferior a
la media considerada (playas de Guadalhorce, Los Álamos, La Carihuela y La Rada), playas en la media
considerada (Las Gaviotas, El Cable, El Cristo, Playa Ancha y Sabinillas) y playas con valores superiores
a la media considerada (Los Boliches y Dunas de Artola).
250
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Susceptibilidad de afección por enjambres de medusas (Pelagia noctiluca) en las playas del litoral occidental de la provincia de Málaga
Una vez comprobado que con el cálculo de intervalos regulares no se aprecia de manera conveniente
las diferencias entre playas, para una mejor apreciación de las diferencias entre playas y en un intento de
homogeneizar los datos de presencia de medusas, se ha optado por la realización de intervalos relativos
al máximo y mínimo porcentaje de la muestra. El resultado del análisis previo comparativo entre datos
registrados de presencia y ausencia de medusas en base 100 se puede ver en la Figura 9.
Figura 9. Porcentaje de presencia/ausencia de medusas por playas. Datos relativos
Fuente: InfoMedusa, 2020. Elaboración propia
Los resultados de los cálculos relativos de los días con medusas se presentan en la Tabla 10, en la cual
se resaltan los valores superiores a la media.
Tabla 10. Porcentaje de días con medusas con intervalos relativos a la muestra de la zona de estudio
Playa
Presencia de medusas
Guadalhorce
36,84
Guadalmar
29,37
Los Álamos
34,48
La Carihuela
35,61
Las Gaviotas
39,58
Los Boliches
39,38
Dunas de Artola
43,75
El Cable
54,74
La Rada
50,00
El Cristo
40,74
Playa Ancha
57,03
Sabinillas
53,79
Ʃ
515,32
42,94
σ
8,95
1/2 σ
4,48
Elaboración propia
En el análisis del porcentaje de días con medusas con intervalos relativos a la muestra de la zona de
estudio se han definido tres intervalos. En la Figura 10 se representan los resultados ordenados de menor
a mayor y por tonalidades de color. En este análisis se observan, a grandes rasgos, menos diferencias
entre playas que en la ponderación por superficies. De entre los intervalos definidos, como playas con
menos presencia de medusas, se encuentran Guadalmar, Los Álamos y La Carihuela. A continuación, en
el intervalo intermedio se presentan cinco playas, estas son, Guadalhorce, Los Boliches, Las Gaviotas,
Investigaciones Geográficas, nº 77, pp. 239-258.
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El Cristo y Dunas de Artola. En el último intervalo, con más días de medusas por playas, están La Rada,
Sabinillas, El Cable y Playa Ancha.
Figura 10. Porcentaje de días con medusas por playa con intervalos relativos a la muestra de la zona de estudio
Fuente: InfoMedusa, 2020. Elaboración propia
El análisis con datos porcentuales relativos permite observar, con mayor claridad, un patrón de distribución espacial de la peligrosidad bastante definido, con una disposición este-oeste en la que aumenta
progresivamente la presencia de medusas (Figura 11).
Figura 11. Intervalos con datos relativos de presencia de días con medusas por playas
Fuente: InfoMedusa, 2020. Elaboración propia
4. Discusión
La bibliografía sobre los ciclos de aparición de floraciones de medusas en el mediterráneo y su llegada
a la costa está poniendo de manifiesto un acortamiento progresivo de los ciclos. La mayoría de los autores,
a la hora de buscar causas explicativas al incremento de la frecuencia en zonas costeras, han trabajado
252
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a escala regional. Entre las causas aludidas para esta tendencia, se citan la presión antropogénica sobre
las zonas litorales y su impacto directo en los mares y océanos (Condon et al., 2013; Duarte et al., 2013;
Purcell, 2011). Otros autores apuntan como los efectos del cambio climático inciden directamente en este
hecho (Purcell, 2011) con lo que podría entenderse que es un efecto global y común en todo el entorno
Mediterráneo.
El motivo del presente estudio es observar si existen diferencias apreciables entre áreas, ante un hecho universal, en esta tendencia generalizada al aumento de frecuencia y severidad de las floraciones de
medusas, en cuyo caso las razones de la variabilidad entre zonas habría que investigarlas en las características estructurales de la costa. Se considera fundamental entender el comportamiento de estas floraciones
de medusas, ya que afectan negativamente a actividades relacionadas con estos espacios litorales como la
pesca, la acuicultura o el turismo (Richardson, Bakun, Hays y Gibbons, 2009).
Para analizar la incidencia y severidad se ha utilizado una aplicación de registro de medusas, InfoMedusa, abordando el problema mediante la colaboración de la población, la cual aporta, en esta y otras materias, conocimiento con uso científico que puede ser incorporado en las políticas públicas y, en este caso
particular, puede ayudar en la implantación de medidas preventivas y/o paliativas del riesgo. Además, la
contribución de la ciudadanía, puede ser muy beneficiosa para la comunidad, ya que siente formar parte
del proceso (Danielsen, Burgess, Jensen y Pirhofer-Walzl, 2010). Los resultados que aportan este tipo de
metodologías se equiparan a otro tipo de técnicas, más tradicionales, en las que intervienen profesionales del tema en cuestión, lo que argumenta a favor de esta técnica en estudios científicos (Cohn, 2008;
Irwin, 1995; Theobald et al., 2015). Silvertown (2009) matiza la utilidad de esta técnica de investigación,
indicando la necesidad de validar los datos que aporta el público participante y la necesidad de un buen
diseño y estandarización para que los resultados no pierdan calidad.
En lo que se refiere a la calidad de la fuente utilizada en la investigación en relación a otras fuentes
con similar aplicación, un análisis comparado de las prestaciones y limitaciones permite observar ventajas notables de InfoMedusa sobre otras opciones. Sus principales ventajas son la totalidad de observaciones por playa que aporta y la extensión temporal de los registros válidos (periodo 2015 a 2020). Como
inconveniente puede mencionarse la detallada labor de interpretación de los datos que requiere, hecho
que se relaciona con el carácter libre de las observaciones y comentarios que conforman la base de datos.
Estas características exigen una labor extensa de traducción de los comentarios libres de la ciudadanía,
para interpretarlos y obtener resultados útiles para el propósito del inventario.
Existen investigaciones, en el marco del litoral andaluz, que toman como referencia otras fuentes de
información, cuya calidad y criterios de homogeneización es discutible (Rubio y Gutiérrez, 2020). Entre
las otras aplicaciones y bases de datos de medusas disponibles en la Costa del Sol se encuentra la proporcionada por la Red de Información Ambiental de Andalucía (Rediam), que ofrece una disponibilidad
temporal que abarca desde 1994 a 2017, para toda la costa andaluza. Esta base de datos podría, a priori,
considerarse complementaria a InfoMedusa, ya que comienza con anterioridad a la serie 2015-2020 con
la que se ha trabajado en este estudio. Un análisis de detalle de la fuente, sin embargo, descarta esta
expectativa ya que Rediam, que se basa en la investigación de Prieto y Navarro (2013), no trabaja con
datos en tiempo real proveniente de visitantes, lo que se traduce en un menor volumen de información.
La aportación de tan sólo 12, 7 y 2 avistamientos en los años concurrentes de ambas bases de datos, esto
es 2015, 2016 y 2017 respectivamente, se muestra como claramente insuficiente para la pretensión del
presente estudio. Otra limitación de esta base de datos es que, además, carece de actualización, ya que
está activa sólo hasta 2017.
Mayor similitud en prestaciones a InfoMedusa tiene la aplicación Medusapp, que aporta datos diarios
y opera en el ámbito espacial requerido en este trabajo. Uno de los aspectos negativos de esta aplicación
es el intervalo de años que comprende, que comienza en 2018, lo que la limita en este aspecto, además
de que los registros u observaciones que muestra sean mucho menos abundantes. Si comparamos el año
2018, que constituye el que más avistamientos posee en ambas aplicaciones, la diferencia de datos es considerable, ya que Medusapp contiene 194 comentarios en comparación a los 98.004 de InfoMedusa, en
ambos casos para el conjunto del tramo occidental de la Costa del Sol. Otras aplicaciones similares como
MedJelly, Grumering u Oboradar operan fuera del ámbito de análisis, por lo que se descarta en un primer
momento como fuentes de trabajo.
El análisis comparado de las prestaciones ofrecidas por la base de datos utilizada respecto a las otras
fuentes mencionadas muestra con claridad que, a pesar de sus inconvenientes, las posibilidades que
ofrecen los datos de InfoMedusa para el análisis de detalle de la frecuencia de avistamientos en la costa
es incuestionablemente de mejor calidad. En este contexto, el inventario resultante de la presente investigación constituye una aportación especialmente valiosa, dada la inexistencia de una fuente de similar
calidad y características en la zona de estudio.
Investigaciones Geográficas, nº 77, pp. 239-258.
253
de la Fuente Roselló, A., Sortino Barrionuevo, J.F., Reyes Corredera, S. J. y Perles Roselló, M.J.
Tanto InfoMedusa como Medusapp, registran los datos a nivel de playa. El desarrollo del trabajo ha
mostrado que el uso de la playa como unidad de trabajo básica para la representación y cálculo estadístico
de la peligrosidad presenta ventajas e inconvenientes. El principal inconveniente es la extensión variable
de la misma, hecho que distorsiona la interpretación de la alta o baja frecuencia de avistamientos, dado
que no se parte de un espacio potencial de frecuentación de usuarios similar. La base de datos de Rediam,
por el contrario, al obtener los datos mediante recopilación de información y el procesado de imágenes de
satélite, carece de este tipo de problemática. Sin embargo, como ventaja fundamental del uso de la playa
como unidad de trabajo, puede señalarse que constituye un marco para la evaluación y cartografía de la
susceptibilidad, coherente con la unidad fisiográfica, geomorfológica y funcional litoral básica, y facilita,
por ello, la interpretación de las relaciones entre el patrón de comportamiento espacial de las medusas
con las causas precisas que lo puedan estar generando.
En lo que se refiere a la periodicidad de la toma de datos y la duración del periodo disponible, la
fuente de datos explotada e inventariada ofrece una secuencia más sistemática que Medusapp y Rediam,
con un ritmo menos frecuente y un periodo de datos menor, en el caso de Medusapp y poco actualizado
con respecto a Rediam.
Por último, con respecto al formato de información ofrecida por estas tres fuentes, la más operativa
es, sin lugar a dudas, Rediam, que ofrece datos en formato Shape (.shp) de fácil manejo y tratamiento
mediante Sistemas de Información Geográfica (SIG). Medusapp, por su parte, requiere la captura de datos
procedente de la aplicación y su tratamiento para poder importarlos al SIG, tarea que se realizó para verificar su utilidad en el estudio y cuyo uso se descartó por la insuficiente suma de registros anteriormente
comentada. Por último, la base de datos de InfoMedusa, si bien es la más amplia con diferencia, también
es la más exigente en dedicación y necesidades de tratamiento de los datos brutos (comentarios cualitativos en formato libre).
Por consiguiente, aunque la escala de trabajo sea similar en estos tres casos analizados, tanto el intervalo temporal como la disponibilidad y calidad de los datos, descarta a Rediam y Medusapp como fuente
sistemáticas para comprender el comportamiento espacial de las medusas. A partir de la observación
comparada de las potencialidades y limitaciones de la fuente de datos, puede afirmarse que el inventario
de datos obtenido a partir de su explotación constituye, por sus características, una aportación valiosa y
excepcional en el contexto de trabajo, fiable para generar indicadores de distribución espacial del fenómeno.
Respecto a los indicadores calculados para evaluar la propensión de las distintas playas a recibir medusas, como se ha comentado, se ha constatado en los mismos una limitación relativa a la diferencia de
tamaño de la unidad espacial de observación (la playa) y la posible diferencia asociada a su frecuentación
por usuarios. Este hecho puede proporcionar una interpretación confusa de la alta o baja presencia de
días con medusas, ya que el dato puede estar motivado por una alta o baja frecuentación de usuarios en
la playa. Para aquilatar este problema, se ha propuesto el indicador Número de días con medusas ponderado
por la superficie de la playa y Número de días sin medusas ponderado por la superficie de la playa. Mediante
la ponderación por la superficie se ha intentado suavizar el hecho probable de que los valores altos de
días con medusas de una playa estén motivados por la alta frecuentación de usuarios en la misma. Se ha
considerado una relación directa entre la extensión de la playa y la frecuentación de usuarios. Este factor
de ponderación se ha ajustado con un coeficiente de minoración (superficie de la playa* 0,5) en los casos en los que la playa esté muy alejada de los circuitos de acceso urbano. Se intenta discriminar, de esta
forma, las playas de grandes dimensiones urbanas (alto potencial de frecuentación tanto por el espacio
disponible como por la cercanía a la ciudad como punto emisor de usuarios), respecto a las grandes playas alejadas de la ciudad y poco frecuentadas. En cualquier caso, en avances futuros de la investigación,
se están calculando indicadores empíricos de frecuentación de las playas para afinar la representación del
dato estadístico. Otros estudios han intentado resolver el problema de la homogeneización de datos planteado a través de distintas soluciones. En este sentido, Rubio y Gutiérrez (2020) elaboran una cartografía
de los avistamientos basada en una cuadrícula de 5x5 km sobre la que representan la cuantificación de
las medusas. Este procedimiento puede ser apropiado para la escala de su investigación, ya que la unidad
espacial de cálculo y representación es el municipio, pero no sería útil para el trabajo a nivel de playa. En
otro estudio en la costa catalana, Benedetti-Cecchi et al. (2015) estudian los brotes de medusas por año,
es decir, se analiza el patrón temporal, no el espacial, lo que hace innecesario calibrar los datos por áreas.
Los resultados estadísticos y cartográficos obtenidos permiten deducir que la distribución espacial
de la llegada de medusas a la costa no presenta un patrón aleatorio en las playas del tramo estudiado. Al
analizar los resultados en su conjunto, se aprecia una clara tendencia al aumento de los avistamientos de
medusas conforme se avanza hacia las playas más occidentales, siendo éstas por lo tanto las que presentan
una mayor peligrosidad de afección por medusas. En el lado opuesto, las playas más orientales disfrutan
de más registros de ausencia de medusas, siendo, por consiguiente, menos proclives a su presencia.
254
Investigaciones Geográficas, nº 77, pp. 239-258.
Susceptibilidad de afección por enjambres de medusas (Pelagia noctiluca) en las playas del litoral occidental de la provincia de Málaga
Algunos autores han avanzado en la búsqueda de un patrón espacial de la distribución de los enjambres. A una escala regional, de mucho menos detalle, Canepa et al. (2014) atribuyen los avistamientos
costeros en el mar Adriático a la dirección y velocidad del viento y al efecto de las mareas, ya que se trata
de aguas costeras poco profundas, lo que favorece grandes acumulaciones de medusas. Estos mismos autores, en un estudio del litoral de Cataluña, concluyen que, si bien las observaciones de presencia de medusas están extendidas a lo largo de toda la costa, tienen a concentrase con mayor frecuencia en las playas
cercanas a los cañones submarinos, que actúan como “superautopistas” desde las aguas más profundas. A
escala de la Costa del Sol, Gutiérrez-Estrada et al. (2021) identifican, al igual que Canepa et al. (2014), la
velocidad y la dirección del viento como factores clave que determinan el momento y la intensidad de la
llegada de las medusas a las playas. En el mismo entorno geográfico, Rubio y Gutiérrez (2020), que analizan la totalidad de la costa andaluza, relacionan una posible causa de los avistamientos con accidentes
geográficos como bahías, ensenadas y costas altas con presencia de calas. Estos autores identifican la localización hacia levante, en el tramo que se extiende desde el municipio malagueño de Torrox hasta Motril
en Granada como el más proclive a recibir medusas. En nuestro estudio, sin embargo, no se percibe esta
tendencia, sino la contraria, en la que la peligrosidad de las playas aumenta hacia el oeste. Posiblemente
motivado por el mayor nivel de detalle de nuestro estudio, puede observarse una explicación coherente
a estos hechos, en principio, contradictorios. Debido a la propia incurvación del litoral mediterráneo
andaluz, las playas que muestran una mayor propensión en nuestro análisis se posicionan en las franjas
de costa orientadas al flujo de levante, desde dónde los estudios más generales afirman que provienen los
enjambres, que se trasladan hacia la orilla desde el mar de Alborán (Bellido et al., 2020).
A nivel de detalle, un análisis preliminar de la distribución espacial observada en nuestro estudio, permite asociar la distribución la mayor frecuencia de avistamientos de medusas a una serie de factores que
pueden estar actuando como condicionantes. Más que la localización hacia el sector oriental de la costa,
se han identificado como posibles causas de la mayor peligrosidad de algunas playas aspectos como su
orientación sureste, el mayor ángulo de apertura de la playa y la ausencia de una protección lateral de la
playa respecto al flujo de levante. Según se ha constatado, las playas de ángulo más abierto y orientadas
hacia el este y sudeste, presentan una mayor frecuencia de enjambres. Este hecho pone de manifiesto que,
cuando el análisis se realiza a nivel de detalle, la orientación de la embocadura de la playa hacia la zona
de procedencia predominante de los enjambres, puede determinar la susceptibilidad, en mayor medida,
que la propia localización de la playa. De las playas muestreadas en este estudio, El Cristo, más cerrada
y con una orientación suroeste, que además se encuentra resguardada del flujo de levante por una barra
de protección lateral en su extremo este, presenta un menor grado de peligrosidad que las de su entorno.
La hipótesis que explica la distribución de la peligrosidad observada, como resultante de la orientación del tramo de costa en el que se ubica la playa y su apertura al flujo de levante, se comprueba,
igualmente, al analizar el sector oriental del tramo analizado, que presenta playas menos proclives a la
aparición de medusas. La protección frente al viento del este que le otorga la propia bahía de Málaga,
abierta hacia el poniente, puede explicar esta menor propensión en las playas más orientales. Los valores
más bajos de frecuencia de medusas en este sector costero también podrían relacionarse con la proximidad de las playas a fuentes de aporte de agua dulce, debido al río Guadalhorce. Autores como Bellido
et al. (2020), citan la posible incidencia de este variable en la distribución espacial de las floraciones de
medusas en el litoral y su llegada hasta las zonas de playa desde mar adentro.
5. Conclusiones
La base de datos ofrecida por InfoMedusa proporciona una información esencial en lo que se refiere
al conocimiento y gestión del problema de la afección por medusas. Su uso con propósitos científicos,
no obstante, requiere de un tratamiento detallado y profuso de la información bruta para, después de un
proceso de depuración y filtrado, obtener datos fiables. Sus potencialidades como fuente para el análisis
del patrón espacial de afección de medusas en el litoral se muestran superiores a las ofrecidas por otras
bases de datos de propósito similar, que resultan muy insuficientes. En este contexto, el inventario de
datos aportado en la investigación adquiere una especial relevancia, dado la inexistencia de inventarios
de datos de similares prestaciones en el ámbito costero andaluz.
La variabilidad en los resultados de presencia o ausencia de medusas entre las distintas playas a lo
largo del periodo analizado permite afirmar que existe un patrón de distribución de la peligrosidad no
aleatorio. La pauta de distribución observada refleja una tendencia al aumento de la presencia de medusas
conforme la playa se localiza en sectores más occidentales de la zona de estudio. Factores como la orientación abierta de las playas más proclives hacia las corrientes provenientes de levante, por la orientación
general de la costa, puede relacionarse con el incremento de la susceptibilidad en las playas en el sector
occidental de la zona de análisis. La orientación a sotavento respecto a los flujos de levante de las playas
Investigaciones Geográficas, nº 77, pp. 239-258.
255
de la Fuente Roselló, A., Sortino Barrionuevo, J.F., Reyes Corredera, S. J. y Perles Roselló, M.J.
más orientales puede explicar, de igual modo, la menor frecuencia de medusas en estas playas. Este patrón espacial no aleatorio y la coherencia general, entre el modelo de distribución espacial de la aparición
de enjambres y factores explicativos asociados a la morfología y funcionamiento del litoral, permiten
observar, a pesar de sus limitaciones, la potencialidad como fuente del inventario que se ha elaborado, así
como la validez de los indicadores de peligrosidad.
El comportamiento diferenciado de las playas objeto de estudio, que se ha constatado en este trabajo,
anima a plantear futuras hipótesis, más complejas, en lo que se refiere a otros posibles factores subyacentes al patrón de susceptibilidad observado, que puedan estar causando las diferencias de afección, para
avanzar hacia la generación de un modelo predictivo. La explotación de los datos sobre presencia de medusas y la metodología de análisis aplicada en la investigación, han permitido concluir aportaciones de
carácter inédito y de interés para el análisis de la afección de medusas en el litoral. Los resultados ofrecen
una base de partida sólida para extender el área de estudio e indagar con mayor profundidad en la identificación de factores condicionantes, al objeto de realizar cartografías predictivas de la susceptibilidad en
otras zonas costeras.
Agradecimientos
Los autores agradecemos a J. Bellido (Aula del Mar. Málaga) el acceso a los datos que constituyen la
fuente en que se fundamenta el trabajo (InfoMedusa).
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The use of scenarios in climate policy planning: an assessment of actors’ experiences and lessons learned in Finland
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Climate Policy ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/tcpo20 Full Terms & Conditions of access and use can be found at
https://www.tandfonline.com/action/journalInformation?journalCode=tcpo20 Kalle Aro, Jyrki Aakkula, Ville Lauttamäki, Vilja Varho, Pim Martens & Pasi
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experiences and lessons learned in Finland, Climate Policy, DOI: 10.1080/14693062.2022.2123773 To link to this article: https://doi.org/10.1080/14693062.2022.2123773 © 2022 The Author(s). Published by Informa
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View Crossmark data CLIMATE POLICY
https://doi.org/10.1080/14693062.2022.2123773 ABSTRACT Scenarios are often used to depict the possible outcomes of alternative future
developments as part of the evaluation of climate and energy policy measures. In
Finland, scenarios have become a standard practice in climate-related policy
planning. However, scenario planning often results in a single cohesive narrative,
which lacks transparency in why certain developments and solutions are included
and others left out. This article focuses on how scenarios created during the
planning of Finland’s Medium-term Plan for Climate Change Policy (KAISU) were
built, and how the actors involved assessed the characteristics and quality of these
scenarios. We interviewed a total of 18 participants from research, administration,
and policymaking domains involved in the creation of the plan and the scenarios it
contains. Semi-structured interviews provided an insight into the creation and use
of scenarios as part of policy formulation, as well as points of further improvement
for the process. The KAISU plan was constructed as a cross-sectoral collaborative
effort between policymakers, public officials, and researchers. Despite the variety of
actors involved in the process, the resulting scenarios were perceived as well-
executed by the participants. However, national scenario foresight could be
improved in three dimensions: 1) the process where actors collaborate to build
scenarios, 2) the scenario methodology, where solutions and developments are
included or excluded, and 3) use of the scenarios after their construction. KEYWORDS
Scenario planning; emissions
scenarios; climate change;
Finland; stakeholder
participation; qualitative
content analysis Key policy insights:
h . The scenario approach used in Finland corresponds with needs at the political
level. However, the approach could be improved without compromising this goal. . The underlying scenario assumptions and the compatibility of the models used
lack transparency. Many actors involved in the scenario process have a limited
understanding of the models used and measures proposed outside their
respective sector. p
. Construction of more than one policy scenario would allow a larger variety of
possible future trajectories to be evaluated in national policymaking. In this
case, the aim and role of the scenario process would have to be adjusted and
clarified accordingly. . Communication regarding the role and applicability of foresight methods should
be improved for citizens and politicians alike. The use of scenarios in climate policy planning: an assessment of actors’
experiences and lessons learned in Finland Aakkulaa, Ville Lauttamäki
b, Vilja Varhoa, Pim Martensc and Pasi Rikkonend Kalle Aro
a, Jyrki Aakkulaa, Ville Lauttamäki
b, Vilja Varhoa, Pim Martensc aNatural Resources Institute Finland, Helsinki, Finland; bFinland Futures Research Centre (FFRC), Turku, Finland; cMaastricht
University, Campus Venlo, Venlo, Netherlands; dNatural Resources Institute Finland, Mikkeli, Finland ARTICLE HISTORY
Received 22 November 2021
Accepted 7 September 2022
KEYWORDS
Scenario planning; emissions
scenarios; climate change;
Finland; stakeholder
participation; qualitative
content analysis ARTICLE HISTORY
Received 22 November 2021
Accepted 7 September 2022 © 2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group
h
l
d
b
d
d
h
f h
b
h
l
b
CONTACT Kalle Aro
kalle.aro@luke.fi
Natural Resources Institute Finland, Latokartanonkaari 9, 00790 Helsinki, Finland © 2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group
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.
CONTACT Kalle Aro
kalle.aro@luke.fi
Natural Resources Institute Finland, Latokartanonkaari 9, 00790 Helsinki, Finland 1. Introduction Current climate action around the globe covers mitigation, adaptation, and other aspects of climate policy, and
ranges in scale from individual choices and activities to globally significant actors and large cross-border initiat-
ives (Hale et al., 2021). The European Union has continuously increased the ambition level of climate and energy CONTACT Kalle Aro
kalle.aro@luke.fi 2 K. ARO ET AL. 2 policy in the twenty-first century. Recently, as part of the European Green Deal, the Commission proposed on
September 2020 the raising of the 2030 greenhouse gas (GHG) emission reduction target, including emissions
and removals, to at least 55% compared to 1990 (EU Commission, 2021). Furthermore, the EU aims to be
climate-neutral by 2050. The EU has examined the actions required across all sectors, including increased
energy efficiency and renewable energy, and started the process of making detailed legislative proposals in
June 2021 to implement and achieve the increased ambition. This will enable the EU to move towards a
climate-neutral economy and implement its commitments under the Paris Agreement. In 2020, the European
Union mandated member countries to prepare and submit their national energy and climate plans. gy
In Finland, climate policy is framed through a series of plans covering the various sectoral and temporal
aspects of the climate domain. Government planning mostly revolves around the Energy and Climate strategy
(Huttunen, 2017) and the Medium-term Climate Change Policy Plan, KAISU1 (Ministry of the Environment, 2017),
which both utilize scenarios as the basis for their policy. Together, these documents form the practical basis of
national climate policy in Finland by being the most regularly updated (Table 1). Of the two documents, KAISU
holds a more formal position in policymaking, because its mandate comes directly from the Finnish Climate
Change Act (609/2015). KAISU outlines the expected impact of existing policy measures outside the EU Emis-
sions Trading System (ETS) on the projected evolution of GHG emissions, renewable energy, and energy
efficiency up to 2040. The plan projects the effects of the planned policy measures on the energy system,
GHG emissions and sinks, economic development, the environment, and public health. The KAISU plan also
assesses the possible impact of planned and existing policy measures on investment (Ministry of Economic
Affairs and Employment (MEAE) et al., 2019). 1. Introduction y
The use of scenario planning in the preparation of climate actions helps evaluate the overall impacts of chosen
policy packages, and at best shows the feasible pathways to stated goals. Scenarios are typically made in conjunc-
tion with foresight processes that assist policymaking. The literature often differentiates between the process
where scenarios are constructed, including the chosen scenario framework, actors involved and models used
(e.g. Wright et al., 2019), and the use of the scenarios as a tool in policy planning (e.g. Cook et al., 2014). However, the line between scenario creation and scenario-inspired decision making becomes blurred when
the two processes involve common actors with political motives and decision-making power. In other words,
the political dimension can hardly be absent from the scenario-building process which sets the framework for
furtherpolicyplanning,causing overlapbetweenthe two processes. Nationalemissionsscenarios can be strongly
influenced by decision makers, who may approach the scenarios as a framework for applicable emissions
reduction means or, as a strategic planning process (Tyler et al., 2013). Especially when the latter approach is
adopted, a scenario-building process becomes inherently political, as the actors involved are negotiating and
selecting the emissions reduction means to be implemented nationally. Wright and Goodwin (2009) found
thatactorschosentoconductthescenario-buildingprocesscanhavea majorinfluenceonthescenario’scontents
and the resulting level of predictability. Ambiguous framing of the scenario, individual biases, and erroneous per-
ceptions of causality between variables contributed heavily to the low predictability of the whole scenario. National policy planning also acts as a vital mechanism for advocacy groups to legitimize, strengthen, and
promote their solutions to the public agenda (Meadowcroft, 2011). Scenarios and projections can thus be under-
stood as forums in which alternative low-carbon solutions compete for recognition and publicity in the national
future narrative. As politicians, stakeholders and citizens have different views concerning the probable, preferred,
and possible future scenarios, the processes of creating and using scenarios become intertwined. Hence, how
the alternative scenarios are formed and discussed is a central question, both in environmental studies of delib-
erative democracy and in the deliberative processes in the real world (Renn, 2006). Hale et al. (2021) pinpoint that
policymakers need to understand which approaches are working and which are not, promoting the diffusion of
best practices and creating conditions for stronger action in the future. In Finland, the scenarios created in the national energy and climate strategy have been an integral part of
the policy process. The acronym was formed from the Finnish name of the plan Keskipitkän aikavälin ilmastopolitiikan suunnitelma. 1. Introduction According to the KAISU plan, the scenarios were formed by compiling sectoral estimations of
possible emission reduction measures (Ministry of the Environment, 2017). The measures were evaluated based
on their impact and cost-effectiveness to form a cohesive set of additional policy measures. However, the 3 CLIMATE POLICY 3 Table 1. Administrative framework for Finnish climate policy. The framework consists of multiple interlinked documents, which vary in their
sectoral and temporal scope. Document
Purpose
Latest entry
Instructive
Climate Change Act (609/2015)
To strengthen the governance of climate policy planning and
monitoring
2015
Regulation of the Governance of the
Energy Union and Climate Action (EU/
2018/1999)
To unify governance of climate policy and policy goals within
the EU in accordance with the Kyoto Agreement, Paris
Agreement, and the EU Climate and Energy Package
2018
Governmental Plan
Includes goals in national energy and climate policy to be
promoted in the current government’s term
(Finnish Government, 2019)*
Executive
Energy and Climate Strategy
To provide outlines, goals, and measures that the government
deems necessary to accomplish the national goals. Updated
once every parliamentary term. (Huttunen, 2017)
Medium-term Plan for Climate Change
Policy (KAISU)
To specify policy measures outside the ETS to 2030. Updated once every parliamentary term
(Ministry of the Environment,
2017)
National Energy and Climate Plan
To present national policy measures for energy efficiency,
renewables, GHG reductions, interconnections, and research
and innovation in 2021–2030
(Ministry of Economic Affairs and
Employment (MEAE) et al.,
2019)
Adaption Plan for Climate Change
To address measures and their sensitivities for climate change
adaptation. Updated at least once every 10 years
(Ministry of Agriculture and
Forestry, 2014)**
Long-term Plan for Climate Change
Policy
To outline national policies for climate change mitigation and
adaptation to 2050. Updated at least once every 10 years
n/a
Monitoring
Annual Climate Report
Annually examines the meeting of the targets set for emission
reduction obligations
(Lounasheimo et al., 2021)
* During the first KAISU process, the active governmental plan was that of the government of Juha Sipilä, published in 2015
** The latest adaptation plan was created prior to the Climate Change Act, but displays clear linkages with the upcoming act. scenario-building process is poorly documented in terms of the internal choices made during the process. Bist-
line et al. 1. Introduction (2021) propose that transparency should not only be understood as accessibility of data and models
used, but should also include a read on the structural assumptions made during the scenario process. They
suggest that efforts to increase transparency should be done especially for non-modellers, such as policymakers
and stakeholders, in mind. Hence, to improve scenario work and increase the understanding of the national
scenario-building processes, it is important to evaluate how the administration, policymakers, and scenario
makers interact, which background assumptions guide the process and scenario models, and how scenarios
influence policy decisions. This paper inspects the creation of the Finnish Medium-term Plan for Climate Change Policy (KAISU), focus-
ing on the construction and use of the scenarios. It gives an in-depth portrayal of a national cross-sectoral scen-
ario-building process largely absent in the current literature, and acts as a point of comparison when studying
other national scenario processes. It also presents several suggestions on how to improve the scenario process,
thus providing useful insights on the potential benefits and issues related to them also outside of the Finnish
context. The research questions in this article are the following: RQ 1) How were the scenarios in the creation of the KAISU plan formed and used? RQ 2) How was the scenario process perceived by the KAISU process participants? 4
K. ARO ET AL. Second, 10 respondents represented public officials and other actors directly involved with the KAISU
process as part of the working group. These respondents’ roles in the KAISU process were diverse, as they rep-
resented multiple ministries and other organizations. Moreover, they were responsible for different actions in
the working group, in which some were more involved in the KAISU plan’s coordination, management, and
external communications, and others more specifically involved in scenario building by representing their cor-
responding sector. Finally, four respondents represented political actors then involved in the climate policy domain. These four
participants included members of parliament and their policy advisors. While neither directly involved in the
scenario building nor part of the official KAISU working group, they were regularly contacted by public
officials to ensure political acceptance of the measures proposed for inclusion in the KAISU plan. In certain
cases, politicians also provided specific goals for inclusion in the KAISU plan, which correspondingly shaped
the scenario-building process. The interviews were conducted to backtrack the KAISU process and to discover key participants’ perceptions
of scenario building in the KAISU process. The interviews were semi-structured: three different interview frames
were created, one for each respondent group. The interview frames contained largely similar questions, but
some topics varied, based on respondent group’s expertise and involvement in the KAISU process. The inter-
view frames also followed the KAISU process in chronological order, first covering the starting point and the
framework for scenario building, followed by the creation of the scenarios, and finally addressing the presen-
tation and further use of the finalized KAISU plan. Although the interviews’ focus was on the formulation and
use of scenarios created in the KAISU process, respondents’ perceptions of the general process were also dis-
cussed. Whereas interviews held with public officials followed a largely uniform pattern in line with the inter-
view frames, more variance between interviews was discovered when interviewing representatives of research
groups and policymakers. The interviews were held between late 2020 and early 2021 as remote video meet-
ings that were recorded and transcribed. The materials were analysed using inductive content analysis (Hsieh & Shannon, 2005; Schreier, 2012) with
NVivo 12 for Windows software. The purpose of the analysis was to map the KAISU process from its creation to
policy use, and to discover how the participants perceived scenario formulation and use. 4
K. ARO ET AL. The interview frames
were utilized as the starting point of the analysis. The transcribed interviews were first divided and segmented
to correspond to different parts of the scenario process. As respondents often commented on multiple items of
interest simultaneously and referred to their previous answers throughout the interviews, excerpts from other
parts of the interview were added to the corresponding segment group. A more nuanced analysis was con-
ducted within the segmented groups by coding the excerpts by their content. Answers with a shared
meaning were coded together inductively, and new codes were created where no existing code within a
segment group could accurately grasp the answer’s meaning. Some answers were coded into two or more seg-
mented groups, as they provided an insight that could benefit different parts of the analysis. Finally, the result-
ing codes were organized into thematic categories within the segment groups. Thematic groups were used as
the main unit for the analysis, and the study’s results on the scenario process (section 3.1) and the lessons
learned (section 3.2) were compiled based on a chronological and thematic reading of the resulting groups. Two rounds of coding were conducted with three months in between. The results of the two rounds were
compared and evaluated against one another to validate the findings. The final thematic categories thus com-
prise elements from both coding rounds. 2. Methods and materials In this study, we interviewed 18 participants involved in the creation of the Medium-term Plan for Climate
Change Policy. The respondents were chosen to represent different sectors involved in KAISU planning, and
their involvement in KAISU process varied as follows: First, four respondents represented research institutions and modelling groups involved mainly in scenario
creation. Their role centred around utilizing modelling tools and providing scientific insights and guidance
based on their knowledge of the field and the models in use. Moreover, modelling group representatives quan-
tified the effect of selected policy measures. 4
K. ARO ET AL. 4
K. ARO ET AL. 3.1. Creation of medium-term plan for climate change The first Medium-term Plan for Climate Change policy was prepared and made public less than a year after the
revised national Energy and Climate Strategy was published. While the two were not directly linked, it was
expected that they would share a similar landscape and compatible measures across emission sectors, and
many actors were involved in creating both documents. As with earlier energy and climate strategies, a quan-
titative emission scenario was created by the Ministry of Economic Affairs and Employment for emission CLIMATE POLICY
5 CLIMATE POLICY
5 Figure 1. Simplified overview of the KAISU process based on the interviews. Multiple actor groups (rectangles) and documents (ellipses)
influence the scenario building. (Created by authors, generated in diagrams.net). Figure 1. Simplified overview of the KAISU process based on the interviews. Multiple actor groups (rectangles) and documents (ellipses)
influence the scenario building. (Created by authors, generated in diagrams.net). projection purposes. This formed the ‘with existing measures’ (WEM) scenario, against which the policy scenario
was to be contrasted in the KAISU plan. According to respondents, this WEM scenario’s assumptions and pro-
jections were not really discussed during the KAISU process. Instead, the WEM scenario was presented, and the
group responsible for the modelling described the models’ general logic. Respondents recalled that the WEM
scenario was to follow outlines provided from the EU level, which partly limited the modelling options. Additionally, as most of the modelling for the WEM scenario was conducted earlier during the creation of
the Energy and Climate Strategy, the discussions took place before the KAISU process. projection purposes. This formed the ‘with existing measures’ (WEM) scenario, against which the policy scenario
was to be contrasted in the KAISU plan. According to respondents, this WEM scenario’s assumptions and pro-
jections were not really discussed during the KAISU process. Instead, the WEM scenario was presented, and the
group responsible for the modelling described the models’ general logic. Respondents recalled that the WEM
scenario was to follow outlines provided from the EU level, which partly limited the modelling options. Additionally, as most of the modelling for the WEM scenario was conducted earlier during the creation of
the Energy and Climate Strategy, the discussions took place before the KAISU process. The ‘with additional measures’ (WAM) scenario’s creation differs significantly from the former. 2The Effort Sharing Regulation is part of the EU’s climate framework, which sets binding targets to national emission reductions outside of the
Emissions Trading System. For Finland, the target set in the ESR was -39% compared to the emission level in 2005 (Ministry of the Environ-
ment, 2022). 3.1. Creation of medium-term plan for climate change Whereas the
WEM scenario was a mostly technical emission projection created during the Energy and Climate Strategy
project, public officials’ and politicians’ roles were more central in the WAM scenario (Figure 1). Public
officials were to draft a policy scenario including measures to meet the national emission reduction target
set in the EU’s Effort Sharing Regulation (ESR).2 The discrepancy between projected emissions and the target
was estimated to be 6 Mt CO2-eq. in 2030 (Ministry of the Environment, 2017). The WAM scenario’s goal
was therefore to present sufficient policy measures to meet this target in the Finnish ESR sectors. The WAM
scenario’s outlines were prompted by the government working group overseeing the plan alongside the con-
temporary government plan. Additionally, an independent research project (KEIJU) was created to evaluate the
measures in both Energy and Climate Strategy and the KAISU plan. gy
gy
p
Four ministries were especially involved in KAISU’s creation: the Ministry of the Environment was the pro-
cess’s coordinating ministry, as the preparation of KAISU plan was officially assigned to them. The Ministries
of Agriculture and Forestry, Economic Affairs and Employment, and Transport and Communications partici-
pated in the central working group and were responsible for measures and policy proposals in their sectors. Each administrative sector was tasked with the creation of a sectoral plan, which was constructed indepen-
dently within sectors in collaboration between corresponding ministries and research institutions. The resulting
sectoral plans were originally supposed to list all available and even remotely feasible policy measures
accompanied by their GHG emission potential for sectors in the ESR, forming KAISU’s backbone. The Finnish 6
K. ARO ET AL. 6 K. ARO ET AL. Climate Change Panel3, as an external advisor to the process, provided a common framework in which all
measures collected could be presented uniformly, thus helping evaluate different measures within and
across sectors. The framework set cost-effectiveness as the foundation against which different measures
were presented and evaluated. Cost-effectiveness therefore became the guiding feature in the WAM scenario’s
initial creation. The common framework was generally perceived as helpful, though there were some concerns
about social and larger ecological issues being somewhat side-lined due to this techno-economic emphasis. The atmosphere in the working group was highly praised by the participants. 3.1. Creation of medium-term plan for climate change Although the working group
consisted of members from a variety of ministries and organizations with different practices, respondents felt
that all the participants were genuinely invested in and keen to prepare KAISU to the greatest possible detail. However, the creation of KAISU was also described as rather conservative. Outlines, targets, and the practical
scenario modelling work were strongly guided by the EU Climate Framework and the contemporary govern-
mental plan, which limited the scenario planning. Moreover, it was felt that public officials were somewhat
reluctant to include measures they deemed as lacking political support. Similarly, public officials were very cau-
tious when including emerging technologies and measures with limited estimations for their effectiveness to
avoid shortfalls if the WAM scenario was to be realized. This left some respondents speculating whether certain
possibly effective but unproven measures were disregarded due to a conservative attitude. The scenarios’ initial formulation was largely done within ministries. As the sectors covered in the ESR are
highly diverse, policy measures were generally evaluated by research institutions and public officials within
administrative sectors. The identified measures were then presented to and approved by the corresponding
minister’s staff. This effectively meant that the KAISU working group was more about coordinating responsibil-
ities between ministries than evaluating individual policy measures in detail. Most of the reported topics for
discussion within the working group were more involved in the general framing of the KAISU plan: how to
account for future development of emissions sectors outside of the ESR, and how to develop a just distribution
of the burden between sectors within ESR. Although the division of labour was originally designed for all
sectors to collect measures to reduce a similar gross percentage of emissions, this was found to be difficult
in practice. Respondents especially mentioned the agriculture and transport sectors. Whereas the public
officials in the transport sector had adopted a somewhat proactive stance on climate policy by evaluating
the effectiveness of different climate policy measures before the KAISU plan, agricultural sector representatives
felt that not many politically feasible measures would be found in agriculture, and therefore showed more
reluctance about the process. Ultimately, the transport sector was assigned to an ambitious −50% emission
reduction target by 2030. The choice was above all political but relieved pressure from the other sectors,
especially agriculture. This implies that political feasibility and policy goals were ultimately prioritized over
cost-effectiveness. 3The Finnish Climate Change Panel is an independent advisory institution that supports and evaluates climate change policymaking. The Panel
consists of 15 distinguished academic scientists, and its official position is established in the Climate Change Act (Finnish Climate Change
Panel, 2021). 3.1. Creation of medium-term plan for climate change Active political lobbying was considered part of a healthy democratic process, and it could bring
new ideas and perspectives to the table. The KAISU plan was accepted in late 2017 and presented to parliament. At the time of its public release, it
received limited attention in the media, but it soon had a major impact on the Finnish national climate policy
framework. It outlined the national climate policy’s direction, including technologies and division of labour
between different emission sectors. The WAM scenario constructed in KAISU was considered the most probable
and politically feasible future trajectory and has greatly influenced Finnish climate policy. Outside parliamentary
policymaking, other actors in Finland take an interest in KAISU, often using the plan in their rhetoric and policy
documents. Additionally, KAISU’s WAM scenario is often studied and referred to as the Finnish parliament’s
official statement on the goals and means it actively works to achieve. 3.2. Lessons learned and further improvements In general, the KAISU plan’s formulation was considered well executed. Relevant ministries and research organ-
izations provided sufficient high-quality estimations of the potential GHG mitigation measures and their effec-
tiveness. Moreover, respondents found the process insightful and effective. Many also praised the working
group’s positive and genuinely collaborative atmosphere, despite the many organizations and ministries par-
ticipating. However, respondents identified multiple points where the scenario-building process, the scenarios’
content, and the eventual use of the KAISU plan could be improved (see Table 2). 3.1. Creation of medium-term plan for climate change Politically, the scenarios were discussed in a ministerial working group, which ensured the political feasibility
of the overall scenario and provided a formal discussion forum for politicians and the KAISU working group. Additionally, respondents noted the importance of the political advisory level, which acted as a more pragmatic
link between politicians and public officials. Political advisors thus pre-assessed the means proposed for the
KAISU plan as clearing the burden from ministers who were more focused on the plan’s broader outlines. Citizens, non-governmental organizations, and other stakeholders were encouraged to participate in the
KAISU process during the plan’s preparation: workshops were organized during the creation of sectoral
plans, and a public hearing round was held for the KAISU draft. While public participation was deemed impor-
tant by the respondents, many of them considered these public involvement efforts as largely insignificant. As
actors in respective policy fields were often well acquainted with each other, respondents felt these participa-
tory efforts added little to what was already known and discussed in the ministries or in the KAISU working
group. In particular, the consultation round was considered ill-timed: by the time it was held at the end of
the process, there was little enthusiasm for modifying the draft on the working group’s behalf. While the
respondents had not encountered attempts to affect the scenario-building process, some speculated that if CLIMATE POLICY
7 Improve coordination across different processes during scenario building
Provide transparent reading of models and sectoral plans used
Ensure sufficient resources and time allocation
Expand group of experts involved
Provide transparent assessment of sensitivities and uncertainties
Enhance the role of qualitative assessments
Utilize a wider range of scenarios
Improve communication of scenarios to wider audience
Clarify role of scenarios to policymakers
Utilize ex post assessments
Update scenarios regularly there had been any external forces in play, those efforts would have happened outside and before the formal
process. Politically, lobbying was not considered harmful to the process, but a normal two-way interaction with
stakeholders. Active political lobbying was considered part of a healthy democratic process, and it could bring
new ideas and perspectives to the table. there had been any external forces in play, those efforts would have happened outside and before the formal
process. Politically, lobbying was not considered harmful to the process, but a normal two-way interaction with
stakeholders. 3.2.1. Scenario process
d
b Coordination between processes was somewhat discussed by respondents. As the KAISU plan shared strong and
obvious linkages with the Energy and Climate Strategy, some respondents wondered why the processes were
not constructed and coordinated more closely. Similar remarks were made concerning the numerous research
projects and scenario exercises conducted for the KAISU and Energy and Climate strategy. Understanding the
roles, linkages, and aims of different processes were found confusing, especially by those only involved in one
process. Respondents proposed that the creation of KAISU and Energy and Climate strategy should be further
integrated in future to avoid obvious overlaps in the two processes and generate a more holistic stance on
Finnish climate policy and the available measures across the three pillars. Of course, a strict boundary between the Effort-Sharing Sector and Emission-Trading Sector keeps emerging as problematic. It’s the same with the Effort-Sharing Sector and Land-Use Sector, as the constant motion [of emissions] across boundaries is
obvious (Respondent B6). Transparency of data and sectoral plans were mentioned several times during the interviews. As every admin-
istrative sector formulated its own sectoral plan mostly independently before the integrated KAISU plan,
respondents from other administrative sectors often felt unable to comment on the selected measures and
their rationale. Respondents also implied that they trusted their colleagues to be experts in their respective 8 K. ARO ET AL. 8 fields, thus reducing enthusiasm to challenge their assumptions and policy proposals. However, it was noted
that more transparency in the selection of measures might have generated a more holistic and trans-sectoral
approach to the KAISU plan, instead of treating each administrative sector independently. It’s rather hard to question representatives of other sectors over, say, whether certain cultivation methods could reduce emis-
sions. In this case, the knowledge would be on the Ministry of Agriculture and Forestry’s side (Respondent B7). The compatibility and limitations of the models used were also mentioned in the interviews. As models are
complex systems requiring expertise to operate and interpret, few respondents felt qualified to evaluate
their functionality and effectiveness. However, the use of multiple largely disconnected models within the
same scenario exercise raised some concerns about possible systematic errors resulting from differences in
data and general assumptions. While this problem was somewhat addressed by maintaining the shared
assumptions and communication efforts between modelling teams, it was a time-consuming process requiring
significant resources. 3.2.1. Scenario process
d
b However, the idea of a single, more holistic, and interconnected assessment model was
deemed too difficult and complex to be practical. A model this large was deemed to easily turn against its
purpose, as it would require many compromises and generalizations in its internal logic to be practical,
risking the loss of many nuances embedded in the current models. Notable criticism was given to the short timeframe, where scenarios had to be modelled, evaluated and
compiled. Especially the modelling tasks for WAM scenario and the impact assessment (KEIJU) were conducted
with short notice and given a limited timeframe. Respondents hoped that expanding the group involved in scen-
ario making could help manage the problem if more people were acquainted with the scenario tools and
models in both the research and administrative domains. As an added benefit, it would allow a wider range
of actors to conduct scenario work more independently, thus reducing the heavy workload of current
experts and generating a wider range of scenarios with different assumptions. Operating heavy-duty models and soft-linking between them is very resource-intensive, and would require multiple iteration
rounds to find a stronger, broader linkage to connect them. In my opinion, a lot of questions remained regarding the coher-
ence between different analyses (Respondent C4). 3.2.2. Scenario components Addressing sensitivities and uncertainties within the results lacked transparency according to several respon-
dents. Respondents found that uncertainties regarding the effectiveness of particular policy measures were
poorly explored and described, and done only for the final set of measures. Some respondents feared a
large number of small fluctuations across different measures might cause a stark gap between the targeted
GHG reduction level and the proposed measures. They felt a more careful assessment of different measures’
sensitivities could help generate a more robust climate plan, in which the risk resulting from certain estimation
errors could be better nullified with a selection of measures not strongly interlinked with one another. It should
be noted that some respondents recalled that sensitivities and their causes were presented during the process,
and felt that confusion over them was due to limited understanding on how scenarios work in general. Many
respondents also emphasized that scenarios can never present an accurate future, and the existence of sensi-
tivities should therefore be understood as a default from the outset. Some sensitivity assessment was done at the end of the process. However, I found it somewhat vague to be honest (Respon-
d
B9) Some sensitivity assessment was done at the end of the process. However, I found it somewhat vague to be honest (Respon-
dent B9). Some respondents felt the creation of a wider range of scenarios would help highlight the uncertainties of the
future and overall scenario work. It was suggested that presenting only a single policy scenario was a potential
cause of harmful lock-ins to certain developments and technologies, which might lead to suboptimal policy-
making in an uncertain future. Moreover, some respondents suggested that multiple groups acting indepen-
dently should undertake the initial scenario modelling. Comparing independently created scenarios would
help to pinpoint commonly agreed trajectories across experts and possible risks regarding certain policy pro-
posals, as well as to discover any new climate policy developments. Some respondents suggested the creation
of multiple scenarios, portraying diverging paths to the future using different sets of policy measures. A CLIMATE POLICY CLIMATE POLICY 9 perceived benefit of multiple detached scenarios was their ability to better clarify the role of scenarios as visions
of possible future pathways rather than being addressed solely as estimations of the future. I would, without a doubt, consider using a scenario spectrum, where we would try to recognize the most effective and certain
measures. 3.2.2. Scenario components In a way, we would take the uncertainty of the future as a fact and try to incorporate this uncertainty into the
process rather than excluding it (Respondent B7). A similar division was found in qualitative assessments in scenario work. As scenarios were built mostly on stat-
istical data and estimations, qualitative assessment regarding the measures and assumptions used was found
lacking by some respondents. Respondents deemed qualitative assessment helpful for discovering underlying
social, cultural, and environmental conditions and trajectories, which could help analyse the preconditions and
effects of the proposed policy measures. Many respondents felt that numerical estimations were easier to
digest by policymakers and were thus an expected result of scenario work. Questions were, however, raised
about the increased level of uncertainty when including more qualitative assessments in the scenario frame-
work. Qualitative assessments, regardless of how well conducted and thorough they were, would still have
to be transformed into numerical values for them to be properly included in the predominantly quantitative
scenario. If this was not done, it was often the case that qualitative estimations were largely side-lined in
the scenarios included in KAISU, and received only a little attention from policymakers, who were more inter-
ested in the quantitative techno-economic assessments of larger trajectories. Qualitative understanding is highly important, of course, because using numbers always has a simplifying effect. So I’d say
that it’s more important to understand the effects of the selected measures, what we’re changing, and what the effects of
those changes are. And here it’s important to have a qualitative understanding. Yet it might be something we often don’t
take seriously enough (Respondent B1). 4. Discussion Scenario planning approaches have become a widely accepted way of understanding and communicating
climate change outcomes (Rogelj et al., 2012). Over the years, such scenarios as the Representative Concen-
tration Pathways (RCPs) were developed to provide internally consistent sets of projections of the components
of radiative forcing for use in analysis by both climate models and IAMs (van Vuuren et al., 2011). Shared socio-
economic pathways (SSPs) were developed ex post to be consistent with the RCPs by integrating the descrip-
tions of socioeconomic development with climate change projections and assumptions about climate mitiga-
tion and adaptation policies (Ebi et al., 2014). Broader scenario planning use within climate mitigation and
adaptation could be seen as supporting the evaluation of both required actions and their societal impacts. One way to improve climate policy planning is to ensure that the insight of such global scenario studies can
be included more closely in policy planning to provide foresight information during the planning process. The experts who produce evidence-based research information for planning and decision making can be
seen as representatives of a wider scenario setup, and thus a wider future alternative insight than just the
WEM and WAM scenario approaches. In the KAISU process, the scenarios were used as a platform for integrating
the understanding of the different trajectories of climate policy effects. They also serve as a medium of internal
and external communication on the needed and expected additional climate policy measures. Other potential
benefits of scenario work, such as the ability to prepare and manage uncertainties, identify discontinuities
(Volkery & Ribeiro, 2009) or weak signals, did not appear as priorities in this process. Wright et al. (2020) identify the challenge of understanding risks and opportunities following emerging tech-
nological development in policy formulation. In particular, the impact of emerging technologies on different
domains in society can be difficult to grasp by policymakers, and this difficulty may lead to disregarding emer-
ging issues when designing policies. In a similar fashion, Pye et al. (2021) propose that current modelling
approaches often lack radicalism in the solutions included, as many potentially effective, yet politically unfea-
sible, solutions are left unaccounted for in the models or side-lined in the scenario process. 3.2.3. Use of scenarios ARO ET AL. 10 10 multiple scenarios with different assumptions, some respondents felt that generating multiple policy scenarios
would cause only more confusion and would easily reduce the role of scenarios from a strategic tool to a narra-
tive battleground. A single, well-considered, and carefully assessed policy scenario was therefore found more
appealing, as it streamlined the policy process and made the KAISU plan more approachable overall. multiple scenarios with different assumptions, some respondents felt that generating multiple policy scenarios
would cause only more confusion and would easily reduce the role of scenarios from a strategic tool to a narra-
tive battleground. A single, well-considered, and carefully assessed policy scenario was therefore found more
appealing, as it streamlined the policy process and made the KAISU plan more approachable overall. 3.2.3. Use of scenarios Several respondents suggested more frequent revisiting and ex post assessment of previously created scenarios. Respondents found that scenario work would greatly benefit from regular revisions where the scenarios could
be re-evaluated against contemporary developments. This might enable regular subtle adjustments instead of
generating new scenarios every four years. Moreover, some respondents found scenario work in its current
form inadequate in providing significant help to policymakers. KAISU plan scenarios were only done once in
a parliamentary term. As the level of uncertainty in scenario projections increased significantly with every
year, scenarios might lose their practicality quite quickly. To accumulate further understanding from the pre-
vious modelling efforts, scenarios should be exposed to ex post analysis. Comparing previous scenarios with
contemporary developments would highlight discrepancies between the two. This in turn could help under-
stand limitations within the models and data used, and create more robust future scenario modelling. If they hit us once every four years with this vast project, where we then rush to formulate WEM and WAM scenarios, it’s
bound to be challenging (Respondent C1). Finally, external communication regarding the scenarios was found lacking, especially for wider audiences such
as citizens and the media. While respondents regarded the participation efforts during the KAISU process and
external relations after its publication as better than average, they also felt more resources should be allocated
to external briefing in future. It was deemed quite difficult for an ordinary citizen to holistically understand the
policy framework in climate- and energy-related affairs, and accurately situate KAISU scenarios in this
framework. From a communication perspective, it is key that citizens understand the outlines [of climate policy]. Currently, it’s quite hard
to grasp, as the pieces are in silos all over the place (Respondent B8). From a communication perspective, it is key that citizens understand the outlines [of climate policy]. Currently, it’s quite hard
to grasp, as the pieces are in silos all over the place (Respondent B8). Moreover, the respondents found it hard to explain the applicability and limitations of scenarios for policy-
makers, who were not familiar with the methodology. Policymakers were prone to treat the WAM scenario as
a deterministic narrative, which downplayed the uncertainty embedded in the foresight method. Additionally,
policymakers sometimes pinpointed a certain estimation or causality as incorrect, thus generating a reserved
posture toward the rest of the scenario. While this phenomenon could be somewhat addressed by utilizing K. 4. Discussion In our findings, lack
of radicalism can be found in the strict modelling boundaries, which gave hardly any leeway to imagine vastly
different economic or energy systems, and the precautionary tendencies of the participants to avoid measures
with higher levels of uncertainty. As a solution, Wright et al. (2020) suggest forming scenarios accompanied by
comparative analyses with regard to different policy aspects and policy recommendations from stakeholders. Hanna and Gross (2021) point to several instances where energy scenario modelling has failed to anticipate
changes that have taken place within the energy domain. Similar shortfalls were apparent in the KAISU process
as, especially where the WAM scenario is concerned, the pressure to quantify GHG impacts may lead to distor-
tion when selecting policy measures. If the models available for the scenario work cannot be used for a quan-
titative impact analysis of a certain type of policy measure, there is an obvious temptation to replace such policy
measures with measures that can be modelled quantitatively more easily. This underlines the potential disparity
in the applicability of qualitative and quantitative scenario approaches, when goals steering the scenario design
are set in quantitative terms. Another difficulty with scenarios arises from the modelling and quantification of the impact of proposed
measures. In this case, many of the interviewees felt that in the scenario work various models which were
used to quantify the impacts of applied policy measures or GHG emission reduction techniques were difficult
to perceive and not very transparent in terms of their embedded causality. Of course, this is to some extent
an inevitable outcome, because modelling becomes very complex when all the sectors in the economy and
their GHG emissions and sinks are included. Not even the researchers and experts responsible for the use of
models can master all the models simultaneously, but rather the ones in which they happen to be specialized. This means actors in all the different preparation spheres (research, administration, policymaking) must trust
each other. This was the case in the KAISU process, which was indicated in several interview responses. CLIMATE POLICY 11 The idea of having a reference scenario based on currently applied policy measures (WEM) and a policy scenario
including policy measures required to achieve the new policy goals set (WAM) is doubtless a practical way to
prepare an action plan. 5. Conclusions The complexity of combining cross-sectoral climate policy, the uncertainty inherently embedded in scenario
studies, and value-based policymaking demand a high level of planning, cooperation, and context-driven
approaches to scenario building. Despite this complexity, the KAISU process appears quite well executed,
based on this study’s empirical evidence. The researchers, public officials and political actors involved described
the KAISU working group as highly motivated and genuinely engaged with the scenario work, thus mediating
the complexity of an intersectoral process. The 2017 KAISU plan generated the framework for Finnish climate
policy for several years, creating a more predictable policy landscape for citizens, investors, and other stake-
holders. Despite the success of the initial process, many issues were mentioned for potential future improve-
ment. Practical scenario building could benefit from better overall integration, transparency, and additional
allocated resources. In terms of the scenarios themselves, more variety in scenarios and scenario-specific assess-
ments might prepare better for uncertainties. Concerning scenario use, approaching scenarios as a continuous
learning and communication process might strengthen their role and practicality as policy planning tools. However, it should be remembered that it is vital to maintain the practicality of scenarios created for policy-
making. Holding high academic ideals in a policy process may prompt new ideas for imagining the future, but it
may also result in the scenarios being unusable in political decision making and evaluation processes. The scen-
ario approach selected for KAISU appeared quite straightforward with its emphasis on quantitative assessment
and a baseline - policy scenario dynamic. While the approach may seem low in creativity and leaves room for
further improvement, the selected approach and the fundamental goals set for its creation have given policy-
makers a cohesive and consistent roadmap to follow. Improving the use of scenarios as part of a policy process
thus requires balancing of scientifically considered ideals with the practical realities of representative politics. Finding a middle-road between the two calls for unambiguous goal setting and transparent methodology, as
well as understanding on how the scenarios will be used in the context of policy planning. Acknowledgments We thank the interviewees for sharing their time and expertise. We also wish to thank the three anonymous reviewers for their con-
structive feedback. 4. Discussion However, with only one alternative WAM scenario, the choice of policy options and instru-
ments becomes limited and may lead to an emphasis on already existing, and thus evident, policy measures. According to Koskimaa et al. (2021) the central role of semi-autonomous public officials strengthens the long-
term perspective in environmental policy planning, as it balances out the more short-term perspective stemming
from representative and party politics. However, our results suggest that this emphasis on long-term develop-
ments can also act as a restraint on new emerging climate solutions. Constructing a single alternative scenario
is likely to amplify this restraining effect when public officials are more inclined to apply a stricter principle of pre-
caution. Keeping this in mind, we can conclude that, although most interviewees agreed that the WEM-WAM scen-
ario approach was beneficial in the KAISU process as such, a more extensive array of policy scenarios would be
desirable when the long- and medium-term climate policy goals and related measures are to be determined. Disclosure statement No potential conflict of interest was reported by the author(s). We thank the interviewees for sharing their time and expertise. We also wish to thank the three anonymous reviewers for their con-
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http://orcid.org/0000-0002-4171-4304
Ville Lauttamäki
http://orcid.org/0000-0002-0688-5618 Funding This study was funded by the Academy of Finland in projects Towards deliberative climate and energy foresight (DECENT) under
grant numbers 325209 and 325207, and Determinants of successful foresight system in preventing food crisis (DEFORFO) under
grant number 335648. 12
K. ARO ET AL. 12
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Transport and Communications, and Ministry of Finance. . (2019). Finland’s Integrated Energy and Climate Plan. 2019:66. Ministry of
Economic Affairs and Employment. http://urn.fi/URN:ISBN:978-952-327-478-5
Ministry of the Environment. (2017). Government Report on Medium-Term Climate Change Plan for 2030 – Towards Climate-Smart
Day-to-Day Living. Ministry of the Environment. http://urn.fi/URN:ISBN:978-952-11-4752-4
Ministry of the Environment. (2022). “EU Climate Policy.” Retrieved August 12, 2022 (https://ym.fi/en/eu-climate-policy). Pye, S., Broad, O., Bataille, C., Brockway, P., Daly, H. E., Freeman, R., Gambhir, A., Geden, O., Rogan, F., Sanghvi, S., Tomei, J., Vorushylo,
I., & Watson, J. (2021). Modelling Net-zero emissions energy systems requires a change in approach. Climate Policy, 21(2), 222–31. https://doi.org/10.1080/14693062.2020.1824891 Ministry of Agriculture and Forestry. (2014). References Global warming under Old and New scenarios using IPCC climate sensitivity range
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and ‘LCDS.’. Climate Policy, 13(6), 770–76. https://doi.org/10.1080/14693062.2013.823334 Tyler, E., Boyd, A., Coetzee, K., Gunfaus, M. T., & Winkler, H. (2013). Developing country perspectives on
and ‘LCDS.’. Climate Policy, 13(6), 770–76. https://doi.org/10.1080/14693062.2013.823334 13 van Vuuren, D. P., Edmonds, J., Kainuma, M., Riahi, K., Thomson, A., Hibbard, K., Hurtt, G. C., Kram, T., Krey, V., Lamarque, J.-F., Masui, T.,
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Change, 154, 119972. https://doi.org/10.1016/j.techfore.2020.119972 Wright, G., Cairns, G., O’Brien, F. A., & Goodwin, P. (2019). Scenario analysis to support decision making in addressing
wicked problems: Pitfalls and potential. European Journal of Operational Research, 278(1), 3–19. https://doi.org/10.1016/j.ejor. 2018.08.035 Wright, G., Cairns, G., O’Brien, F. A., & Goodwin, P. (2019). Scenario analysis to support decision making in addressing
wicked problems: Pitfalls and potential. European Journal of Operational Research, 278(1), 3–19. https://doi.org/10.1016/j.ejor. 2018.08.035 Wright, G., & Goodwin, P. (2009). Decision making and planning under Low levels of predictability: Enhancing the scenario method. International Journal of Forecasting, 25(4), 813–25. https://doi.org/10.1016/j.ijforecast.2009.05.019 Wright, G., & Goodwin, P. (2009). Decision making and planning under Low levels of predictability: Enhancing the scenario method. International Journal of Forecasting, 25(4), 813–25. https://doi.org/10.1016/j.ijforecast.2009.05.019
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https://www.nature.com/articles/srep46576.pdf
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Neurodevelopmental Changes in Excitatory Synaptic Structure and Function in the Cerebral Cortex of Sanfilippo Syndrome IIIA Mice
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Scientific reports
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Neurodevelopmental Changes in
Excitatory Synaptic Structure and
Function in the Cerebral Cortex of
Sanfilippo Syndrome IIIA Mice
Chrissa A. Dwyer1, Samantha L. Scudder2, Ying Lin1, Lara E. Dozier2, Dustin Phan1,
Nicola J Allen3 Gentry N Patrick2 & Jeffrey D Esko1 received: 06 December 2016
accepted: 17 March 2017
Published: 18 April 2017 Sanfilippo syndrome, MPS IIIA-D, results from deficits in lysosomal enzymes that specifically degrade
heparan sulfate, a sulfated glycosaminoglycan. The accumulation of heparan sulfate results in
neurological symptoms, culminating in extensive neurodegeneration and early death. To study the
impact of storage in postnatal neurodevelopment, we examined murine models of MPS IIIA, which
lack the enzyme sulfamidase. We show that changes occur in excitatory postsynaptic structure and
function in the somatosensory cortex prior to signs of neurodegeneration. These changes coincide with
accumulation of heparan sulfate with characteristic non-reducing ends, which is present at birth in the
mutant mice. Accumulation of heparan sulfate was also detected in primary cultures of cortical neural
cells, especially astrocytes. Accumulation of heparan sulfate in cultured astrocytes corresponded with
augmented extracellular heparan sulfate and glypican 4 levels. Heparan sulfate from the cerebral cortex
of MPS IIIA mice showed enhanced ability to increase glutamate AMPA receptor subunits at the cell
surface of wild type neurons. These data support the idea that abnormalities in heparan sulfate content
and distribution contribute to alterations in postsynaptic function. Our findings identify a disease-
induced developmental phenotype that temporally overlaps with the onset of behavioral changes in a
mouse model of MPS IIIA. All cell types in vertebrates and invertebrates, including cells in the central nervous system (CNS), produce
plasma membrane and extracellular matrix heparan sulfate proteoglycans (HSPGs). HSPGs consist of a pro-
tein core with one or more covalently attached heparan sulfate (HS) chains. HS is comprised of alternating glu-
cosamine and uronic acids (d-glucuronic and l-iduronic) variously modified by sulfate groups1. Studies of animal
models deficient in HS biosynthesis show that HS has a role in nearly every developmental process in the brain,
including neural progenitor cell proliferation and differentiation2–4, axon guidance5,6 and synapse formation7,8. HS in the adult brain also plays a role in maintaining synaptic function9. These processes depend on the binding
of numerous growth factors, receptors and guidance proteins to sulfated domains within the chains1. HS turnover occurs through several mechanisms, including proteolytic shedding of proteoglycans from
the cell surface, limited endolytic cleavage of the chains by heparanase, and endocytosis, which culminates in
lysosomal degradation. Degradation of HS in the lysosome occurs in a sequential manner beginning at the
non-reducing end of the chain. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports SCIeNTIfIC REPortS | 7:46576 | DOI: 10.1038/srep46576 Results 1I),
showing that excitatory postsynaptic strength is reduced in Sgshh/h mice. No change in mEPSC inter-event inter-
val (Fig. 1J) was observed, consistent with the absence of change in the number of dendritic spines. These find-
ings reflect reduced glutamatergic excitatory synaptic strength in layer II/III pyramidal cortical neurons without
alterations in synapse number or presynaptic neurotransmitter vesicle release. Evaluation of α-amino-3-hydroxy-
5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit levels, GluA1 and GluA2, showed no change in
Sgshh/h mice by Western blotting (Fig. S1D,E) and surface expression by biotinylation (Fig. S1F,G). Thus, reduced
glutamatergic synaptic strength does not reflect a loss of GluA1 or GluA2 receptors. g
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To determine whether changes in synaptic function occurred in other brain regions, we also assessed a form
of short-term synaptic plasticity in the hippocampus as measured by excitatory synaptic basal transmission and
paired pulse facilitation. Basal synaptic transmission in the Schaffer collateral pathway in acute hippocampal
slices had normal field excitatory postsynaptic potentials (fEPSPs) in response to increasing stimulation intensi-
ties (Fig. S2A). Paired-pulse facilitation in these hippocampi was intact, although a small reduction in facilitation
with 50 msec separation of pulses was observed (Fig. S2B). Thus, MPS IIIA mice exhibit specific deficits in excita-
tory postsynaptic structure and function in the developing postnatal primary somatosensory cortex. Lysosomal expansion, but not astrocytosis and ganglioside accumulation, are present in the
developing postnatal somatosensory cortex. To determine whether changes in postsynaptic structure
and function might be due to lysosomal expansion in mutant animals, we analyzed the lysosomal marker, LAMP-
1, in 14, 21 and 61 day old mice. Enhanced LAMP-1 immunostaining was detected throughout the cortical layers
of the primary somatosensory cortex at all ages in Sgshh/h mice relative to littermate Sgsh+/h controls (Fig. 2A,B). We also evaluated astrogliosis and secondary accumulation of GM3 ganglioside, traits associated with neuro-
degenerative stages of disease progression in the aged MPS IIIA brain. Staining tissue sections with antibodies
against glial fibrillary protein (GFAP) showed reactive astrocytes along the pial basement membrane (P), a subset
of blood vessels (BV) and white matter (WM) tracks in control mice (Fig. 2C). Staining in the vicinity of the blood
vessels and white matter tracts was elevated in Sgshh/h mice (Fig. 2C), culminating at 61 days of age in widespread
distribution of GFAP-positive astrocytes throughout the somatosensory cortex (Fig. 2C,D). Results Alterations in excitatory synaptic structure and function occurs in the developing brain of Sgsh
mutant mice. To determine whether synaptic development was altered in MPS IIIA mice, we used a nat-
urally occurring hypomorphic mutant (Sgshh/h)16,17. This line contains a missense mutation in the sulfamidase
gene, Sgsh, reducing enzyme activity by >95%. Sgshh/h mice exhibit hyperactivity and signs of reduced anxiety as
early as 3 weeks of age, which is exaggerated by repeated behavioral testing18,19. Early behavioral abnormalities in
human patients and mouse models are consistent with manifestation of disease pathology in the somatosensory
cortex, a region of the cerebral cortex involved in the integration of sensory input. Formation and refinement of
the neural circuitry in this region of the brain occurs from postnatal day 7 to 28 in mice. Staining of sections of
postnatal day 21 somatosensory cortex with the excitatory postsynaptic marker postsynaptic density 95 (PSD-95)
showed enhanced puncta number in cortical layers I, II/III and V in Sgshh/h mice compared to heterozygous mice
(Sgsh+/h), which do not store HS or present any disease symptoms (Fig. 1A,B; Fig. S1A,B). Western blot analysis
on whole tissue homogenates and synaptosomes prepared from the cerebral cortex confirmed the accumulation
of PSD-95 in Sgshh/h mice (Fig. 1C). The increase in PSD-95 was not observed in 14-day old mice (Fig. S1C). In
the healthy brain, the postsynaptic apparatus containing PSD-95 localizes to the head of membranous protrusions
called dendritic spines. Golgi silver-impregnation of layer II/III pyramidal neurons in the primary somatosensory
cortex of Sgshh/h mice at day 21 showed that the number of dendritic spines was not altered (Sgsh+/h 0.84 ± 0.12,
Sgshh/h 0.79 ± 0.11 protrusions/μm, p > 0.05), but a modest increase in spine head width (Fig. 1D,E) and the per-
centage of mushroom-shaped spines was observed (Fig. 1F). These findings suggest that synapse architecture is
altered by the end of postnatal neurodevelopment in Sgshh/h mice. y
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Synapse strength closely correlates with dendritic spine morphology (reviewed in ref. 20). To determine
whether excitatory synaptic function was altered, electrophysiological recordings were performed in layer II/III
pyramidal neurons in the primary somatosensory cortex of acutely isolated brain slices from 21-day old Sgshh/h
mice (Fig. 1G). Evaluation of total miniature excitatory postsynaptic current (mEPSC) events revealed fewer large
events in the mutant (Fig. 1H). Average mEPSCs amplitude was also significantly reduced in the mutant (Fig. Neurodevelopmental Changes in
Excitatory Synaptic Structure and
Function in the Cerebral Cortex of
Sanfilippo Syndrome IIIA Mice
Chrissa A. Dwyer1, Samantha L. Scudder2, Ying Lin1, Lara E. Dozier2, Dustin Phan1,
Nicola J Allen3 Gentry N Patrick2 & Jeffrey D Esko1 Thus, inactivating mutations in any of the degradative enzymes arrests the pro-
cess, causing accumulation of HS fragments in the lysosome. In humans and animal models, storage results in
mucopolysaccharidosis (MPS), a lysosomal storage disorder10. Sanfilippo syndromes (MPS IIIA-D) arise from
deficits in enzymes that hydrolyze or modify the non-reducing terminal glucosamine unit. MPS IIIA (OMIM
252900) is a homozygous recessive, congenital disorder caused by the lack of the enzyme sulfamidase (SGSH),
which removes the N-sulfate group from terminal N-sulfoglucosamine units. Failure to degrade the chains and/
or accumulation of chains with terminal N-sulfoglucosamine units results in cell death and neurodegeneration. 1Department of Cellular and Molecular Medicine, Glycobiology Research and Training Center, University of California,
San Diego, La Jolla, CA, 92093-0687, USA. 2Section of Neurobiology, Division of Biological Sciences, University of
California, San Diego, La Jolla, CA 92093-0347, USA. 3Salk Institute for Biological Studies, Molecular Neurobiology
Laboratory, La Jolla, CA 92037, USA. Correspondence and requests for materials should be addressed to C.A.D. (email: cdwyer@ucsd.edu) or J.D.E. (email: jesko@ucsd.edu) SCIeNTIfIC REPortS | 7:46576 | DOI: 10.1038/srep46576 1 www.nature.com/scientificreports/ The syndrome also manifests behavioral changes during childhood11,12. MPS IIIB (OMIM 252920), MPS IIIC
(OMIM 252930) and MPS IIID (OMIM 252940), caused by deficiencies in other enzymes involved in lysosomal
processing of the non-reducing terminal glucosamine residue in HS, have similar clinical consequences. Recently,
mutation of a fifth enzyme involved in processing of terminal glucosamine residues (3-sulfoglucosamine sulfa-
tase) was shown to result in lysosomal storage and neuropathology in mice13.if y
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Sanfilippo syndrome affects the central nervous system and is considered a childhood neurodegenerative
disorder. MPS III children present typically with developmental delay that can be accompanied by severe hyper-
activity, autistic-like social behaviors and insomnia. In animal models of MPS III, the appearance of abnormal
neurological behaviors precedes neuronal cell loss and nervous system atrophy14,15. This observation suggests that
changes in neuronal circuit function may give rise to neurological dysfunction at disease onset. Here we show that
HS accumulation occurs at birth in the developing MPS IIIA brain and impacts neuronal function at early time
points in disease pathogenesis. The results demonstrate that MPS IIIA mice exhibit deficits in synaptic function
in the developing somatosensory cortex, which may explain early behavioral abnormalities in MPS III patients. Results (B) Quantification
of PSD-95 puncta in cortical layers I, II/III, and V of the primary somatosensory cortex of 21d unaffected
Sgsh+/h and Sgshh/h mice. Mean ± SEM. N = 5–6 animals per genotype. (C) Western blot for PSD-95 levels in
cortical homogenate and synaptosome preparations from 21d unaffected and hypomorphic MPS IIIA mice. Band pixel intensities were measured in ImageJ and normalized to β-actin. Mean ± SEM. N = 3 animals per
genotype, spanning 2 litters. (D) Representative maximum intensity projection z-stacks of dendritic segments
from pyramidal neurons in layer II/III of the somatosensory cortex of 21d unaffected and hypomorphic MPS
IIIA mice. (E) Quantification of dendritic spine head width and shaft length of pyramidal neurons in layer II/III
of the somatosensory cortex of 21d unaffected and hypomorphic MPS IIIA mice. Mean ± SEM. N = 3 animals
per genotype. (F) Quantification of dendritic spine type. Mean ± SEM. (G) Representative mEPSC recordings
from pyramidal neurons in layer II/III of the somatosensory cortex of 21–22d unaffected and hypomorphic
MPS IIIA mice. Scale bar depicts 20 pA, 200 msec. (H) Cumulative probability distributions of all mEPSC event
amplitudes recorded from pyramidal neurons in layer II/III of the somatosensory cortex of 21–22d unaffected
and hypomorphic MPS IIIA (mice, depicting reduced amplitude in mutant animals. N = 1883, 2362 events. p < 0.001, Kolmogorov-Smirnov test. (I) Mean mEPSC amplitude. Mean ± SEM. (J) Mean mEPSC inter-event
interval. N = 5 mice per genotype, 16–19 cells total. p = 0.015, Student’s t test. Heparan sulfate storage occurs at birth and increases coincident with postnatal brain
growth. Accumulation of LAMP-1 stained lysosomes is consistent with storage of HS fragments. HS that
accumulates in MPS IIIA has diagnostic terminal non-reducing end (NRE) glycan structures that serve as bio-
markers of the disease21. Digestion of the chains with bacterial lyases releases these NRE glycans, which can
be readily quantified by liquid chromatography/mass spectrometry (LC/MS)22. Accumulation of NRE gly-
cans in MPS disorders is usually quite dramatic in comparison to overall HS or glycosaminoglycan (GAG)
accumulation because the biomarker is typically present at very low levels in normal tissues, whereas all cells
make HS. Results GM3 ganglioside
also accumulated in cortical layers II/III and V of the somatosensory cortex of 61-day-old adult mutant mice
(Fig. 2C,E), but not in younger or unaffected mice. Thus, lysosomal expansion and post-synaptic dysfunction
precedes widespread astrocytosis and secondary storage of GM3 ganglioside. SCIeNTIfIC REPortS | 7:46576 | DOI: 10.1038/srep46576 2 www.nature.com/scientificreports/ Figure 1. Excitatory postsynaptic structure and function of layer II/III pyramidal neurons from the
primary somatosensory cortex of postnatal MPS IIIA mice is abnormal. (A) Representative confocal images
show an increase in PSD-95 puncta (red) in layer II/III of the primary somatosensory cortex in 21d (day-old)
hypomorphic MPS IIIA (Sgshh/h) mice. Scale, 50 μm. Nuclei are stained with DAPI (blue). (B) Quantification
of PSD-95 puncta in cortical layers I, II/III, and V of the primary somatosensory cortex of 21d unaffected
Sgsh+/h and Sgshh/h mice. Mean ± SEM. N = 5–6 animals per genotype. (C) Western blot for PSD-95 levels in
cortical homogenate and synaptosome preparations from 21d unaffected and hypomorphic MPS IIIA mice. Band pixel intensities were measured in ImageJ and normalized to β-actin. Mean ± SEM. N = 3 animals per
genotype, spanning 2 litters. (D) Representative maximum intensity projection z-stacks of dendritic segments
from pyramidal neurons in layer II/III of the somatosensory cortex of 21d unaffected and hypomorphic MPS
IIIA mice. (E) Quantification of dendritic spine head width and shaft length of pyramidal neurons in layer II/III
of the somatosensory cortex of 21d unaffected and hypomorphic MPS IIIA mice. Mean ± SEM. N = 3 animals
per genotype. (F) Quantification of dendritic spine type. Mean ± SEM. (G) Representative mEPSC recordings
from pyramidal neurons in layer II/III of the somatosensory cortex of 21–22d unaffected and hypomorphic
MPS IIIA mice. Scale bar depicts 20 pA, 200 msec. (H) Cumulative probability distributions of all mEPSC event
amplitudes recorded from pyramidal neurons in layer II/III of the somatosensory cortex of 21 22d unaffected Figure 1. Excitatory postsynaptic structure and function of layer II/III pyramidal neurons from the Figure 1. Excitatory postsynaptic structure and function of layer II/III pyramidal neurons from the
primary somatosensory cortex of postnatal MPS IIIA mice is abnormal. (A) Representative confocal images
show an increase in PSD-95 puncta (red) in layer II/III of the primary somatosensory cortex in 21d (day-old)
hypomorphic MPS IIIA (Sgshh/h) mice. Scale, 50 μm. Nuclei are stained with DAPI (blue). Results Quantification of total GAG (as uronic acid) and the NRE biomarker characteristic of MPS IIIA
(N-sulfoglucosamine) from whole brains of wild type, Sgsh+/h and Sgshh/h mice at birth and postnatal days 8, 10,
14, 37, and 58–60 showed that GAG accumulation in the brain was apparent at birth (Fig. 3A), with striking accu-
mulation of the diagnostic NRE biomarker at all time points (Fig. 3B). In mice, brain mass increases as cells are
added during the first several postnatal weeks, a process that did not differ significantly between genotypes at any SCIeNTIfIC REPortS | 7:46576 | DOI: 10.1038/srep46576 3 www.nature.com/scientificreports/ Figure 2. Lysosomal expansion but not astrocytosis and ganglioside accumulation are present in the
developing postnatal primary somatosensory cortex. (A) Representative confocal images from unaffected
(Sgsh+/h*) and hypomorphic MPS IIIA (Sgshh/h) mouse primary somatosensory cortex stained with LAMP-1
(green). Left panels show low magnification images, scale bar 100 μm. Right panels show high magnification
of layer II/III (boxed region), scale bar 50 μm. Insets in lower right corner show single cells. Nuclei were
stained with DAPI (blue). (B) Quantification of LAMP-1 stained vesicle area from high magnification
images. (C) Representative confocal images stained with a marker for reactive cortical astrocytes, GFAP (red)
and ganglioside GM3 (green), as described in (A). Arrows in low magnification images point to cells with
intracellular staining of GM3, denoted by asterisks in high magnification images. P, pial basement membrane;
WM, white matter; BV, blood vessel. (D) Quantification of GFAP immunoreactivity in the parenchyma of the
somatosensory cortex. (E) Quantification of GM3 immunoreactivity in the parenchyma of the somatosensory
cortex. N = 3 animals per genotype per age, 2 sections per animal, 2 regions of interest per section were
quantified. All brains were collected, stained, and analyzed at the same time. Findings were confirmed on a
separate cohort of N = 3 animals per genotype per age. Mean ± SD. Figure 2. Lysosomal expansion but not astrocytosis and ganglioside accumulation are present in the
developing postnatal primary somatosensory cortex. (A) Representative confocal images from unaffected
(Sgsh+/h*) and hypomorphic MPS IIIA (Sgshh/h) mouse primary somatosensory cortex stained with LAMP-1
(green). Left panels show low magnification images, scale bar 100 μm. Right panels show high magnification
of layer II/III (boxed region), scale bar 50 μm. Insets in lower right corner show single cells. Nuclei were
stained with DAPI (blue). Results (B) Quantification of LAMP-1 stained vesicle area from high magnification
images. (C) Representative confocal images stained with a marker for reactive cortical astrocytes, GFAP (red)
and ganglioside GM3 (green), as described in (A). Arrows in low magnification images point to cells with
intracellular staining of GM3, denoted by asterisks in high magnification images. P, pial basement membrane;
WM, white matter; BV, blood vessel. (D) Quantification of GFAP immunoreactivity in the parenchyma of the
somatosensory cortex. (E) Quantification of GM3 immunoreactivity in the parenchyma of the somatosensory
cortex. N = 3 animals per genotype per age, 2 sections per animal, 2 regions of interest per section were
quantified. All brains were collected, stained, and analyzed at the same time. Findings were confirmed on a
separate cohort of N = 3 animals per genotype per age. Mean ± SD. age (Fig. S3A). The extent of accumulation of NRE biomarker, when normalized to brain weight, did not change
significantly over the 8-week observation period. gi
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Disaccharide analysis also was performed using LC/MS, which provides compositional data as well as an
independent means for assessing total HS content23. HS was significantly elevated in cerebral cortical homoge-
nates prepared from Sgshh/h mice and increased modestly from 14 days to 58–60 days (Fig. 3C), as did NRE
levels (Fig. 3D). NRE levels were elevated to a greater extent in cortical samples compared to whole brain (11 ± 2
(14d) and 16 ± 4 (58d) nmol NRE/g tissue wet weight in cortical samples versus 5 ± 1 (14d) and 7 ± 1 (58d) nmol
NRE/g tissue wet weight in whole brain). The NRE biomarker was elevated to a similar extent at 14 days of age
in the cerebral cortex of a novel MPS IIIA mouse strain bearing a null allele for Sgsh (Sgsh−/−) (see methods;
compare Fig. 3E and B). Based on disaccharide analysis, the degree of 2-O-sulfation increased over time in Sgshh/h
mice (Fig. S3B), with corresponding changes in 2-O-sulfated disaccharides at 58 days (Fig. S3C). In contrast, no
changes in chondroitin/dermatan sulfate (CS/DS) levels or disaccharide composition were observed between
genotypes (Fig. S3D–F). These results show that the developing postnatal cerebral cortex is heavily burdened with
a pathological form of HS having an altered composition and characteristic NRE, which we refer to as “patholog-
ical HS”. Importantly, HS accumulates in the absence of secondary pathology and precedes synaptic dysfunction. Results (C) Cerebral cortex heparan sulfate levels measured by LC/MS. (D) Cerebral
cortex NRE levels by LC/MS. (E) Sgsh null (Sgsh−/−) cerebral cortex NRE levels by LC/MS. Values normalized
to wet tissue mass. Unaffected wild type and heterozygous (Sgsh+/h* open bars) and hypomorphic MPS IIIA
(S hh/h l
d b
)
l
th
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f bi th
0 d
N
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t p p Figure 3. Accumulation of undegraded heparan sulfate occurs in the developing postnatal MPS IIIA
mouse brain and cerebral cortex. (A) Whole brain glycosaminoglycan uronic acid levels by carbazole assay. (B) Whole brain NRE levels (N-sulfoglucosamine) by LC/MS. (A,B) values are normalized to brain wet weight,
which is presented in Fig. S3A. (C) Cerebral cortex heparan sulfate levels measured by LC/MS. (D) Cerebral
cortex NRE levels by LC/MS. (E) Sgsh null (Sgsh−/−) cerebral cortex NRE levels by LC/MS. Values normalized
to wet tissue mass. Unaffected wild type and heterozygous (Sgsh+/h* open bars) and hypomorphic MPS IIIA
(Sgshh/h closed bars), unless otherwise specified. Day of birth = 0 day. N = 3 animals per genotype per age. Transcardial perfusion with PBS was performed on all animals to eliminate contamination from residual blood
in tissue. In all cases graphs show mean ± SD. Figure 3. Accumulation of undegraded heparan sulfate occurs in the developing postnatal MPS IIIA
mouse brain and cerebral cortex. (A) Whole brain glycosaminoglycan uronic acid levels by carbazole assay. (B) Whole brain NRE levels (N-sulfoglucosamine) by LC/MS. (A,B) values are normalized to brain wet weight
which is presented in Fig. S3A. (C) Cerebral cortex heparan sulfate levels measured by LC/MS. (D) Cerebral
cortex NRE levels by LC/MS. (E) Sgsh null (Sgsh−/−) cerebral cortex NRE levels by LC/MS. Values normalized
to wet tissue mass. Unaffected wild type and heterozygous (Sgsh+/h* open bars) and hypomorphic MPS IIIA
(S hh/h l
d b
)
l
h
fi d
f b
h
d
l Figure 3. Accumulation of undegraded heparan sulfate occurs in the developing postnatal MPS IIIA mouse brain and cerebral cortex. (A) Whole brain glycosaminoglycan uronic acid levels by carbazole assay. (B) Whole brain NRE levels (N-sulfoglucosamine) by LC/MS. (A,B) values are normalized to brain wet weight,
which is presented in Fig. S3A. (C) Cerebral cortex heparan sulfate levels measured by LC/MS. (D) Cerebral
cortex NRE levels by LC/MS. (E) Sgsh null (Sgsh−/−) cerebral cortex NRE levels by LC/MS. Results Values normalized
to wet tissue mass. Unaffected wild type and heterozygous (Sgsh+/h* open bars) and hypomorphic MPS IIIA
(Sgshh/h closed bars), unless otherwise specified. Day of birth = 0 day. N = 3 animals per genotype per age. Transcardial perfusion with PBS was performed on all animals to eliminate contamination from residual blood
in tissue. In all cases graphs show mean ± SD. Sgshf/f mice to strains carrying different cell-specific Cre drivers (Fig. 4D) resulted in extensive recombination
of the floxed Sgsh allele based on PCR analysis of DNA derived from cerebral cortex (Fig. S4A,B). However,
inactivation of Sgsh in neurons (Sgshf/fSyn1-cre+), astrocytes (Sgshf/fGFAP-cre+) or endothelia/myeloid cells
(Sgshf/fTie2-cre+) did not result in NRE biomarker accumulation in the cerebral cortex (Fig. 4E), most likely due to
cross correction by secreted sulfamidase25. Consistent with this hypothesis, Sgshf/fNes-cre+ animals, which results
in Sgsh inactivation in the majority of cells in the brain, accumulated NRE biomarker (Fig. 4E) and LAMP-1
immunoreactive lysosomes, albeit to a lesser extent than Sgshh/h (Fig. 2A) and Sgsh−/− mice (Fig. S4C). y
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Although we were unable to assess the contribution of astrocyte accumulation to the neurodevelopmental
phenotype in vivo, we demonstrated that pathological HS that accumulates in the cerebral cortex affects the sur-
face expression of AMPA receptors in the postsynaptic membrane of rat cortical neurons cultured in vitro. HS
chains from the cerebral cortex of Sgsh+/h or Sgshh/h mice, which mostly derives from resident astrocytes, were
purified and added to cultures of mature wild type rat cortical neurons in equal “brain equivalents,” which mirrors
the two-fold increase in overall brain HS in the mutant. Plasma membrane AMPA receptor subunit levels were
assessed by cell surface biotinylation followed by precipitation with streptavidin beads and immunoblotting for
AMPA receptor GluA2 or GluA1 subunits. The addition of HS from either the mutant or the unaffected animals
did not affect the total level of GluA2 subunits (Fig. 5A), but plasma membrane associated GluA2 subunits were
increased when HS from the mutant was added or when higher concentrations of HS were added (Fig. 5B). Note
that the horizontal line with grey shading shows control levels of GluA receptor subunits in untreated samples. The addition of HS chains did not alter GluA1 levels, suggesting the elevated HS expressed in the mutant has a
specific effect on AMPA receptor GluA2 subunits (Fig. Results Pathological heparan sulfate alters cell surface AMPA GluA2 subunits. To determine the cellular
origin of HS bearing the NRE biomarker, primary cultures of highly enriched neurons or astrocytes (>95%
purity) were prepared. Dramatic accumulation of NRE biomarker was present in cell lysates prepared from mixed
primary cultures containing neurons and glia isolated from the cerebral cortex of newborn Sgshh/h mice (Fig. 4A). Most of this material derives from astrocytes based on 60-fold more NRE in enriched astrocyte preparations
compared to isolated neurons (Fig. 4B). Evaluation of Sgsh transcript levels in available RNAseq datasets showed
significantly higher expression of Sgsh transcripts in astrocytes than neurons, an observation that we confirmed
in the astrocyte and neuronal cell cultures by qPCR (Fig. 4C)24. Several HS-degrading lysosomal hydrolases and
genes that regulate lysosomal biogenesis, including transcription factor EB (TCFEB) also were enriched in astro-
cytes compared to neurons (Table S1). y
p
Synapse formation has both a cell autonomous neuronal component and non-cell autonomous glial compo-
nent. We attempted to measure the contribution of different cell types to disease pathogenesis in vivo by selective
deletion of a “floxed” allele of Sgsh (Sgshf/f) in astrocytes and other cell types found in the brain. Crossbreeding SCIeNTIfIC REPortS | 7:46576 | DOI: 10.1038/srep46576 4 www.nature.com/scientificreports/ Figure 3. Accumulation of undegraded heparan sulfate occurs in the developing postnatal MPS IIIA
mouse brain and cerebral cortex. (A) Whole brain glycosaminoglycan uronic acid levels by carbazole assay. (B) Whole brain NRE levels (N-sulfoglucosamine) by LC/MS. (A,B) values are normalized to brain wet weight,
which is presented in Fig. S3A. (C) Cerebral cortex heparan sulfate levels measured by LC/MS. (D) Cerebral
cortex NRE levels by LC/MS. (E) Sgsh null (Sgsh−/−) cerebral cortex NRE levels by LC/MS. Values normalized
to wet tissue mass. Unaffected wild type and heterozygous (Sgsh+/h* open bars) and hypomorphic MPS IIIA
(Sgshh/h closed bars), unless otherwise specified. Day of birth = 0 day. N = 3 animals per genotype per age. Transcardial perfusion with PBS was performed on all animals to eliminate contamination from residual blood
in tissue. In all cases graphs show mean ± SD. Figure 3. Accumulation of undegraded heparan sulfate occurs in the developing postnatal MPS IIIA
mouse brain and cerebral cortex. (A) Whole brain glycosaminoglycan uronic acid levels by carbazole assay. (B) Whole brain NRE levels (N-sulfoglucosamine) by LC/MS. (A,B) values are normalized to brain wet weight,
which is presented in Fig. S3A. Results (B) NRE levels in intracellular preparations from mature primary
cortical cultures of enriched neurons or astrocytes from unaffected and hypomorphic MPS IIIA mice. Note
that differences are presented using a logarithmic scale. N = 3 individual animals per genotype spanning two
litters. Cell preparations of neurons and astrocytes were obtained from different animals. (C) Relative Sgsh
transcript expression by quantitative RT-PCR in wild type primary cultures enriched for neurons or astrocytes. N = 3–4 individual animals spanning two litters. (D) Genomic arrangement of conditional Sgsh allele and
Cre-recombinase drivers. Table shows the affected cell types and developmental stage when cre recombinase is
expressed for each strain. (E) NRE levels in the cerebral cortex from mice at 182 days of age. N = 3 animals per
genotype. Figure 4. Contributions of neurons and astrocytes to HS accumulation in the cerebral cortex. (A) NRE Figure 4. Contributions of neurons and astrocytes to HS accumulation in the cerebral cortex. (A) NRE
levels in cell lysates prepared from primary mature cortical neural cultures containing a mixture of neurons
and glia isolated from unaffected (Sgsh+/h*) and hypomorphic MPS IIIA (Sgshh/h) mice. N = 3 individual
animals per genotype spanning two litters. (B) NRE levels in intracellular preparations from mature primary
cortical cultures of enriched neurons or astrocytes from unaffected and hypomorphic MPS IIIA mice. Note
that differences are presented using a logarithmic scale. N = 3 individual animals per genotype spanning two
litters. Cell preparations of neurons and astrocytes were obtained from different animals. (C) Relative Sgsh
transcript expression by quantitative RT-PCR in wild type primary cultures enriched for neurons or astrocytes. N = 3–4 individual animals spanning two litters. (D) Genomic arrangement of conditional Sgsh allele and
Cre-recombinase drivers. Table shows the affected cell types and developmental stage when cre recombinase is
expressed for each strain. (E) NRE levels in the cerebral cortex from mice at 182 days of age. N = 3 animals per
genotype. is sufficient to increase GluA2 levels and shows a dependence on concentration and that astrocytes may be the
primary source of HS that contributes to alterations in post-synaptic function. is sufficient to increase GluA2 levels and shows a dependence on concentration and that astrocytes may be the
primary source of HS that contributes to alterations in post-synaptic function. Results 5C).h pif
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The high level of HS accumulation in astrocytes and the predominance of astrocytes in the brain suggest that
these cells are the primary source of HS. Analysis of HS distribution in astrocytes by radiolabeling with 35SO4
showed that cell surface and secreted GAG levels did not differ in unaffected and mutant cells, but accumula-
tion of intracellular GAG was obvious in the mutant (Fig. 5D). In a label-chase experiment, which tracks the
turnover of metabolically labeled HS, both intracellular and cell surface GAG was increased in Sgshh/h astrocytes
(Fig. 5E). Purification of HS from these samples confirmed elevation of HS at the cell surface (Fig. 5F). Glypican
4 levels were also modestly elevated in astrocyte lysates derived from Sgshh/h mice, although the difference did
not reach significance (Sgsh+/h 1.0 ± 0.37, Sgshh/h 1.36 ± 0.56) (Fig. 5G). These findings show HS supplementation SCIeNTIfIC REPortS | 7:46576 | DOI: 10.1038/srep46576 5 www.nature.com/scientificreports/ p
Figure 4. Contributions of neurons and astrocytes to HS accumulation in the cerebral cortex. (A) NRE
levels in cell lysates prepared from primary mature cortical neural cultures containing a mixture of neurons
and glia isolated from unaffected (Sgsh+/h*) and hypomorphic MPS IIIA (Sgshh/h) mice. N = 3 individual
animals per genotype spanning two litters. (B) NRE levels in intracellular preparations from mature primary
cortical cultures of enriched neurons or astrocytes from unaffected and hypomorphic MPS IIIA mice. Note
that differences are presented using a logarithmic scale. N = 3 individual animals per genotype spanning two
litters. Cell preparations of neurons and astrocytes were obtained from different animals. (C) Relative Sgsh
transcript expression by quantitative RT-PCR in wild type primary cultures enriched for neurons or astrocytes. N = 3–4 individual animals spanning two litters. (D) Genomic arrangement of conditional Sgsh allele and
Cre-recombinase drivers. Table shows the affected cell types and developmental stage when cre recombinase is
expressed for each strain. (E) NRE levels in the cerebral cortex from mice at 182 days of age. N = 3 animals per
genotype. Figure 4. Contributions of neurons and astrocytes to HS accumulation in the cerebral cortex. (A) NRE
levels in cell lysates prepared from primary mature cortical neural cultures containing a mixture of neurons
and glia isolated from unaffected (Sgsh+/h*) and hypomorphic MPS IIIA (Sgshh/h) mice. N = 3 individual
animals per genotype spanning two litters. Discussion In this report, we show that excitatory postsynaptic structure and function is abnormal in the developing
somatosensory cortex in MPS IIIA mice, that HS accumulation is present at birth and remains at an elevated
steady-state level in the developing postnatal brain, and that HS accumulation occurs prior to astrocytosis and
secondary storage of ganglioside GM3. Furthermore, we show that HS accumulation mostly derives from astro-
cytes and that exogenous HS is sufficient to alter the level of AMPA receptor, GluA2, on the cell surface. The
reduced excitatory synaptic strength observed in 21 day-old MPS IIIA mice coincides with the appearance of
behavioral changes that have been published by other research groups18, suggesting a potential causal relation-
ship. Together these findings provide the first evidence that neurodevelopmental abnormalities contribute to
disease pathogenesis in MPS III. To our knowledge, previous studies have not addressed whether pathological HS accumulates early in brain
development and prior to histological hallmarks of neurodegeneration. In part, this lack of information reflects
a technological limitation for assessing HS (and other GAGs) in small samples, a problem solved using LC/MS
assessment of the MPS IIIA NRE biomarker, N-sulfoglucosamine. This method has a wide dynamic range, can
detect storage in as little as 104 cells or ≤1 mg wet weight of tissue, and has a signal (mutant) to noise (wildtype)
ratio that cannot be achieved using methods that simply measure total GAG by dye binding or chemical methods. Using LC/MS NRE analysis, we show that storage of pathological HS occurs in the brain at birth and did not
change over the first 8 postnatal weeks when normalized to brain weight, potentially explaining why the expected
increase in NRE is not observed over this period. We also show that HS accumulates differentially in isolated SCIeNTIfIC REPortS | 7:46576 | DOI: 10.1038/srep46576 6 www.nature.com/scientificreports/ Figure 5. HS alters AMPA receptor subunit GluA2 recruitment to the cell surface. (A–C) Mature wildtype
rat cortical neurons were treated for 72 hours with different brain equivalents of HS purified from the cerebral
cortex of unaffected and hypomorphic MPS IIIA mice. Note that the mutant brain contains twice the amount of
HS as the unaffected mice. (A) Western blots for GluA2 AMPA receptor subunit shows cell surface GluA2 levels
are enhanced following addition of HS, while total GluA2 levels are unchanged. (B) Quantification of surface
GluA2 levels normalized to total GluA2 from panel A. Mean ± SEM. Discussion (C) Quantification of surface GluA1
levels normalized to total GluA1 for the same samples in (B). Mean ± SEM. N = 3–6 wells per concentration
from N = 3 experiments. (B,C) Horizontal line with grey shading shows control levels in untreated samples
as mean ± SEM used for statistical comparison. (D) Purified GAGs from intracellular, cell surface, and media
fractions from primary astrocyte cultures from unaffected (Sgsh+/h) and hypomorphic MPS IIIA (Sgshh/h) mice
following a 48-hour radiolabeling with 35SO4. (E) Purified GAGs from corresponding fractions following a 48-
hour chase. (F) Purified HS from the fractions shown in panel E. Mean ± SD. N = 2 animals per genotype, 2
wells per animal. Results were confirmed on a separate cohort of N = 2 animals per genotype. (G) Western blot
for glypican 4 in cell lysates of cultured primary astrocytes. Buffer control (−), heparin lyase treated (+). re 5. HS alters AMPA receptor subunit GluA2 recruitment to the cell surface. (A–C) Mature wildtype fi Figure 5. HS alters AMPA receptor subunit GluA2 recruitment to the cell surface. (A–C) Mature wildtype
rat cortical neurons were treated for 72 hours with different brain equivalents of HS purified from the cerebral
cortex of unaffected and hypomorphic MPS IIIA mice. Note that the mutant brain contains twice the amount of
HS as the unaffected mice. (A) Western blots for GluA2 AMPA receptor subunit shows cell surface GluA2 levels
are enhanced following addition of HS, while total GluA2 levels are unchanged. (B) Quantification of surface
GluA2 levels normalized to total GluA2 from panel A. Mean ± SEM. (C) Quantification of surface GluA1
levels normalized to total GluA1 for the same samples in (B). Mean ± SEM. N = 3–6 wells per concentration
from N = 3 experiments. (B,C) Horizontal line with grey shading shows control levels in untreated samples
as mean ± SEM used for statistical comparison. (D) Purified GAGs from intracellular, cell surface, and media
fractions from primary astrocyte cultures from unaffected (Sgsh+/h) and hypomorphic MPS IIIA (Sgshh/h) mice
following a 48-hour radiolabeling with 35SO4. (E) Purified GAGs from corresponding fractions following a 48-
hour chase. (F) Purified HS from the fractions shown in panel E. Mean ± SD. N = 2 animals per genotype, 2
wells per animal. Results were confirmed on a separate cohort of N = 2 animals per genotype. www.nature.com/scientificreports/ www.nature.com/scientificreports/ considered as hallmarks of the later neurodegenerative phase of the disease. The delay in primary and secondary
pathology likely reflects the progressive nature of neurological deterioration in the aging MPS IIIA brain. p
gy
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Studies to evaluate alterations in synaptic function were performed primarily in the somatosensory cortex,
which is a region of the brain involved in processing sensory information. A striking increase in the number of
PSD-95 positive puncta was observed at 21 days of age prior to astrocytosis and ganglioside accumulation. In the
normal brain, PSD-95 localizes to dendritic spine heads. However, the number of dendritic spines on pyramidal
neurons in layer II/III did not differ from normal littermates and no change in mEPSC inter-event interval was
observed. Together these data indicate that the enhancement in PSD-95 puncta does not reflect an increase in
synapse number. Given the degree of HS accumulation in the 21-day old mouse brain, the absence of substantial
alterations in synapse number is surprising and suggests that the establishment of the appropriate number of
synaptic connections can occur even in the presence of lysosomal storage. y
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We observed a modest but consistent reduction in mEPSC amplitude. Reduced mEPSC amplitude was unex-
pectedly accompanied by enhanced dendritic spine width, suggesting that AMPA receptor function was reduced. Free HS chains are sufficient to alter AMPA receptor subunit levels at the cell surface based on the impact of add-
ing HS isolated from the cerebral cortex to rat cortical neurons. The elevated level of glypican 4 and cell surface
HS in MPS IIIA astrocytes support that astrocyte HS may be the primary source of HS that drives changes in post-
synaptic function in MPS IIIA. Allen et al. recently showed that the addition of HS proteoglycans, glypicans 4 or 6,
to retinal ganglion cells increases mEPSC amplitude and promotes the formation of excitatory synapses, whereas
deletion of glypican 4 reduces mEPSC amplitude7. Irie et al. reported that deletion of the HS co-polymerase Ext1
in post-mitotic neurons led to a reduction in plasma membrane levels of glutamate AMPA receptors, specifically
GluA2, and a modest but significant reduction in mEPSC amplitude9. The sensitivity of this receptor system to
both elevated and reduced HS levels is consistent with the bell shaped response curves reported by Allen and
colleagues7. www.nature.com/scientificreports/ Glypican 4 may be just one of several HSPGs that accumulate in MPS III, based on the observation
that MPS IIIB mice have elevated levels of glypican 1 in the medial entorhinal cortex28. g yp
Sambri and colleagues recently reported lysosomal impairment in 10-month-old MPS IIIA mice causes a
loss of presynaptic function in the hippocampus by inhibiting synaptic vesicle trafficking29. Thus, we suggest
that different forms of synaptic impairment likely cause changes in neurological function in the developing and
aged MPSIIIA brain, consistent with progressive neurological deterioration associated with this disorder. Future
studies to develop therapeutic treatments will benefit from this enhanced understanding of the disease process.f p
pi
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Only limited information is available on the contribution of different neural cell types to disease pathology
in MPS III30,31. Towards this goal, we examined a new mouse model carrying a conditional allele of Sgsh, driving
deletion of Sgsh in specific cell types by intercrossing Sgshf/f mice to animals expressing Cre recombinase in differ-
ent neural cell types, including neurons, endothelial cells, and astrocytes. The lack of NRE biomarker accumula-
tion in conditional knockout mice shows that cross-correction by secreted sulfamidase apparently occurs across
different cell types in the brain. Lower, but significant levels of biomarker were observed in Sgshf/fNestin-Cre mice,
suggesting that a non-neuroepithelial cell type (microglia, meningeal fibroblasts, and ependymal cells) may also
participate in cross-correction in the brain. p
p
In summary, we show for the first time that MPS IIIA has a neurodevelopmental component most likely
induced by accumulation of pathological forms of HS. Further studies are warranted to explore the relationship of
HS content and composition in synaptic biology not only in MPS disorders, but also in other neurodevelopmental
and neurodegenerative disorders that have shared characteristics with MPS III32. Discussion (G) Western blot
for glypican 4 in cell lysates of cultured primary astrocytes. Buffer control (−), heparin lyase treated (+). Figure 5. HS alters AMPA receptor subunit GluA2 recruitment to the cell surface. (A–C) Mat Figure 5. HS alters AMPA receptor subunit GluA2 recruitment to the cell surface. (A–C) Mature wildtype
rat cortical neurons were treated for 72 hours with different brain equivalents of HS purified from the cerebral
cortex of unaffected and hypomorphic MPS IIIA mice. Note that the mutant brain contains twice the amount of
HS as the unaffected mice. (A) Western blots for GluA2 AMPA receptor subunit shows cell surface GluA2 levels
are enhanced following addition of HS, while total GluA2 levels are unchanged. (B) Quantification of surface
GluA2 levels normalized to total GluA2 from panel A. Mean ± SEM. (C) Quantification of surface GluA1
levels normalized to total GluA1 for the same samples in (B). Mean ± SEM. N = 3–6 wells per concentration
from N = 3 experiments. (B,C) Horizontal line with grey shading shows control levels in untreated samples
as mean ± SEM used for statistical comparison. (D) Purified GAGs from intracellular, cell surface, and media
fractions from primary astrocyte cultures from unaffected (Sgsh+/h) and hypomorphic MPS IIIA (Sgshh/h) mice
following a 48-hour radiolabeling with 35SO4. (E) Purified GAGs from corresponding fractions following a 48-
hour chase. (F) Purified HS from the fractions shown in panel E. Mean ± SD. N = 2 animals per genotype, 2
wells per animal. Results were confirmed on a separate cohort of N = 2 animals per genotype. (G) Western blot
for glypican 4 in cell lysates of cultured primary astrocytes. Buffer control (−), heparin lyase treated (+). cortical neurons and astrocytes. Other secondary markers of disease, including GM326, CS/DS27, 2-O-sulfated
HS derived disaccharides15, and astrocytosis are not present in the developing MPS IIIA brain, and should be SCIeNTIfIC REPortS | 7:46576 | DOI: 10.1038/srep46576 7 www.nature.com/scientificreports/ i
For staining of PSD-95 in tissue sections, PBS perfused whole brains were dissected and cut down the mid-
line. The right hemisphere was flash frozen in OCT in a bath of isopentane that had been pre-cooled in liquid
nitrogen. Tissue was transferred to dry ice and frozen at −80 °C for a minimum of one day. Sagittal sections (12 μ
m) were cut using a cryostat and mounted directly onto glass slides. Glass slides were fixed for 8 min at −20 °C in
methanol, and then washed and fixed for 4 min in 4% PB-PFA at room temperature. Sections were stained with
an anti-PSD95 antibody (LifeTech 51-6900) as previously described34.i Species-specific Alexa Fluor-conjugated secondary antibodies were used to visualize primary antibody stain-
ing. Confocal images were collected on a Nikon Ti microscope equipped with the Nikon A1R confocal system
using a high NA 63X oil objective. The ND acquisition tool was used to acquire Z stacks in three channels. Quantification of PSD95 was performed using the puncta analyzer ImageJ plugin on 5 μm max projection confo-
cal images constructed from z-stacks. Data from Sgshh/h sections were normalized to unaffected littermates that
were sectioned and stained at the same time. N = 5–6 animals per genotype, 2 sections per animal, 2 images per
section, 2 regions of interest per image. Golgi impregnation and dendritic spine analysis and quantification were
performed exactly as described35. Western blot analysis. SDS-PAGE gels were cast manually. After electrophoresis of samples, bands
were transferred to either nitrocellulose or PVDF membrane. Western blotting was performed as previously
described36. Glycosaminoglycan Purification. Mice were heavily anesthetized using a ketamine/xylazine mixture, and
transcardial perfusion with Dulbecco’s PBS (dPBS) was performed. Whole brains were removed and dissected
into anatomical sub regions, when specified. Organs were homogenized in ice-cold buffer containing 50 mM
sodium acetate (pH 6.0) and 0.2 M sodium chloride. The tissue was dissociated with a Polytron homogenizer, and
the resulting homogenates were digested overnight at 37 °C with 0.1 mg/ml Pronase (type XIV from Streptomyces
griseus, Sigma Aldrich) containing 0.1% Triton X-100. Tissue homogenates were filtered through 0.45 μm syringe
filters or centrifuged at 10,000× g for 10 min to remove insoluble debris. Glycosaminoglycans were purified from
the supernates by anion exchange chromatography as described previously37. Non-reducing end and glycosaminoglycan analysis. The disaccharides resulting from enzymatic
depolymerization were dried and tagged with [12C6] aniline as described23. www.nature.com/scientificreports/ prior to decapitation and brains were incubated at 4 °C in modified artificial cerebrospinal fluid (ACSF) contain-
ing sucrose (83 mM NaCl, 2.5 mM KCl, 1 mM NaH2PO4, 26.2 mM NaHCO3, 22 mM glucose, 72 mM sucrose,
0.5 mM CaCl2, and 3.3 mM MgSO4). Coronal slices were prepared (350 μm) using a Leica VT1200 vibratome
and recovered in standard ACSF (119 mM NaCl, 5 mM KCl, 1 mM NaH2PO4, 26 mM NaHCO3, 11 mM glucose,
2 mM CaCl2, and 1 mM MgSO4). The slices were incubated at 34 °C for 30 min and at room temperature prior to
recordings. Slices were transferred to a submerged recording chamber and perfused with 30 °C oxygenated ACSF
(containing 0.1 mM picrotoxin). For miniature excitatory postsynaptic current (mEPSC) recordings, 1 μM tetro-
dotoxin (TTX) was included in the circulating ACSF. g
For mEPSC recordings, neurons displaying pyramidal-like morphology in layer II/III of primary somatosen-
sory cortex were whole-cell voltage-clamped at −70 mV with glass pipettes of 2.5–3.5 MΩ resistance filled with
a cesium-based internal solution (10 mM CsCl, 105 mM CsCH3SO3, 0.5 mM ATP, 0.3 mM GTP, 10 mM HEPES,
5 mM glucose, 2 mM MgCl2, and 1 mM EGTA, pH 7.2). mEPSC amplitude and inter-event interval were ana-
lyzed from 80–150 events per cell using Clampfit 10.3 and differences were determined using unpaired t-test in
GraphPad Prism. Immunohistochemistry. Mice of various ages were heavily anesthetized using a mixture of ketamine/xyla-
zine and transcardially perfused dPBS followed by 4% phosphate-buffered paraformaldehyde (PB-PFA). Tissues
were post-fixed overnight at 4 °C and cryo-protected by sinking in 30% PB-sucrose at 4 °C and cut on a cry-
ostat. Coronal sections (40 μm) were collected and stained as free-floating sections. Primary antibodies used
were LAMP-1 (1D4B, Developmental Studies Hybridoma Bank, University of Iowa), glial fibrillary acidic protein
(GFAP) (Sigma, G3893), and ganglioside GM3 (Cosmo Bio Co., NBT-M101). Confocal images were collected
on a Nikon Ti microscope equipped with the Nikon A1R confocal system using a high NA 63X oil, 10X, or 20X
objectives. The ND acquisition tool was used to acquire Z stacks in three channels. Quantification of LAMP-1 ves-
icle size was performed on high magnification max projection Z-stacks that span 4 μm in depth using the puncta
analyzer Image J plugin. Quantification of area stained by GFAP and GM3 was performed on images set to the
same threshold and area quantification was performed using Image J. Materials and Methods Mouse models. Hypomorphic MPS IIIA mice were purchased from Jackson Laboratory (B6. Cg-Sgshmps3a/PstJ)17. These mice carry a homozygous missense mutation in the Sgsh gene and exhibit <5%
residual activity of sulfamidase. A mouse model of MPS IIIA bearing a conditional “floxed” allele of Sgsh was
obtained from the Knockout Mouse Project Repository (Sgshtm1a(KOMP)Wtsi). The Sgsh “knockout first” allele was
converted to a conditional allele via flippase-mediated recombination, which exposed two lox-P sequences flank-
ing exons 3 through 6 of Sgsh. Subsequent crossing to animals bearing germ-line expression of bacterial Cre
recombinase eliminated these 4 exons and induced a frame-shift mutation at the junction between exons 2 and
7 and a premature stop codon, thus abolishing enzyme function and/or synthesis of the enzyme. Sgshf/f mice
were crossed to Nestin-cre mice (Jackson Laboratory, Tg(Nes-cre)2Wme), Synapsin1-cre (Jackson Laboratory, B6. Cg-Tg(Syn1-cre)671Jxm/J), and a GFAP-cre line (generated by the Messing Laboratory and provided by Don
Cleveland, UCSD33. To generate a mouse line bearing a null allele of Sgsh, Sgshf/+ mice were crossed to EIIA-
cre mice purchased from Jackson Laboratory (Bg.FVB-Tg(EIIa-cre)C5379Lmgd/L). Female offspring of genotype
Sgshf/+EIIA-cre+ were crossed to wildtype C57BL/6 h mice to obtain germline transmission of the null Sgsh allele
and to remove the cre transgene. Heterozygous offspring were bred to produce wild type, Sgsh+/− and Sgsh−/− off-
spring. Supplemental Table S2 contains primer sequences for genotyping.i g
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Plug discovery was counted as embryonic day 0 (E0) and the date of birth was defined as postnatal day 0 (P0). Male and female mutant mice were analyzed and gender was carefully monitored in all experiments. Unaffected
littermate animals of the same gender were analyzed to control for inter-litter and male/female variation. In all
experiments gender did not systematically vary. Data collected in experiments employing different experimental
techniques was generated using separate cohorts of mice. Animals were housed and bred in vivaria approved by
the Association for Assessment and Accreditation of Laboratory Animal Care located in the School of Medicine,
UCSD. All experiments were performed in accordance with relevant guidelines and regulations following stand-
ards and procedures approved by the UCSD Institutional Animal Care and Use Committee. Slice Electrophysiology. Acute hippocampal and cortical slices were prepared from 21–22 day old Sgsh+/h
and Sgshh/h mice with the experimenter blinded to genotype. Mice were deeply anesthetized with isofluorane 8 SCIeNTIfIC REPortS | 7:46576 | DOI: 10.1038/srep46576 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Each sample was mixed with a known
amount of [13C6] aniline tagged standards, including N-sulfoglucosamine, the primary saturated non-reducing
end biomarker for MPS IIIA (Carbosynth). Samples were analyzed by liquid chromatography-mass spectrometry
(LC/MS) using an LTQ Orbitrap Discovery electrospray ionization mass spectrometer (Thermo Scientific) as
described22. Internal disaccharides and the non-reducing end monosaccharide were quantified manually based
on their unique mass signatures relative to internal standards and normalized by either tissue wet weight or pro-
tein concentration, determined by BCA assay (Pierce). Data is presented using a disaccharide structural code38. Primary Neuron and Astrocyte Isolation. Mixed cortical cultures were prepared from single newborn
MPS IIIA pups as described39. Cells were cultured for 21 days prior to NRE and morphological analyses. Enriched
cultures of primary cortical neurons were prepared from single mouse embryos (E15.5) as described40. To select
for post-mitotic neurons, 5 μM cytosine arabinoside (AraC) was added at 1 day and removed with a medium
change at 3 days. cultures of primary cortical neurons were prepared from single mouse embryos (E15.5) as described . To select
for post-mitotic neurons, 5 μM cytosine arabinoside (AraC) was added at 1 day and removed with a medium
change at 3 days. Astrocyte cultures were obtained from the cortex of single newborn mice as described41. For NRE analysis,
cells cultured to maturation (18–21 days) were treated with 0.05% trypsin-EDTA to remove cell surface HSPGs. g
y
Astrocyte cultures were obtained from the cortex of single newborn mice as described41. For NRE anal
cells cultured to maturation (18–21 days) were treated with 0.05% trypsin-EDTA to remove cell surface HSP SCIeNTIfIC REPortS | 7:46576 | DOI: 10.1038/srep46576 9 www.nature.com/scientificreports/ Intact cells containing intracellular storage material were then collected by centrifugation. RNA was isolated
from enriched cultures of wild type or heterozygous neurons and astrocytes and Sgsh expression was assessed by
qRT-PCR (primer sequences, Table S2). Intact cells containing intracellular storage material were then collected by centrifugation. RNA was isolated
from enriched cultures of wild type or heterozygous neurons and astrocytes and Sgsh expression was assessed by
qRT-PCR (primer sequences, Table S2). Data quantification and statistical analyses. In all cases a researcher blinded to genotype performed
image quantification. www.nature.com/scientificreports/ Statistical analyses were performed using GraphPad Prism software Synaptic analyses are
presented as average values ± SEM to take into account the large number of samples and size of datasets, whereas
other data is presented as average values ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. p
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Additional details for experimental procedures including mouse breeding scheme, immunohistochemistry,
glycosaminoglycan purifications, primary cultures for neuron and astrocyte enrichment, slice electrophysiology
for field recordings, Western blot analysis, evaluation of surface AMPA receptor levels, and metabolic label/chase
experiments can be found in Supplementary Information under Materials and Methods. References 1. Xu, D. & Esko, J. D. Demystifying heparan sulfate-protein interactions. Annu Rev Biochem 83, 129–157 (2014). 1. Xu, D. & Esko, J. D. Demystifying heparan sulfate-protein interactions. Annu Rev Biochem 83, 129–157 (2014). 1. Xu, D. & Esko, J. D. Demystifying heparan sulfate-protein interactions. Annu Rev Biochem 83, 129–157 (2014). b
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l 2. Grobe, K. et al. Cerebral hypoplasia and craniofacial defects in mice lacking heparan sulfate Ndst1 gene function. Development 132
3777–3786 (2005). 3. Inatani, M., Irie, F., Plump, A. S., Tessier-Lavigne, M. & Yamaguchi, Y. Mammalian brain morphogenesis and midline axon guidance
require heparan sulfate. Science 302, 1044–1046 (2003).ii q
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4. McLaughlin, D. et al. Specific modification of heparan sulphate is required for normal cerebral cortical development. Mech Dev 120,
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(2010).fii SCIeNTIfIC REPortS | 7:46576 | DOI: 10.1038/srep46576 10 www.nature.com/scientificreports/ 36. Dwyer, C. A., Baker, E., Hu, H. & Matthews, R. T. RPTPzeta/phosphacan is abnormally glycosylated in a model of muscle-eye-b
disease lacking functional POMGnT1. Neuroscience 220, 47–61 (2012). 7. Esko, J. D. In Current protocols in molecular biology (eds Ausubel, F. et al.) 17.12.11–17.12.19 (Greene Publishing and Wiley
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9. Patrick, G. N., Bingol, B., Weld, H. A. SCIeNTIfIC REPortS | 7:46576 | DOI: 10.1038/srep46576 © The Author(s) 2017 Additional Information Supplementary information accompanies this paper at http://www.nature.com/srep Supplementary information accompanies this paper at http://www.nature.com/srep Competing Interests: The authors declare no competing financial interests. Competing Interests: The authors declare no competing financial interests. How to cite this article: Dwyer, C. A. et al. Neurodevelopmental Changes in Excitatory Synaptic Structure and
Function in the Cerebral Cortex of Sanfilippo Syndrome IIIA Mice. Sci. Rep. 7, 46576; doi: 10.1038/srep46576
(2017). How to cite this article: Dwyer, C. A. et al. Neurodevelopmental Changes in Excitatory Synaptic Structure and
Function in the Cerebral Cortex of Sanfilippo Syndrome IIIA Mice. Sci. Rep. 7, 46576; doi: 10.1038/srep46576
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Analytic Pragmatism and Universal LX Vocabulary
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1
Department of Philosophy, The Ohio State University, Columbus, OH, USA
2
Department of Philosophy; Arché Philosophical Research Centre; Centre for Exoplanet Science,
University of Saint Andrews, St Andrews, Fife, UK Philosophia (2017) 45:1803–1827
DOI 10.1007/s11406-017-9866-4 Analytic Pragmatism and Universal LX Vocabulary Richard Samuels1 & Kevin Scharp2 Received: 6 June 2017 /Accepted: 16 June 2017 /
Published online: 24 August 2017
# The Author(s) 2017. This article is an open access publication Received: 6 June 2017 /Accepted: 16 June 2017 /
Published online: 24 August 2017
# The Author(s) 2017. This article is an open access publication # The Author(s) 2017. This article is an open access publication Abstract In his recent John Locke Lectures – published as Between Saying and Doing
– Brandom extends and refines his views on the nature of language and philosophy by
developing a position that he calls Analytic Pragmatism. Although Brandom’s project
bears on an extraordinarily rich array of different philosophical issues, we focus here on
the contention that certain vocabularies have a privileged status within our linguistic
practices, and that when adequately understood, the practices in which these vocabu-
laries figure can help furnish us with an account of semantic intentionality. Brandom’s
claim is that such vocabularies are privileged because they are a species of what he calls
universal LX vocabulary –roughly, vocabulary whose mastery is implicit in any
linguistic practice whatsoever. We show that, contrary to Brandom’s claim, logical
vocabulary per se fails to satisfy the conditions that must be met for something to count
as universal LX vocabulary. Further, we show that exactly analogous considerations
undermine his claim that modal vocabulary is universal LX. If our arguments are
sound, then, contrary to what Brandom maintains, intentionality cannot be explicated as
a Bpragmatically mediated semantic phenomenon^, at any rate not of the sort that he
proposes. Keywords Pragmatism.Conceptualanalysis.Logic.Conditionals.LogicalConstants. Intentionality * Kevin Scharp
kscharp@gmail.com 1 Introduction Over the past three decades or so, Robert Brandom has exerted an enormous influence
on debates regarding the nature of language and philosophy. In his recent John Locke * Kevin Scharp
kscharp@gmail.com * Kevin Scharp
kscharp@gmail.com 1804 Philosophia (2017) 45:1803–1827 Lectures – published as Between Saying and Doing – Brandom further extends and
refines his views on these matters by seeking to develop and defend a position that he
calls Analytic Pragmatism. As we will see, this position combines two commitments
that are exceedingly widely endorsed amongst analytic philosophers; and a careful
discussion of the resulting view is of importance if for no other reason than, to date,
Brandom’s position constitutes the most detailed and systematic endeavor to develop
such a view. Although Brandom’s project bears on an extraordinarily rich array of different philo-
sophical issues, we focus here on a single central strand that runs throughout, and without
which the project as a whole fails. In brief, the strand in question concerns the contention that
certain vocabularies have a privileged status within our linguistic practices, and that when
adequately understood, the practices in which these vocabularies figure can help furnish us
with an account of semantic intentionality. More specifically, we focus on Brandom’s
discussion of two vocabularies that have long figured prominently in analytic philosophy:
logical vocabulary – especially the conditional – and modal vocabulary, such as that used to
express necessity and possibility.1 Brandom’s claim is that such vocabularies are privileged
because they are a species of what he calls universal LX vocabulary –roughly, vocabulary
whose mastery is implicit in any linguistic practice whatsoever. On such a view, then, logical
and modal vocabularies are privileged because they possess a kind of transcendental status. Further, according to Brandom, this transcendental status, at least in the case of modal
vocabulary, is important to characterizing the sort of intentionality essential to thought and
language. This paper has three main aims. First, we show that logical vocabulary per se fails to
satisfy the conditions that must be met for something to count as universal LX
vocabulary. We focus in particular on the case of the conditional, which Brandom
takes to be a paradigmatic example of logical vocabulary. If our argument goes though,
then the conditional is not universal LX, and Brandom’s efforts to defend the privileged
status of logic per se fail. 1 Following Brandom, we are interested here in aleithic modality as opposed to epistemic modality.
2 Brandom (2008: 189). 2 Brandom (2008: 189). 1 Introduction Second, we show that exactly analogous considerations
undermine the claim that modal vocabulary is universal LX. If this is so, then another of
Brandom’s central claims is undermined – what he calls the modal Kant-Sellars thesis –
the thesis that the use of ordinary, empirical vocabulary presupposes those capacities
required for the introduction and deployment of modal vocabulary. Finally, we argue
briefly that given our criticism of the modal Kant-Sellars thesis, Brandom’s conception
of intentionality, which he articulates in the final pages of his Locke Lectures, is also
untenable. If our arguments are sound, then, contrary to what Brandom maintains,
intentionality cannot be explicated as a Bpragmatically mediated semantic
phenomenon^,2 at any rate not of the sort that he proposes. Here is how we proceed. In Section 1, we provide an overview of Brandom’s
Analytic Pragmatism. In Section 2, we focus on the notion of universal LX vocabulary,
and both explain what it is and why is it so important to Brandom’s overarching project. In Section 3, we set out Brandom’s case for the conditional being universal LX. Then in
Section 4, we present our criticisms of this case; and in Section 5, we turn our attention
to modal vocabulary and argue that considerations exactly analogous to those discussed
in Section 4 also undermine the modal Kant-Sellars thesis. Finally, in Section 6, we 1805 Philosophia (2017) 45:1803–1827 draw out the implications of the forgoing discussion for Brandom’s account of
intentionality. draw out the implications of the forgoing discussion for Brandom’s account of
intentionality. 3 Brandom (2008: 1). 2 Analytic Pragmatism Brandom’s overall project – what he calls Analytic Pragmatism – can be thought of as a
consequence of endorsing two commitments. The first is a methodological commit-
ment, which he takes to be central to traditional analytic philosophy: The Analytic Project: A central goal of philosophy ought to be to establish
semantic relations between vocabularies – kinds of linguistic expressions – in
order to Bmake sense of the meanings expressed by one kind of locution in terms
of the meanings expressed by another kind of locution^.3 Paradigmatically, these
semantic relations include translation, paraphrase, a priori entailment, and truth-
making. The history of analytic philosophy is, of course, littered with attempts to produce
such analyses —including, the reduction of arithmetic to logic, the translation of
psychological vocabulary into physical vocabulary, and the analysis of moral vocabu-
lary in non-moral terms. By endorsing this commitment, then, Brandom locates his
position as continuous with the project of traditional analytic philosophy. Brandom’s second central commitment is a general thesis about the metaphysics
of meaning: Semantic Pragmatism: The meaning of a linguistic expression is determined by
its use in a linguistic practice. 4 Terminological note: Brandom tends variously to use the expressions ‘practice’, ‘activity’, ‘ability’, ‘exercise
of a practice’, and ‘practice-or-ability’ more-or-less interchangeably; see Brandom (2008: 9). For simplicity
and ease of exposition we just use the term ‘practice’. Semantic Pragmatism: The meaning of a linguistic expression is determined by
its use in a linguistic practice. This sort of meaning pragmatism has also been prominent in the history of analytic
philosophy. Though we suspect that many proponents of the analytic project have also
endorsed Semantic Pragmatism, far from being a natural complement to the Analytic
Project, such pragmatism poses, as Brandom notes, a serious challenge to it. The
challenge takes a variety of forms, but the rough idea is this: If our practices determine
the meanings of linguistic expressions, then it is highly implausible to suppose that
expressions comprising different vocabularies – and, hence, having different meaning-
determining practices – will tend to bear systematic semantic relations to each other,
such as translation, reduction and paraphrase. Thus by endorsing both the Analytic
Project and Semantic Pragmatism, Brandom seeks to combine a pair of commitments
that seem to be in tension with each other. As a consequence, a central challenge for
Brandom – and one that exerts an enormous influence on the overall shape of his
Analytic Pragmatism – is to reconcile this apparent tension: to show that, contrary to
appearances, one can pursue the goal of philosophical analysis at the same time as
cleaving to Semantic Pragmatism. 3 Brandom (2008: 1). 1806 Philosophia (2017) 45:1803–1827 How is this to be done? Brandom’s solution is to propose that Semantic Pragmatism
requires that we rethink the fundamental semantic relations between the analyzed and
analyzing vocabularies in philosophical analyses. Specifically, he proposes that if
linguistic meaning is determined by use, then the relationship between distinct vocab-
ularies should be mediated by linguistic practices.4 Thus, for Brandom, the way to be a
pragmatist and to pursue the Analytic Project is to take the analyzing vocabulary to
describe those practices that are sufficient to deploy the target vocabulary. If one could
make good on this suggestion, then semantic pragmatists could have their analytic
cake, and eat it too. 3 Universal LX Vocabulary So far we have sketched the overarching shape of Brandom’s project. But why does the
notion of universal LX vocabulary come to play such a central role? 3.1 Why Universal LX Vocabulary Matters 3.1 Why Universal LX Vocabulary Matters 3.1 Why Universal LX Vocabulary Matters There are two quite different roles that the notion of universal LX vocabulary plays in
Brandom’s project. The first, exemplified by its application to logic, is to justify parts of
the analyzing base that everyone must presuppose in order to pursue the Analytic
Project. The second, exemplified by Brandom’s discussion of modality, is to justify or
vindicate the use of philosophically contested notions. In both sorts of case, however,
Brandom maintains that the response should be the same. Roughly, where some
vocabulary – contested or otherwise – is universal LX, we get it for free since the
capacity to master that vocabulary is a necessary condition for the possibility of any
language-use whatsoever. Let us consider the cases of logical vocabulary and modality
in a bit more detail. Logic One way to make vivid the potential significance of the notion of universal LX
vocabulary for analytic philosophy is to note, as Brandom does, that within any project
of analysis – classical or pragmatist – logical vocabulary has a privileged status. Roughly put, no matter what the analysandum, it is taken for granted that logical
vocabulary must be part of the analyzing base. If, for example, one seeks to translate
psychological vocabulary into physical vocabulary, it is not merely that one uses the
substantive terms of physics; one also uses – and indeed must use – such logical
resources as conjunction, disjunction, quantifiers, and of course the conditional. But if
this is so, then as Brandom notes, some justification or explanation is required for why
logical vocabulary should have this privileged status. The notion of universal LX
vocabulary is central to Brandom’s effort to address this challenge. Logical vocabulary
is supposed to be privileged because it is a species of universal LX vocabulary, and
universal LX vocabulary is supposed to be privileged. 1807 Philosophia (2017) 45:1803–1827 Modality At least since Locke, modal vocabulary has been a point of contention
within philosophy, especially amongst those of an empiricist bent.5 Suspicion of alethic
modality traditionally comes in at least three forms. First, there are commonplace
epistemic concerns regarding how we can come to know – or justifiably believe –
modal claims. Second, there are familiar metaphysical concerns about the Bweirdness^
of an ontology that allows for modal truths that are not merely truths of language. 3.1 Why Universal LX Vocabulary Matters Finally, empiricists have long questioned the intelligibility of modal locutions
—what claims to possibility or necessity might even mean. Despite these
familiar concerns there has been, as Brandom notes, something of a sea change
in philosophical attitudes towards modality in recent decades, in large measure
due to developments in the formal semantics of modal logics. Nonetheless,
Brandom argues – and we are inclined to agree – that such developments leave
largely unaddressed the original philosophical challenges to modality. It is here
that the notion of universal LX vocabulary is supposed to enter the picture. For
if modal locutions are universal LX, then no matter what philosophical puzzle-
ment accompanies them, they ought to be construed as holding a privileged and
legitimate status. Modal vocabulary is supposed to be privileged because it is a
species of universal LX vocabulary, and universal LX vocabulary is supposed
to be privileged. 5 The same is, of course, true of normative vocabulary —a topic in which Brandom also invests much time in
his Locke lectures. 3.2 What Universal LX Vocabulary is and Why It Matters We now need to say more about Brandom’s notion of universal LX vocabulary. Here is
a first pass. Universal LX vocabulary is supposed to be a privileged class of linguistic
expressions in that they satisfy the following two conditions: a)
The vocabulary allows one to describe practices that are necessary for having
mastery for any vocabulary whatsoever, and a)
The vocabulary allows one to describe practices that are necessary for having
mastery for any vocabulary whatsoever, and b)
Once one masters the described practice, one can in principle deploy the very
vocabulary used to describe it. b)
Once one masters the described practice, one can in principle deploy the very
vocabulary used to describe it. Crudely put, Universal LX vocabulary describes the necessary conditions for mastery
of any linguistic expression whatsoever, including universal LX vocabulary itself. Crudely put, Universal LX vocabulary describes the necessary conditions for mastery
of any linguistic expression whatsoever, including universal LX vocabulary itself. Even this rough formulation is a bit of a mouthful. But to understand clearly
Brandom’s notion of universal LX vocabulary we need to explain several additional
notions. First, we need to explain two notions of autonomy. The first is the notion of an
autonomous vocabulary. To a first approximation: A vocabulary is autonomous if and
only if one could use it even though one could use no other vocabulary whatsoever. Suppose, for example, that it was possible to master the language of arithmetic and
nothing else. Then the language of arithmetic would be an autonomous vocabulary in
the relevant sense. The second notion of autonomy – an autonomous practice – can be
defined in terms of the previous notion. To a first approximation: an autonomous
practice is any practice that is sufficient for mastery of an autonomous vocabulary. 1808 Philosophia (2017) 45:1803–1827 As we will soon see, the paired notions of an autonomous vocabulary and an
autonomous practice are important because they are required in order to specify
what is it for a LX vocabulary to be universal. But we are getting ahead of
ourselves. Before explaining what makes an LX vocabulary universal, we first
need to explain what makes a vocabulary LX, and this in turn requires that we
characterize a range of what Brandom calls meaning-use relations. 3.2 What Universal LX Vocabulary is and Why It Matters Given the
project of Analytic Pragmatism – to specify relations between vocabularies that
are mediated by practices – Brandom appeals to a number of such relations: 1)
PV-Sufficiency: Where one has mastery of any vocabulary – autonomous or
otherwise – Brandom supposes that there must be some set of practices that
suffices for mastery of that vocabulary. He calls this relation PV-sufficien-
cy. Roughly: a practice is PV-sufficient for a vocabulary just in case one
has mastery of the vocabulary, if one engages in that practice. 2)
PV-Necessity: Now suppose that for some vocabulary there is a practice that one
must engage in to count as having mastery of the vocabulary. Suppose, for
example, that the capacity to identify the successor of any natural number is
required for mastery of the language of arithmetic. Then this practice – identifying
successors – would be PV-necessary for mastery of arithmetic vocabulary. 2)
PV-Necessity: Now suppose that for some vocabulary there is a practice that one
must engage in to count as having mastery of the vocabulary. Suppose, for
example, that the capacity to identify the successor of any natural number is
required for mastery of the language of arithmetic. Then this practice – identifying
successors – would be PV-necessary for mastery of arithmetic vocabulary. 3)
VP-Sufficiency: This relation between vocabularies and practices obtains when a
practice can be specified in a particular vocabulary. That is, a vocabulary is VP-
sufficient for a practice just in case that practice can be described using that
vocabulary.6 4)
PP-Sufficiency: The fourth relation that we consider here obtains between prac-
tices. If, when one engages in a particular practice, one can in principle do
everything needed to engage in some other practice, then the former is PP-
sufficient for the latter. For example, perhaps being able to add any three
numbers between 0 and 9 is PP-sufficient for being able to find the sum of
any two numbers by the usual method of addition. In characterizing PP-
sufficiency, Brandom tends to express a preference for using the framework
of computational theory, but there is no reason to think that this is essential
to the notion.7 5)
PP-Necessity: The final relation we consider is also a relation between practices. One practice is PP-necessary for another if it is impossible to engage in the second
without engaging in the first. 6 We discuss this relation in far greater detail in Section 4.1.
7 7 See Brandom (2008: ch. 2). (a)
P2 is PV-necessary for V2, (b)
P2 is PP-sufficient for P1, 7 See Brandom (2008: ch. 2). (d)
V1 is VP-sufficient for P2. In effect, what this says is that V1 specifies a practice P2 that one must be able to
master in order to be a competent user of another vocabulary V2 and, moreover, the
practice P2 incorporates everything that is required, in principle, in order to engage in a
practice that the use of V1 depends upon –i.e., P1. This set of conditions can be
captured, perhaps more intuitively, in what Brandom calls a meaning-use diagram
(see Fig. 1). Finally, we are in position to characterize the notion of universal LX
vocabulary. A vocabulary is universal LX iff it is an LX vocabulary for any
autonomous vocabulary. We can capture this in terms of an additional meaning-
use relation: (e)
P2 is PP-necessary for any autonomous practice – i.e. a practice that is PV-
sufficient for an autonomous vocabulary. 3.2 What Universal LX Vocabulary is and Why It Matters For example, the practice of asserting is PP-necessary
for the practice of asserting conditionals. 5)
PP-Necessity: The final relation we consider is also a relation between practices. One practice is PP-necessary for another if it is impossible to engage in the second
without engaging in the first. For example, the practice of asserting is PP-necessary
for the practice of asserting conditionals. With this terminology in hand, we can now provide a more explicit formu-
lation of LX vocabulary. A vocabulary V1 is LX for a vocabulary V2 iff there
exists practices P1 and P2, such that: (a)
P2 is PV-necessary for V2, (b)
P2 is PP-sufficient for P1, 1809 Philosophia (2017) 45:1803–1827 (c)
P1 is PV-sufficient for V1,
(d)
V1 is VP-sufficient for P2. (c)
P1 is PV-sufficient for V1,
(d)
V1 is VP-sufficient for P2. (c)
P1 is PV-sufficient for V1, (d)
V1 is VP-sufficient for P2. 4 Logic as Universal LX: The Case of the Conditional It is time to consider how the notion of universal LX vocabulary is supposed to
apply to Brandom’s paradigmatic example of a logical expression: the condi-
tional. In order for the conditional to be universal LX, it must be LX for any
autonomous vocabulary whatsoever. So, applying the definition from
Section 2.2, let us assume that V2 is any arbitrary autonomous vocabulary,
and V1 is the conditional. Moreover, let us follow Brandom as characterizing
P1 as the practice of using the conditional, and P2 as the practice of inferring
one claim from another. In which case, again following Brandom, there are five
conditions—represented by the meaning-use diagram in Fig. 2—that need to be
satisfied. Condition 1. According to the first condition: P2, the practice of inferring one claim
from another, is PV-necessary for the autonomous vocabulary, V2. In other words, one
must be able to infer one claim from another in order to count as using any vocabulary
whatsoever. This is a central tenet of Brandom’s inferentialism on which the capacity to
draw inferences is a metaphysically necessary condition on being a concept-user –
hence language-user – of any sort.9 As Brandom puts it: Assertions are essentially, and not just accidentally, speech acts that can play the
role both of premises and as conclusions of inferences … According to this way
of thinking, inferential practices are PP-necessary components of every autono-
mous discursive practice, hence PV-necessary for the deployment of every
autonomous vocabulary, hence PV-necessary for the deployment of every vocab-
ulary whatsoever.10 Though there are those who might wish to take issue with this claim, in what follows
we grant this assumption, at least for the sake of argument. Though there are those who might wish to take issue with this claim, in what follows
we grant this assumption, at least for the sake of argument. Condition 2. The second condition concerns a relationship between practices. According to Brandom: P2, the practice of inferring, is PP-sufficient for P1, the practice of
using the conditional. In other words, if a person can infer one claim from another, then she
can, at least in principle, do everything that she needs to be able to do to count as having
mastery of the conditional. Again, Brandom’s inferentialism, and the connection it assumes
between asserting and inferring, is central here. 8 Brandom (2008: 52-53).
9 See Brandom (1994).
10 Brandom (2008: 42-43). (e)
P2 is PP-necessary for any autonomous practice – i.e. a practice that is PV-
sufficient for an autonomous vocabulary. (e)
P2 is PP-necessary for any autonomous practice – i.e. a practice that is PV-
sufficient for an autonomous vocabulary. Thus if a vocabulary satisfies the conditions (i.e., (a)-(e)) for being universal LX,
then not only are the practices it describes necessary for being a language user of any
sort, but the practices so described also suffice for mastery of the universal LX
vocabulary itself. As Brandom puts it for the case of logic, Banyone who can talk at
all, hence can deploy any base vocabulary, can already do everything one needs to be Fig. 1 General meaning-use diagram for LX Vocabulary Fig. 1 General meaning-use diagram for LX Vocabulary Fig. 1 General meaning-use diagram for LX Vocabulary 1810 Philosophia (2017) 45:1803–1827 able to do in order in principle to be able to say what logical vocabulary lets one say.^8
We might add that if Brandom is correct, then the same is true for modal (and
normative) vocabulary as well. 4 Logic as Universal LX: The Case of the Conditional According to this view, if we begin with an
agent that can assert and infer, then: …we can teach it also to produce-assertively tokenings of the new form Bif p then
q.^…The system must respond to its assertion of the conditional Bif p then q^ by …we can teach it also to produce-assertively tokenings of the new form Bif p then …we can teach it also to produce-assertively tokenings of the new form if p the
q.^…The system must respond to its assertion of the conditional Bif p then q^ b Philosophia (2017) 45:1803–1827 1811 Fig. 2 Our meaning-use diagrams differ from Brandom’s in that we list the PP-necessity relation as an arrow
instead of as inclusion. See Fig. 1.9 on Brandom (2008: 28) Fig. 2 Our meaning-use diagrams differ from Brandom’s in that we list the PP-necessity relation as an arrow
instead of as inclusion. See Fig. 1.9 on Brandom (2008: 28) treating the inference from p to q as a good one—for instance, by being disposed
to endorse q assertionally if it is disposed to endorse p assertionally… In a clear
sense, then, the capacity to distinguish good from bad inferences involving non-
logical sentences is (PP-) sufficient for the ability to deploy conditionals involving
those sentences.11 Condition 3. According to the third condition that needs to be satisfied for ‘If__,
then__’ to be universal LX: P1, the practice of using conditionals, is PV-sufficient for
mastery of V1, conditional vocabulary. This is true given the definitions of P1 and V1. then__’ to be universal LX: P1, the practice of using conditionals, is PV-sufficient for
mastery of V1, conditional vocabulary. This is true given the definitions of P1 and V1. Condition 4. The fourth condition on ‘If__, then__’ being universal LX is that the
vocabulary of conditionals permits us to specify adequately the practice of inferring. In
other words: V1, the vocabulary of conditionals, is VP-sufficient for P2, the practice of
inferring. This condition is captured by the diagonal arrow in Fig. 2. Notice that the
existence of such VP-relations is crucial to the overall success of Brandom’s Analytic
Pragmatism. For precisely what VP-sufficiency does is establish practice-mediated rela-
tions between pairs of vocabularies. In other words, without VP-sufficiency we would be
unable to provide a practice-mediated analysis of one vocabulary in terms of another. 11 Brandom (2008: 45). 4 Logic as Universal LX: The Case of the Conditional In
the present case, conditional vocabulary is taken to provide a means of specifying the
practice of inference making, and thereby Bmake explicit^ what was only implicit in the
practice of drawing inferences. As Brandom puts it: BConditionals make explicit 1812 Philosophia (2017) 45:1803–1827 something that otherwise was implicit in the practical sorting of non-logical inferences into
good and bad.^12 Condition 5: If a vocabulary meets conditions 1–4, then it is LX. The final
condition, which renders it universal LX, is that the practice of inferring is PP-
necessary for any autonomous practice. In other words, every language-user must be
proficient at inferring. Again, this is controversial; but we grant it here, at least for the
sake of argument. To summarize: Brandom maintains that conditionals are universal LX because they
satisfy all of the above conditions. The burden of the next section is to show that he is
mistaken. 5 Conditionals are Not Universal LX Our complaint against Brandom’s treatment of the conditional is threefold. First,
we maintain that there are two quite different readings of VP-sufficiency and
that there are serious problems with the claim that conditionals are, in the sense
relevant to the Analytic Project, VP-sufficient for the practice of inferring. Second, even if the first objection is waived, we maintain that PP-sufficiency
(i.e., Condition 2) fails. Third, even if one supplements the account so that
Condition 2 holds, Condition 4 fails; and moreover it fails no matter which of
the two readings of VP-sufficiency one adopts. Thus, we maintain that even if
conditionals are VP-sufficient for the practice of inferring, it still follows that
conditionals are not Universal LX. 12 Brandom (2008: 46). 5.1 Descriptive and Expressive Notions of VP-Sufficiency The first problem arises from the fact that Brandom’s discussion incorporates two rather
different construals of VP-sufficiency: what we call the descriptive and expressive
readings. The problem for Brandom is that, on the one hand, only the descriptive
reading will do the work required by Analytic Pragmatism, whilst on other the other
hand, conditionals can, at most, provide the basis for the expressive form of VP-
sufficiency. Let us explain this in more detail. As characterized by Brandom, the objective of Analytic Pragmatism is to provide
pragmatic analyses —i.e., to specify, in one vocabulary, the practices that determine the
content of another vocabulary. This is precisely the role that VP-sufficiency is supposed
to play within the broader project. But talk of Bspecifying^ appears, given the context,
to involve describing a set of practices. Indeed, his paradigm cases of VP-sufficiency
confirm this impression. For example, Brandom glosses what he calls the Kaplan-
Stalnaker semantics for indexicals by saying the following: [It provides a] purely non-indexical vocabulary [that] can serve as an adequate
pragmatic metavocabulary for indexical vocabulary. That is, one can say (that is,
describe), in wholly non-indexical terms, everything one needs to do in order to 1813 Philosophia (2017) 45:1803–1827 use indexical vocabulary. Non-indexical vocabulary is VP-sufficient to specify
practices-or-abilities PV-sufficient to deploy indexical vocabulary.13 In short: VP-sufficiency, on this descriptive construal, involves the description of
linguistic practices. Let us turn to the second, expressive construal of VP-sufficiency. On this view, VP-
sufficiency need not involve the description of practices. Instead it consists in one
vocabulary making explicit what is only implicit in a linguistic practice. Very roughly,
one makes explicit a linguistic practice or ability when one introduces an expression
into the language that allows us to formulate claims about – and rationally assess –
performances of the practice.14 To take a relatively simple example, Brandom claims in
some earlier work that the negation sign renders explicit what would otherwise only be
implicit in our linguistic practices — namely, treating two claims as incompatible. As
he puts it: Negation, as a logical connective supporting formally valid inferences, plays the
same explicitating role with respect to material incompatability relations among
judgeable (that is propositional) contents that the conditional plays with respect to
the material inferential relations among such contents.15 n this expressive construal of VP-sufficiency, then, no description need occur. 5.1 Descriptive and Expressive Notions of VP-Sufficiency p
y,
,
p
The problem is that a vocabulary that only makes explicit some feature of a practice need
not describe that practice. Yet precisely what pragmatic analyses seem to require is a
description of the practices. One way to see the point is to consider the pragmatic Banalysis^
that would result from merely making explicit the practice of treating two claims as
incompatible. If all that were required for VP-sufficiency was that this be made explicit,
then the Banalysis^ would consist in one word: not! By any lights, such a result would not be
an analysis worthy of the name. But it should be clear that conditionals can no more describe
the practice of inference than the negation sign can describe the practice of treating-as-
incompatible. Instead, what it more plausibly does, is make some element(s) or aspect(s) of
the practice explicit. And indeed, this is what Brandom ends up emphasizing when he argues
for the claim that conditionals are universal LX.16 Our complaint, in short, is that Brandom
slides from a descriptive notion of VP-sufficiency that cannot be met by conditional
vocabulary to an expressive conception of VP-sufficiency that can be played by the
conditional but which is not fit for the purposes of Analytic Pragmatism. 13 Brandom (2008: 56).
14 Brandom’s notion of making explicit is a notoriously hard one to articulate precisely. The present way of
formulating the idea might sound as if making a practice explicit consists in producing a name for that practice.
For reasons that we need not go into here, however, it is quite clear that this is not Brandom’s intention.
Moreover, it is clearly false that logical expressions, such as negation, are names for practices.
15 Brandom (1994: 115). 15 Brandom (1994: 115). (
)
16 Brandom (2008: 44-47). 17 Brandom (2008: 42). 5.2 Inferring, Asserting and Inferring-and-Asserting Let us waive the previous objection for the sake of argument. Still, there is another –
and in our view more serious – problem for the claim that conditionals are VP- 1814 Philosophia (2017) 45:1803–1827 sufficient for the practice of inferring. Here the problem concerns how to understand
the bottom right-hand interior box in Fig. 2. According to Brandom’s characterization
of conditionals as universal LX, the practice of inferring is supposed to be PP-sufficient
for the practice of using conditionals. But this cannot be right. For, in order to have
mastery of conditionals, one must also be able to engage in the practice of asserting. This is a point that Brandom would himself readily endorse and for a general and rather
obvious reason: a language user who is incapable of assertion would be no language
user at all. But if this is so, then the practice of inferring is not PP-sufficient for the
practice of using conditionals, and so conditionals (on this construal of the diagram) are
not universal LX. Now, it should be clear what is going on here. Brandom is assuming that the
practices of asserting and inferring go together. As he puts it: Bthe beginning of
wisdom^ in understanding the nature of assertion Bis to see that asserting and inferring
are internally related practices, in the sense that each is PP-necessary for the other.^17
So, in order to accurately capture the meaning-use relations for conditionals, we need to
augment the lower right-hand interior box in the diagram in Fig. 2 by having the
conjunction of inferring and asserting as PP-sufficient for the practice of using condi-
tionals. But now the problem crops up elsewhere. Even if we grant that the conjoint
practice of inferring-and-asserting is PP-sufficient for the practice of using conditionals
and that this practice is, in turn, PV-sufficient for the mastery of the conditional, it is
simply false that the conditional locution is VP-sufficient for the practice of inferring-
and-asserting. Moreover, this is so irrespective of whether one adopts a descriptive or
an expressive conception of the VP-sufficiency relation. Now, it should be clear what is going on here. Brandom is assuming that the
practices of asserting and inferring go together. 5.2 Inferring, Asserting and Inferring-and-Asserting As he puts it: Bthe beginning of
wisdom^ in understanding the nature of assertion Bis to see that asserting and inferring
are internally related practices, in the sense that each is PP-necessary for the other.^17 Consider the options. On the descriptive reading, the ‘if__then__’ locution would
need to describe the practice of inferring-and-asserting, which it clearly does not: any
more than ‘not’ describes the practice of treating two claims as incompatible. On the
alternative expressive reading, the ‘if__then__’ locution would need to make explicit
the practice of inferring-and-asserting. But even if we grant that the conditional makes
the practice of inferring explicit, it surely does not make inferring-and-asserting
explicit. To see this, we need only reflect upon the vocabulary that would make
asserting explicit. According to one familiar proposal, for example, the Fregean
assertion stroke (├) can be used to express assertoric force. But on the face of it, the
conditional, by itself, does not obviate the need for the assertion sign, any more than,
say, disjunction does. When one asserts a conditional one does not thereby assert either
the antecedent or the consequent. Moreover, in expressing conditionals, one does not
thereby resolve the force of the utterance since conditionals can, like most other
expressions, figure in assertions, suppositions, queries, commands, and so on. Thus,
however we construe VP-sufficiency (whether descriptively or expressively) condi-
tionals are not VP-sufficient for the practice of inferring-and-asserting. In sum: The practice of inferring, by itself, is not PP-sufficient for the practice of
using conditionals. Perhaps the practice of inferring-and-asserting is PP-sufficient for
the practice of using conditionals. But even if this is true, conditionals are not VP-
sufficient for the practice of inferring-and-asserting. Thus, either Condition 2 fails or
Condition 4 fails. Either way, conditionals turn out not to be LX, much less universal
LX. Philosophia (2017) 45:1803–1827 1815 6 Modal Vocabulary is Not Universal LX (or Why the Modal Kant-Sellars
Thesis is False) 6 Modal Vocabulary is Not Universal LX (or Why the Modal Kant-Sellars
Thesis is False) In the preceding sections we argued at some length that Brandom’s primary example of
logical vocabulary – the conditional – is not universal LX. &
It is PV-necessary for using any empirical vocabulary; 5.2 Inferring, Asserting and Inferring-and-Asserting Since the claim that logical
vocabulary is universal LX is amongst the central claims of Between Saying and Doing,
and since the conditional is the only piece of logical vocabulary that he considers at any
length, we take this to pose a serious problem for his project. We now propose to
challenge another central thesis of the book – what Brandom calls the modal Kant-
Sellars thesis —roughly, the claim that modal vocabulary (e.g., ‘possible’, ‘necessary’,
and ‘contingent’) is universal LX. More specifically, we maintain that it is subject to
exactly analogous problems to those set out earlier. 18 Brandom (2008: 103).
19 Brandom illustrates the point with the following example: BOne grasps the claim ‘the lioness is hungry’
only insofar as one takes it to have various consequences (which would be true if it were true) and to rule out
some others (which would not be true if it were true). And it is not intelligible that one should endorse as
materially good an inference involving it, such as the inference from ‘the lioness is hungry’ to ‘nearby prey
animals visible to and accessible by the lioness are in danger of being eaten,’ but be disposed to make no
distinction at all between collateral premises that would, and those that would not, if true infirm the inference.^
(p. 105) 18 Brandom (2008: 103).
19 6.1 The Modal Kant-Sellars Thesis and Its Philosophical Significance As noted in Section 2.1, a central goal of Brandom’s project in Between Saying and
Doing is to argue for the thesis, which he calls the modal Kant-Sellars thesis, on which
modal vocabulary has a privileged status akin to logical vocabulary. Via an extensive discussion that focuses primarily on Sellars – but takes in Ryle,
Kripke, and Quine along the way – Brandom fashions a characterization of the
modal Kant-Sellars thesis that conforms to the pragmatic conception of meaning
so central to Brandom’s overarching project. In particular, the version of the
modal Kant-Sellars thesis that he endorses is formulated as the conjunction of
two claims: 1. In using ordinary empirical vocabulary, one already knows how to do everything
one needs to know how to do in order to introduce and deploy modal vocabulary. 1. In using ordinary empirical vocabulary, one already knows how to do everything
one needs to know how to do in order to introduce and deploy modal vocabulary. 2. The expressive role characteristic of alethic modal vocabulary is to make explicit
semantic, conceptual connections and commitments that are already implicit in the
use of ordinary empirical vocabulary. As Brandom observes, this formulation of the modal Kant-Sellars thesis is amena-
ble, with minimal modification, to the sort of meaning-use analysis that we have
already encountered. Claim 1 maintains that some set of practices that are PV-
necessary for the use of any empirical vocabulary are PP-sufficient for practices that
are PV-sufficient to deploy modal vocabulary. Claim 2 maintains that modal vocabulary
makes explicit – is VP-sufficient for – those aspects of practices that are implicit in –
i.e., PV-necessary for – the use of any empirical vocabulary whatsoever. Thus, accord-
ing to Brandom’s modal Kant-Sellars thesis, modal vocabulary is universal LX. This is
represented diagrammatically in Fig. 3 (‘CRI’ stands for ‘counterfactually robust
inference’, to be explained below). There is a crucial component of Brandom’s discussion of the modal Kant-Sellars
thesis that we have yet to touch upon. From inspection of Fig. 3, it is clear that the
thesis requires that there be some set of practices – corresponding to the bottom-right
interior box – that satisfies three conditions: Philosophia (2017) 45:1803–1827 1816 Fig. 3 A meaning-use diagram for modal vocabulary Fig. 6.2 Why Brandom’s Modal Kant-Sellars Thesis is False Although we do not deny the general thesis that modal notions are in some way implicit
in ordinary descriptive, linguistic practices, we do reject the claim that modal vocab-
ulary is universal LX, and with it Brandom’s specific formulation of the modal Kant-
Sellars thesis. Moreover, our line of criticism is exactly analogous to the objection we
leveled against the claim that the conditional is universal LX in Section 4.2. In order to have mastery of modal locutions, one must also be able to engage in the
practice of asserting —i.e. PModal in Fig. 3 must involve the ability to make assertions. This is a point that Brandom himself would readily endorse and for precisely the same
reason mentioned in the case of the conditional. But if this is so, then the practice of
counterfactually robust inferring (hereon CRI) is not PP-sufficient for the practice of
using modal notions, and so modal vocabulary (on this construal of the diagram) is not
universal LX. Now, as with the case of the conditional, one might respond to this problem by
maintaining that the capacity for asserting is PP-necessary for the capacity for CRI and
vice versa. In which case, to capture the meaning-use relations for modals, we need to
augment the lower interior right-hand box in the diagram by having the conjunction of
CRI and asserting as PP-sufficient for the practice of using modal locutions. But just as
before, the problem now crops up elsewhere. Even if we grant that the conjoint practice
of CRI-and-asserting is PP-sufficient for the practice of using conditionals, and that this
practice is, in turn, PV-sufficient for the mastery of modals, it is simply false that modal
locutions are VP-sufficient for the practice of CRI-and-asserting. Moreover, this is so
irrespective of whether one adopts a descriptive or an expressive conception of the VP-
sufficiency relation. Consider the options. On the descriptive reading, modal locutions would need to
describe the practice of CRI-and-asserting, which they clearly do not: any more than
‘not’ describes the practice of treating two claims as incompatible. On the alternative
expressive reading, modal locutions would need to make explicit the practice of CRI-
and-asserting. But even if we grant that modals make the practice of CRI explicit, they
surely do not make CRI-and-asserting explicit. To see this, we need only reflect again
upon the vocabulary that would make asserting explicit. 6.1 The Modal Kant-Sellars Thesis and Its Philosophical Significance 3 A meaning-use diagram for modal vocabulary &
It is PP-sufficient for those practices PV-sufficient for deploying modal vocabulary
and &
It is PP-sufficient for those practices PV-sufficient for deploying modal vocabulary;
and &
It is PP-sufficient for those practices PV-sufficient for deploying modal vocabulary;
and &
Modal vocabulary is VP-sufficient for these practices. &
Modal vocabulary is VP-sufficient for these practices. Yet as formulated no commitment is made regarding what these practices
are. This is a gap that Brandom fills in the course of arguing for the modal
Kant-Sellars thesis. In brief, his argumentative strategy is to justify the thesis by showing that
there is a practice that satisfies the above conditions. What might it be? His
answer is that it is the capacity for what he calls Bcounterfactually robust
inference^: the capacity to associate a range of Bcounterfactual robustness^
with each materially good inference. 18 In other words, he maintains that
mastery of any empirical vocabulary requires that one be able to distinguish
the counterfactual conditions in which an inference would fail from those in
which it would go through. For otherwise one would not count as possessing
the relevant empirical concepts.19 Philosophia (2017) 45:1803–1827 1817 6.2 Why Brandom’s Modal Kant-Sellars Thesis is False According to one familiar
proposal, for example, the Fregean assertion stroke can be used to express assertoric
force. But on the face of it, modal vocabulary, by itself does not obviate the need for the
assertion sign, any more than, say, the conditional does. Thus, whichever way we
construe VP-sufficiency – whether it be descriptively or expressively – modals are not
VP-sufficient for the practice of CRI-and-asserting. In sum: The practice of CRI, by itself, is not PP-sufficient for the practice of using
modals. Perhaps the practice of CRI-and-asserting is PP-sufficient for the practice of
using modals. But even if this is true, modals are not VP-sufficient for the practice of
CRI-and-asserting. Thus, either Condition 2 fails or Condition 4 fails. Either way
modals turn out not to be LX, much less universal LX. 6.3 The Normative Kant-Sellars Thesis We have argued that the modal Kant-Sellars thesis fails. But those familiar with
Between Saying and Doing might wonder whether any of this applies to what Brandom 1818 Philosophia (2017) 45:1803–1827 calls the normative Kant-Sellars thesis: the claim that in order to apply ordinary,
empirical, descriptive vocabulary – and hence to deploy any autonomous vocabulary
whatsoever – one must already be able to do everything needed to introduce normative
vocabulary.20 This thesis is defended in tandem with the modal version of the Kant-
Sellars thesis. Moreover, Brandom appears to suppose that the two theses are strongly
analogous, being answers to two variants of a single issue: Kant read Hume's theoretical and practical philosophies as raising variants of a
single question. On the side of theoretical reasoning, Hume asks what our warrant
is for moving from descriptions of what in fact happens to characteriza-
tions of what must happen, and what could not happen. How, he wants to
know, can we rationally justify the move from descriptions of matter-of-
factual regularities to formulations of necessary laws? On the side of
practical reasoning, Hume asks what our warrant is for moving from
descriptions of how things are to prescriptions of how they ought to be. How, he wants to know, can we rationally justify the move from ‘is’ to
‘ought’? … As we have seen, on the modal side, Kant's response is that Hume's predicament
is not a real one. One cannot in fact fully understand the descriptive, empirical
employment of ordinary determinate concepts such as cat without at least im-
plicitly understanding also what is made explicit by the modal concepts that
articulate laws. Kant mounts a corresponding line of thought on the side of
normative or practical necessity. Normative concepts make explicit commitments
that are implicit in any use of concepts, whether theoretically in judgment or
practically in acting intentionally.21 In view of this apparently tight connection between the two Kant-Sellars theses one
might wonder whether the normative version is subject to the same problems we level
against the modal version. We suspect that the normative Kant-Sellars thesis is unscathed by the sorts of
considerations raised above. In order to see this, consider the meaning-use diagram
that Brandom provides for the normative Kant-Sellars thesis (‘GAR’ stands for ‘the
game of giving and asking for reasons’) Fig. 4. 20 Anyone familiar with Brandom’s earlier work, Making It Explicit, should recognize that such a thesis about
normative vocabulary is central to Brandom’s philosophical position.
21 Brandom (2008: 109-110).
22 This is not, of course, to deny that some other objection might apply. But we put such issues to aside here. 6.3 The Normative Kant-Sellars Thesis In order for normative vocabulary to be universal LX, it must be VP-sufficient for
some practice that is, in turn, PP-sufficient for the practice of using normative
vocabulary. According to Brandom the practice in question is giving and asking
for reasons. But this practice is – and this is the crucial point – one that
incorporates the practice of assertion. Giving a reason just is a kind of
asserting. But if this is so, then we have no reason to doubt that it is PP-
sufficient for the practice of using normative vocabulary. Thus no analog of our
earlier objections can be run for the present case.22 1819 Philosophia (2017) 45:1803–1827 Fig. 4 A meaning-use diagram for normative vocabulary Fig. 4 A meaning-use diagram for normative vocabulary 7 Intentionality as a Pragmatically Mediated Relation The final central thesis of Brandom’s Between Saying and Doing concerns the nature of
intentionality – the world-directed character of language and thought – that we call the
pragmatically mediated conception of intentionality. In our view, this proposal is of
profound importance to the overarching project of Analytic Pragmatism. Yet
Brandom’s discussion is underdeveloped and, at times, frustratingly obscure. With this
in mind, we propose to start with a careful and charitable reconstruction of the proposal. Having done so, we show that the arguments of the foregoing sections provide reason
to reject the proposal. 23 See Rosen (2001) for example. As David Lewis once wrote in this spirit, Bsemantics with no treatment of
truth conditions is not semantics^ (Lewis 1983: 190).
24 Brandom labels this principle P3; Brandom (2008: 178). 7.1 Motivation: A Persistent Criticism of Semantic Pragmatism One way to appreciate the central motivation for – and significance of – Brandom’s
pragmatically mediated conception of intentionality is to start by focusing on a
persistent criticism of the semantic assumptions of Analytic Pragmatism. More or less
by definition, Analytic Pragmatism is committed to Semantic Pragmatism: the thesis
that linguistic meaning is determined by use. Moreover, the sorts of analyses that
Brandom seeks to provide consist in specifying the linguistic practices on which our
mastery of various vocabularies are thought to depend. But as Brandom himself readily
acknowledges, there is a worry that such analyses are, in fact, not semantic at all; and
not for any reason that has anything specific to do with the particular analyses that he
advocates. Rather, the worry is quite general. Such analyses fail to be semantic – and
hence to do the work required of them by Analytic Pragmatism – because they conform 1820 Philosophia (2017) 45:1803–1827 to the general precepts of Semantic Pragmatism, and Semantic Pragmatism is unsatis-
factory as an account of semantic properties. On such a view, meaning is ultimately to
be understood in terms of the linguistic practices in which we engage, not the relations
that terms bear to aspects of the world. But according to many, semantics, if it is
anything, is concerned with precisely such word-world relations – e.g., with represen-
tation, truth, and reference.23 In which case, it may seem that to the extent that Analytic
Pragmatism seeks to provide semantic analyses, it is doomed to fail. Thus whatever else
the value of pragmatic analysis, it cannot be a satisfactory extension of the project
initiated by Frege, Russell and Moore. 7.2 The Pragmatist Responds One obvious response to the above concern is simply to deny that semantics is centrally
concerned with word-world relations. Yet clearly this is not Brandom’s response. Rather, he thinks that there is something importantly right about the idea that semantic
properties involve word-world relations. The challenge, then, is to explain why this is
so in a manner consistent with Semantic Pragmatism. This is precisely the role that his
account of intentionality is supposed to fill. Step 1: The Nature of Practices Brandom’s response can be viewed as having two
main steps. The first is driven by what he takes to be a very general fact about the
nature of practices, which he views as a central claim of the pragmatist tradition. Of
course language use qua practice involves relations between subjects and the world
because in general practices involve aspects of the world: they are thick.24 To take some
simple examples, the practice of cooking cannot be adequately characterized absent
such things as food items and kitchen utensils; and the practice of drinking tea cannot
be adequately characterized without reference to such things as cups, saucers, and, of
course, tea leaves. But such components of the practices are also aspects of the world,
and thus the existence of these practices carry a commitment to subject-world relations. And what goes for cooking and tea drinking is also true of language use. Qua practice,
it too ineliminably involves subject-world relations. So there is a sense in which, according to Brandom, the worry with which we started
is no objection at all. That is, if the concern is that Semantic Pragmatism severs the
connection between word and world, then the concern is misplaced. At best it is an
artifact of assuming that the relevant practices ought to be characterized Bthinly^, in
such a way as to avoid reference to aspects of the world. But as Brandom clearly
recognizes, this does not settle the matter. The real problem for the semantic pragmatist
is not that there are no relations between language-using subjects and the
world. On the contrary, there are a great many such relations. Such relations
are cheap. The real problem for semantic pragmatists is specifying the right
relation(s) – those relevant for the purposes of characterizing the intentional
character of language use; and this, according to Brandom, requires a more
detailed, pragmatist account of intentionality. 7.2 The Pragmatist Responds 1821 Philosophia (2017) 45:1803–1827 Step 2: Combining the Accounts of Modal and Normative Vocabulary To appre-
ciate Brandom’s account of intentionality one needs first to make three general points
clear. The first, already hinted at above, is that he views intentionality of the sort
manifested by the practice of language use as a specific instance of a broader phenom-
enon, manifested by all human practices, which he calls practical intentionality. Practices quite generally, you may recall, involve relations between subjects and
aspects of the world. Further, as Brandom notes, practices invariably involve what he
calls practical intentionality: feedback-governed relations in which the results of earlier
actions are used as guides to future, goal-directed behavior. This is true, for example, of
cooking and tea drinking, but also football kicking and everything else for that matter,
including language use. According to Brandom, then, the semantic intentionality
characteristic of language use is best thought of as a specific instance of practical
intentionality.25 The second point concerns the order of explanation one ought to adopt in explaining
semantic intentionality. According to Brandom, some traditions26 purport to start with
independent conceptions of the states of the subject that represent and of the entities so
represented, and then seek to characterize the nature of the intentional relation that
holds between them —to somehow Bbolt^ them together.27 Brandom maintains that such an approach is wrong-headed in that it gets the proper
order of explanation exactly backwards, and thereby ignores precisely that which
allows for intentionality in the first place: Bthe thick, essentially world-involving
practices^ in which we engage.28 In its place, Brandom proposes that should start, as
traditional pragmatists have advocated, with the world-involving practices themselves;
and only characterize intentionality and its relata as a kind of abstraction from these
practices.29 Thus it is only by taking our rich, thickly characterized linguistic practices
as primary and subjecting them to careful enquiry that, according to Brandom, we have
any hope of understanding the general phenomenon of semantic intentionality.30 The final general point concerns the specific practices most central to explaining
intentionality. According to Brandom, the most central are those concerned with our
use of normative and modal vocabularies. Indeed his strategy is, in effect, to explain
intentionality by combining the two Kant-Sellars Theses discussed in Section 5, and
seeking to draw out certain consequences of their joint endorsement. 25 Brandom labels this claim P4; Brandom (2008: 179).
26 Though he does not explicitly mention any philosophical theories that cleave this methodology, we suspect
that he has in mind, amongst other things, causal theories of content, such as those advocated by Fodor and
Dretske; see Dretske 1981) and Fodor (1992).
27 Brandom (2008: 179).
28 Brandom (2008: 180).
29 Historical note: This sort of view was characteristic of the views of pragmatists, such as Dewey and James,
but similar views can also be found in Russell (1945: 812) among others.
30 Superficial appearances not withstanding, Brandom is not claiming that there could be no objective world
without the existence of our linguistic practices. His purpose is not to advocate some kind of linguistic
idealism, but instead to defend an approach to the study of intentionality. 26 Though he does not explicitly mention any philosophical theories that cleave this methodology, we suspe
that he has in mind, amongst other things, causal theories of content, such as those advocated by Fodor an
Dretske; see Dretske 1981) and Fodor (1992).
27
d 25 Brandom labels this claim P4; Brandom (2008: 179).
26 (
)
29 Historical note: This sort of view was characteristic of the views of pragmatists, such as Dewey and James,
but similar views can also be found in Russell (1945: 812) among others.
30 Superficial appearances not withstanding, Brandom is not claiming that there could be no objective world
without the existence of our linguistic practices. His purpose is not to advocate some kind of linguistic
idealism, but instead to defend an approach to the study of intentionality. 28 Brandom (2008: 180).
29 25 Brandom labels this claim P4; Brandom (2008: 179).
26 7.2 The Pragmatist Responds In Section 6.3 we
argue that this strategy does not work. But first some further unpacking is in order. Recall: for Brandom, though an explanation of semantic intentionality requires an
account of the relevant subject-world relations, such an account is to be provided by
abstraction from the relevant linguistic practices. The primary task, then, is to supply an
adequate account of the relevant practices; and for Brandom the two central practices 1822 Philosophia (2017) 45:1803–1827 are those concerning modal vocabulary and normative vocabulary, since they inform us
about the relata of semantic intentional relations: the world and the rational subject,
respectively. The practices made explicit by modal vocabulary (CRI) impose con-
straints on the modal structure of reality. Roughly, they reflect constraints, implicit in
our thought and language, on ways that the world could be and must be. In contrast, the
practices made explicit by normative vocabulary (GAR) impose constraints on rational
agency. Roughly, they tell us what is it to be a rational subject – a producer and
consumer of reasons. Thus for Brandom these linguistic practices, though not them-
selves the poles of intentional relations, are crucial to explicating the nature of
intentionality, because it is by providing appropriately perspicuous characterizations
of them that we are able, by abstraction, to reconstruct the nature of the relata. So far so good. But an account of intentionality must do more than merely explicate
those practices that concern the relata. Rather, one needs to explain how each of these
practices are related to each other so as to yield the larger practice on which semantic
intentionality depends. In effect, Brandom must address an analog of the problem that
confronts more traditional approaches to intentionality of the sort that he rejects. For as
we have already noted, on some approaches to the explanation of intentionality, the
challenge is to specify the relevant relation between the relata: i.e., subject and world. For Brandom, the challenge is to specify the relationship between two practices: the
world-oriented practices made explicit by modal vocabulary and the subject-oriented
practices made explicit by normative vocabulary. How is this to be done? Brandom’s answer is that the two practices intersect in what
he calls rational rectification, which is the practice by which new commitments are
integrated into one’s cognitive economy – one’s system of beliefs, desires, goals, and
other intentional states. 7.2 The Pragmatist Responds Such a practice involves the modification of one’s
beliefs – often in the light of new information – so as to eliminate inconsis-
tencies among one’s current commitments. But the capacity to do so presup-
poses a capacity to recognize inconsistencies, which in turn presupposes a
capacity to engage in inferences that draw out the consequences of one’s
current beliefs. According to Brandom, all of these cognitive capacities are
aspects of the practice made explicit by use of modal vocabulary; and thus are
implicit in the practice of rational rectification. But there is more to rational rectification than this. Rational rectification requires
more of us than the mere capacity to recognize inconsistency – and whatever inferential
capacities this, in turn, presupposes. Among other things, this alone would be compat-
ible with continuing to endorse inconsistent commitments. In addition to this, rational
rectification requires that one take oneself to have an obligation to eliminate such
inconsistencies – to structure one’s commitments in such a way as not to violate facts
about the modal structure of the world. And according to Brandom, the practice of
having one’s commitments conform to such constraints are amongst those made
explicit by our use of normative vocabulary. It is time to bring all the pieces together. What we have said so far on the topic
intentionality is quite impressionistic; and indeed most of Brandom’s own discussion is
similarly gestural. Nevertheless, in accord with his practice elsewhere in the book,
when Brandom seeks to spell out his views precisely, he presents a meaning-use
diagram. In the case of intentionality, the diagram that he uses to distill his views is
reproduced as Fig. 5. Philosophia (2017) 45:1803–1827 1823 Fig. 5 This figure has three arrows numbered for ease of reference below. See Fig. 6.1 on Brandom (2008:
183) Fig. 5 This figure has three arrows numbered for ease of reference below. See Fig. 6.1 on Brandom (200 Fig. 5 This figure has three arrows numbered for ease of reference below. See Fig. 6.1 on Brandom (2008:
183) Although there is a lot going on here, we restrict ourselves to the following
observations. First, the diagram aims to depict a kind of practice – indeed any practice
of using an autonomous vocabulary – as an object for which intentional vocabulary is
VP-sufficient – i.e., either made explicit by or describable by intentional vocabulary. 31 Brandom uses the term ‘discursive’ as a general term for any activity in which only rational entities can
engage. 7.3 An Objection In view of our earlier discussion, one problem with Brandom’s proposed account of
intentionality should be clear. As we have already seen, modal vocabulary is
not universal LX. So, there might be a language user that cannot already do
everything needed to be able to use modal vocabulary; and if that is right, then
the account of semantic intentionality, as represented in Fig. 5, obviously cannot
be right. How deep does this objection go? To see the significance of the objection, we need
to rehearse some aspects of our earlier discussion, and introduce some features of
Brandom’s theory of content. First, recall that we showed earlier that the practice of
counterfactually robust inference is not PP-sufficient for the practice of using modal
vocabulary. But this is just to deny that arrow 1 in Fig. 5 holds. In other words, the
practice labeled Pobj-modal in Fig. 5 is not PP-sufficient for the practice of using modal
vocabulary; and this means that Brandom has failed to characterize adequately the
nature of the practice that, on his view, mediates the intentionality relation. In particular,
if our arguments from Section 5 are correct, then the aspect of the practice that is
supposed to explicate the Bworld pole^ of the intentionality relation has not have been
accurately characterized. Of course, this is not the end of the matter. Following the dialectic set out in
Section 5, Brandom might seek to preserve the connection between Pobj-modal and the
practices sufficient for the use of modal vocabulary by enriching Pobj-modal. Specifically,
he might add the assumption that Pobj-modal is constituted, in part, by the ability to make
assertions. If such a maneuver is pursued, then our earlier objection to arrow 1 fails. But
now – just as before – the problem crops up elsewhere. Though arrow 2 holds more or
less by definition, arrow 3 fails. That is, as argued earlier, we no longer have any reason
to suppose that modal vocabulary is VP-sufficient for Pobj-modal. In which case, modal
vocabulary still fails to be universal LX, and the modal Kant-Sellars Thesis is false. As
a consequence, even if one pursues this escape route, Brandom’s pragmatic theory of
intentionality, at least as represented by Fig. 5, is false. 7.2 The Pragmatist Responds Second, the diagram represents any autonomous discursive practice as incorporating
two essential components: a practice made explicit by modal vocabulary and a practice
made explicit by normative vocabulary.31 Thus the diagram represents mastery of such
practices as necessary for being a language-user at all. Finally, the diagram represents anyone who can engage in these two practices as
being capable, in principle, of deploying the vocabularies that make these practices
explicit – i.e., modal and normative vocabulary. In other words, the diagram represents
the fact that Brandom’s account of intentionality – in conjunction with the earlier
discussed claims regarding modal and normative vocabulary – entails the modal and
normative Kant-Sellars theses. This is important for present purposes, because by tying
his account of intentionality to his previous claims about modality and normativity,
Brandom provides a partial explication of the nature of the practices on which
intentionality depends. As we noted earlier, the real challenge for a pragmatic account
of intentionality of the sort advocated by Brandom is to explicate the word-world
relations that is relevant to intentionality; and with respect to this task, the primary
significance of the Kant-Sellars theses is that they permit Brandom is specify central 1824 Philosophia (2017) 45:1803–1827 aspects of the relevant practices. Brandom is thus relying on his earlier discussion of
modality and normativity in order to flesh out his account of intentionality. aspects of the relevant practices. Brandom is thus relying on his earlier discussion of
modality and normativity in order to flesh out his account of intentionality. 7.3 An Objection One might, however, accept our criticism and yet insist that the objection is easily
met so as to preserve the spirit, though not the letter, of Brandom’s account of
intentionality. Specifically, it seems eminently reasonable to ask why the account of
intentionality as such should require that modal vocabulary be VP-sufficient for Pobj-
modal. Clearly, on anything like Brandom’s view, Pobj-modal should be PP-sufficient for
Pmodal. Absent this connection Brandom would, by his own lights, have failed to
explicate the Bworld pole^ of the intentionality relation. Further, on anything like
Brandom’s account, a relation of PV-sufficiency must hold between Pmodal and Vmodal. After all, on his view, the existence of such a connection obtains more or less by
definition. In contrast, it is utterly unclear why the account of intentionality requires that
Vmodal be VP-sufficient for Pobj-modal and, hence, that modal vocabulary be universal LX. To the extent that we concerned only with the account of intentionality, then, such a
commitment seems entirely optional. 1825 Philosophia (2017) 45:1803–1827 There is a sense in which we think that this response is entirely correct. Brandom’s
approach to intentionality, need not – and indeed should not – demand that Vmodal be
VP-sufficient for Pobj-modal. But we think that the assumption that it should is deeply
engrained in Brandom’s views about semantics and, hence, that preserving the
Brandonian approach to intentionality requires that one give up a deep-seated commit-
ment of Brandom’s philosophical worldview, sometimes called expressive equilibrium. In our experience, the issue is not easily appreciated; and so we propose to creep up on
the relevant assumption by first considering another assumption – expressive complete-
ness – also deep-seated in Brandom’s philosophy, that does not suffice to mandate the
commitment to arrow 3, but that does motivate the endorsement of expressive
equilibrium Expressive Completeness Brandom is, of course, a semantic pragmatist, and so his
theory of content takes the form of a theory of those discursive practices that are
adequate to confer semantic content on the expressions involved. Expressive complete-
ness is invoked as a condition of adequacy on such a theory. To a first approximation,
expressive completeness requires that a theory of discursive practices should, in
principle, be available to its practitioners. 32 Wittgenstein (1922). 7.3 An Objection To see the significance of this notion for Brandom’s semantic project we need to
rehearse briefly the overall structure of the approach to semantic properties that
Brandom adopts. Roughly put, he advocates a two-part approach: 1)
The basic theory: This describes what might be called basic discursive practices –
i.e. those involving no logical vocabulary. This theory will, of course, use logical
vocabulary, but it will not explain how such expressions get their meanings. 2)
The extended theory: This describes the extended discursive practices in which
language-users engage – i.e., those involving logical vocabulary. More specifically,
it does so by showing how basic discursive practices can be extended by the
introduction of logical vocabulary. 2)
The extended theory: This describes the extended discursive practices in which
language-users engage – i.e., those involving logical vocabulary. More specifically,
it does so by showing how basic discursive practices can be extended by the
introduction of logical vocabulary. Now as we have seen, the overall theory – comprised of both the basic and extended
theories – needs to be expressively complete. Failing that, one risks self-defeat prob-
lems of sort that stymied verificationism. Yet clearly, participants in the basic practice
lack the resources to state the basic theory because they posses no logical
vocabulary at all. In which case, the basic theory is self-defeating. That is
where the extended theory enters the picture. This theory explains how to
introduce logical vocabulary – including modal and normative vocabulary –
into the basic practice. So, participants in the extended practice – i.e., the
practice described by the extended theory – possess the vocabulary required
to state the basic theory. But now the problem recurs. In order to ensure
expressive completeness of the extended theory, such practitioners must not
merely have access to the basic theory, but to the extended theory as well. Otherwise, we (once more) risk regress. Brandom’s proposed solution is that logical vocabulary is in expressive equilibrium
—that it makes explicit not only those norms that confer content on non-logical
vocabulary, but those norms that confer content on logical vocabulary as well. And
in the parlance of Between Saying and Doing, this is just the claim that logical
vocabulary, broadly construed to include normative and modal vocabulary, makes
explicit the very practices that one needs to master in order to possess logical vocab-
ulary itself. 7.3 An Objection In other words, an adequate semantic theory
should explain how linguistic expressions acquire their content in such a way that
practitioners are, in principle, able to use the theory in order to explain the practices in
which they participate. A corollary of this – one that is central to appreciating the
significance of this constraint – is that such a theory will apply to itself. That is, an
expressively complete semantic theory will correctly explain how the very linguistic
expressions that comprise the theory acquire their meanings. Why endorse such a commitment? Though we will not explore the issue in detail
here, the central motivation is to avoid the sorts of self-refutation worries that notori-
ously confront various historically influential theories of meaning, most notably early
versions of verificationism, but also Wittgenstein’s Tractarian account of meaning.32
Clearly, an expressively complete theory will not confront this problem since such
theories are self-applicable – that is, they provide a meaning theory for the expressions
in the theory itself. Be that as it may, the assumption of expressive completeness alone fails to explain
Brandom’s commitment to the VP-sufficiency of Vmodal for Pobj-modal. This is because
the mere commitment to expressive completeness very obviously does not demand that
modal expressions make explicit the practices on which they depend. On the face of it,
all that is required is that the theory not be self-defeating – that the theory possess the
resources to explain how the expressions of the theory themselves acquire their
meanings. What does require VP-sufficiency, however, is the strategy that Brandom
adopts in order to ensure the expressive completeness of his preferred approach to
semantic theorizing – what he calls expressive equilibrium. Expressive Equilibrium To a first approximation, the requirement for expressive
equilibrium is the requirement that the broad category of logical vocabulary – which,
for Brandom, includes traditional logical connectives and quantifiers, but also norma-
tive and modal notions – makes explicit not only those norms that confer content on 1826 Philosophia (2017) 45:1803–1827 non-logical vocabulary, but those norms that confer content on logical vocabulary as
well. To see the significance of this notion for Brandom’s semantic project we need to
rehearse briefly the overall structure of the approach to semantic properties that
Brandom adopts. Roughly put, he advocates a two-part approach: non-logical vocabulary, but those norms that confer content on logical vocabulary as
well. 7.3 An Objection In short: the notion of universal LX vocabulary is a way of making more
precise the demand for expressive equilibrium: a demand that Brandom thinks is central
to an account of meaning that avoids self-defeat. We are now in a position to summarize our major concerns about Brandom’s view of
intentionality. First, as noted earlier, our objections to the modal Kant-Sellars thesis in
Section 5 show that the view as stated is false. Second, there is no reason internal to the
theory of intentionality to assume that modal vocabulary is universal LX. At most the
theory of intentionality per se requires arrows 1 and 2 – not arrow 3 – because it is the
first two connections that are involved in specifying the discursive practices relevant to
explicating the Bworld pole^ of the intentionality relation. Third, arrow 3 is required if one seeks to endorse both the account of intentionality
and the strategy for ensuring expressive completeness. But this is just because it is
required by the theory of expressive completeness. In other words, arrow 3 comes not
from the theory of intentionality but from Brandom’s strategy for achieving expressive
completeness for his pragmatist theory of meaning. 1827 Philosophia (2017) 45:1803–1827 Finally, once one sees the problem, it should be clear that arrow 3 is demanded by
expressive equilibrium, irrespective of what account of intentionality Brandom en-
dorses. That is, once one adopts expressive equilibrium as a way of attaining expressive
completeness, it needs to be the case that modal vocabulary is VP-sufficient for Pobj-
modal; and more generally, that logical vocabulary, broadly construed, is VP-sufficient
for those practices needed to master exactly that logical vocabulary. What our argu-
ments in the earlier sections of this paper show is that this strategy cannot succeed. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International
License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and repro-
duction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a
link to the Creative Commons license, and indicate if changes were made. Rosen, G. (2001). Brandom on modality, normativity, and intentionality. Philosophy and Phenomenologica
Research, 63, 611–623. Brandom, R. (1994). Making it explicit. Cambridge: Harvard University Press. References Brandom, R. (1994). Making it explicit. Cambridge: Harvard University Press. Brandom, R. (2008). Between saying and Doing: Towards an analytic pragmatism. Oxford: Oxfo
University Press. Dretske, F. (1981). Knowledge and the flow of information. Cambridge: Cambridge University Press. Fodor, J. (1992). A theory of content and other essays. Cambridge: MIT Press. Lewis, D. (1983). General semantics. In Philosophical papers I. Oxford: Oxford University Press. Rosen, G. (2001). Brandom on modality, normativity, and intentionality. Philosophy and Phenomenological
Research, 63, 611–623. Russell, B. (1945). The history of western philosophy. London: George Allen & Unwin. Wittgenstein, L. (1922). Tractatus Logico-Philosophicus. London: Harcourt, Brace & co..
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The Novel Long Noncoding RNA linc00467 Promotes Cell Survival but Is Down-Regulated by N-Myc
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Abstract Competing Interests: The authors have declared that no competing interests exist. Funding: The authors were supported by National Health and Medical Research Council Australia and Cancer Council New South Wa
recipient of an ARC Future Fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or pre Competing Interests: The authors have declared that no competing interests exist. * E-mail: tliu@unsw.edu.au Bernard Atmadibrata1, Pei Y. Liu1, Nicolas Sokolowski1, Lihong Zhang1,2, Matthew Wong1,
Andrew E. Tee1, Glenn M. Marshall1,3, Tao Liu1,4* 1 Children’s Cancer Institute Australia for Medical Research, Randwick, Sydney, Australia, 2 Department of Anatomy, Histology and Embryology, School of Basic Medical
Sciences, Fudan University, Shanghai, China, 3 Kids Cancer Centre, Sydney Children’s Hospital, Randwick, Australia, 4 School of Women’s & Children’s Health, UNSW
Medicine, University of New South Wales, Randwick, Sydney, Australia Abstract The worst subtype of neuroblastoma is caused by MYCN oncogene amplification and N-Myc oncoprotein over-expression. Long noncoding RNAs (lncRNAs) are emerging as critical regulators of gene expression and tumourigenesis. While Myc
oncoproteins are well-known to exert tumourigenic effects by regulating the expression of protein-coding genes and
microRNAs, little is known about which lncRNAs are Myc targets and whether the Myc target lncRNAs play a role in Myc-
induced oncogenesis. Here we performed differential gene expression studies using lncRNA microarray in neuroblastoma
cells after transfection with control or N-Myc-specific small interfering RNA (siRNA), and identified N-Myc target lncRNAs
including the novel lncRNA linc00467, the expression and function of which were completely unknown. RT-PCR, chromatin
immunoprecipitation and luciferase assays showed that N-Myc suppressed linc00467 gene expression through direct
binding to the linc00467 gene promoter and reducing linc00467 promoter activity. While N-Myc suppressed the expression
of RD3, the protein-coding gene immediately down-stream of linc00467 gene, through direct binding to the RD3 gene
promoter and reducing RD3 promoter activity, linc00467 reduced RD3 mRNA expression. Moreover, Affymetrix microarray
analysis revealed that one of genes significantly up-regulated by linc00467 siRNA was the tumour suppressor gene DKK1. Importantly, knocking-down linc00467 expression with siRNA in neuroblastoma cells reduced the number of viable cells and
increased the percentage of apoptotic cells, and co-transfection with DKK1 siRNA blocked the effects. These findings
therefore demonstrate that N-Myc-mediated suppression of linc00467 gene transcription counterintuitively blocks N-Myc-
mediated reduction in RD3 mRNA expression, and reduces neuroblastoma cell survival by inducing DKK1 expression. Citation: Atmadibrata B, Liu PY, Sokolowski N, Zhang L, Wong M, et al. (2014) The Novel Long Noncoding RNA linc00467 Promotes Cell Survival but Is Down-
Regulated by N-Myc. PLoS ONE 9(2): e88112. doi:10.1371/journal.pone.0088112 Editor: Yunli Zhou, Harvard Medical School, United States of America Received June 11, 2013; Accepted January 6, 2014; Published February 19, 2014 Copyright: 2014 Atmadibrata et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors were supported by National Health and Medical Research Council Australia and Cancer Council New South Wales project grants. TL was a
recipient of an ARC Future Fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Citation: Atmadibrata B, Liu PY, Sokolowski N, Zhang L, Wong M, et al. (2014) The Novel Long Noncoding RNA linc00467 Promotes Cell Survival but Is Down-
Regulated by N-Myc. PLoS ONE 9(2): e88112. doi:10.1371/journal.pone.0088112 Introduction intronic and intergenic (lincRNA) [9,10]. lncRNAs are emerging
as important regulators of gene transcription, tumour initiation
and progression [9,10]. For example, lincRNA-p21 is directly
activated by p53 and functions as an inhibitor of the genes that
interfere with apoptosis [11], the lincRNA CTBP1-AS promotes
both hormone-dependent and castration-resistant prostate cancer
growth [12], and the lincRNA MALAT1 and HOTAIR play
critical roles in lung and breast cancer invasion and metastasis
[13,14]. Neuroblastoma is a solid extracranial paediatric cancer that
arises from neural crest cells, and accounts for 15% of cancer-
related death in children [1]. Amplification of MYCN oncogene
and consequent N-Myc oncoprotein over-expression occur in
approximately 40% of high risk neuroblastoma, and is clinically
associated with cancer metastasis, resistance to therapies and poor
patient outcome [1,2]. Myc oncoproteins, including N-Myc and c-Myc, exert biolog-
ical effects through modulating gene transcription. After Myc
oncoproteins dimerize with Max, the Myc-MAX complex binds to
Myc-responsive element E-boxes at target gene promoters, leading
to transcriptional activation [3,4]. On the other hand, Myc
oncoproteins repress gene transcription by forming transcriptional
repressor complexes with histone deacetylases at Sp1-binding sites
of target gene promoters [5,6,7,8]. Identifying N-Myc target genes
and understanding the function of the N-Myc target genes are
important in developing better anticancer therapies. Myc oncoproteins have been extensively shown to modulate the
expression of microRNAs, and targeting the microRNAs is a
promising approach for treating Myc-induced cancers (reviewed in
[15]). However, little is known about which lincRNAs are Myc
targets and whether the Myc target lincRNAs play a role in Myc-
induced cancer. Here, we screened for lincRNA targets of N-Myc
in neuroblastoma cells by noncoding RNA microarray, and
identified linc00467 as an N-Myc target. While linc00467 had not
been studied at all in the literature, we discovered that linc00467
suppressed the expression of its downstream protein-coding gene
RD3, and induced neuroblastoma cell survival by reducing the
expression of the tumour suppressor gene DKK1. Long noncoding RNAs (lncRNAs) are transcripts longer than
200 nucleotides without a functional open reading frame, and can
be divided into five different types: sense, antisense, bidirectional, February 2014 | Volume 9 | Issue 2 | e88112 1 February 2014 | Volume 9 | Issue 2 | e88112 PLOS ONE | www.plosone.org linc00467 Promotes Cell Survival genome project). Linc00467 reduces mRNA expression of the linc00467
neighbouring protein-coding gene RD3 g
g
g g
lincRNAs exert biological functions partly through in cis
regulation of mRNA expression of their neighbouring protein
coding genes through various mechanisms [12,21,22,23]. We
therefore examined whether linc00467 regulated the expression of
RD3, the gene immediately down-stream of linc00467. BE(2)-C
and Kelly cells were transfected with control siRNA, linc00467
siRNA-1 or linc00467 siRNA-2 for 48 hours, followed by RT-
PCR analysis of RD3 mRNA expression. As shown in Figure 2A,
transfection with linc00467 siRNA-1 or linc00467 siRNA-2
reduced linc00467 RNA expression in the neuroblastoma cells. Importantly, knocking-down linc00467 expression up-regulated
RD3 mRNA expression in both BE(2)-C and Kelly cells
(Figure 2B). The data indicate that linc00467 reduces mRNA
expression of its neighbouring protein-coding RD3. We
have
shown
previously
that
N-Myc
represses
gene
transcription by recruiting histone deacetylases to Sp1-binding
site-enriched regions of target gene promoters [5,6,7,8]. To
understand whether N-Myc could directly repress linc00467 gene
transcription, we firstly analysed transcription factor binding sites
at the linc00467 gene promoter with Gene-Regulation software
(http://www.gene-regulation.com/pub/programs/alibaba2/index. html). Results showed that Sp1-binding sites were enriched at
2176 bp to 214 bp upstream of linc00467 gene transcription start
site as well as +7 bp to +426 bp in intron 1 (Figure 1D). We then
examined a c-Myc chromatin immunoprecipitation-sequencing
(ChIP-Seq) dataset, which was generated by Dr. Michael Snyder’s
group at Yale University for the ENCODE/SYDH project (The
Encyclopedia of DNA Elements/Stanford/Yale/USC/Harvard N-Myc suppresses the expression of the long noncoding
RNA linc00467 by direct binding to its gene promoter N-Myc suppresses the expression of the long noncoding
RNA linc00467 by direct binding to its gene promoter Myc oncoproteins exert biological effects by modulating gene
transcription. However, it is unknown whether N-Myc modulates
the transcription of lncRNAs. We therefore performed differential
gene expression analysis using NCodeTM Human Non-coding
RNA Microarray in BE(2)-C neuroblastoma cells 30 hours after
transfection with control siRNA or N-Myc siRNA No. 1 (N-Myc
siRNA-1). As shown in Table 1, the microarray gene expression
study showed that 5 lncRNAs were down-regulated, and 1
lncRNA was up-regulated, by N-Myc siRNA-1 within 30 hours by
more than 2 fold. One of the lncRNAs most significantly up-
regulated by N-Myc siRNA-1 was linc00467, which was identified
by Human Genome Organisation Gene Nomenclature Commit-
tee (HGNC) according to published DNA and cDNA sequencing
data [16,17,18,19,20]. To validate the microarray data, we performed siRNA
transfections with control siRNA, N-Myc siRNA-1 or N-Myc
siRNA-2 for 48 hours in two MYCN oncogene amplified human
neuroblastoma cell lines, BE(2)-C and Kelly, followed by real-time
RT-PCR study of linc00467. As shown in Figure 1A, transfection
with N-Myc siRNA-1 or N-Myc siRNA-2 reduced the expression
of both N-Myc mRNA and protein in the two neuroblastoma cell
lines. Consistent with the microarray data, down-regulation of N-
Myc
expression
resulted
in
increased
linc00467
expression
(Figure 1B). We next performed RT-PCR study of N-Myc and
linc00467 in SHEP-21N neuroblastoma cells, which were stably
transfected
with
a
tetracycline
withdrawal-inducible
N-Myc
expression construct [6,7], 48 hours after incubation with or
without tetracycline. As shown in Figure 1C, withdrawal of
tetracycline induced N-Myc expression, and reduced linc00467
RNA expression. Introduction As shown in Figure 1E, the ChIP-seq data
showed that c-Myc oncoprotein bound to the linc00467 gene core
promoter region matching the Sp1-binding site-enriched region in
K562 leukemia cells. Consistently, our own ChIP assays showed
that an anti-N-Myc antibody efficiently immunoprecipitated the
region of the linc00467 gene core promoter enriched in the Sp1-
binding sites in BE(2)-C neuroblastoma cells (Figure 1F), in
addition to the gene core promoter of ODC1 (Figure S1), a well-
known Myc target gene. To further understand whether the
binding of N-Myc to the linc00467 gene promoter region repressed
linc00467 gene transcription, the Sp1-binding site-enriched region
of the linc00467 gene promoter was cloned into a pLightSwitch_-
Prom construct. Luciferase assays were performed in BE(2)-C cells
after transfection with control siRNA or N-Myc siRNA-1, followed
by transfection with a pLightSwitch_Prom construct expressing
empty vector or the linc00467 promoter region. Results showed
that knocking-down N-Myc expression significantly up-regulated
luciferase activity of the pLightSwitch_Prom construct expressing
the linc00467 promoter region (Figure 1G). Taken together, the
data suggest that N-Myc represses linc00467 gene transcription by
direct binding to the Sp1-binding site-enriched region of the
linc00467 gene promoter and reducing linc00467 promoter
activity. N-Myc represses RD3 gene expression by direct binding
to the RD3 gene promoter (G) BE(2)-C cells were transfected with control siRNA or N-Myc siRNA-1, followed by co-transfection with Cypridina TK control
construct plus empty vector or linc00467 gene promoter pLightSwitch_Prom construct. Luciferase activities were measured with a LightSwitch Dual
Assay System kit, and expressed as a percentage change relative to control siRNA transfected samples. Error bars represented standard error. *, ** and
*** indicated P,0.05, 0.01 and 0.001 respectively. doi:10.1371/journal.pone.0088112.g001
linc00467 Promotes Cell Survival Figure 1. N-Myc represses linc00467 gene expression by direct binding to the linc00467 gene promoter. (A–B). BE(2)-C and Kelly cells
were transfected with scrambled control (Cont) siRNA, N-Myc siRNA-1 or N-Myc siRNA-2 for 48 hours, followed by RNA and protein extraction, real-
time RT-PCR and immunoblot analyses of N-Myc mRNA, protein expression (A) or linc00467 RNA expression (B). (C) SHEP-21N cells were incubated
with or without tetracycline for 48 hours, followed by RNA extraction and RT-PCR analysis of N-Myc and linc00467 RNA expression. (D) Schematic
representation of the linc00467 gene promoter. TSS represented transcription start site, and | represented Sp1-binding sites. (E) ChIP-Seq data from
Dr. Michael Snyder’s group at Yale University for the ENCODE/SYDH project generated from K562 cells. (F) ChIP assays were performed with a control
or anti-N-Myc antibody (Ab) and primers targeting a negative control region or the linc00467 gene core promoter region enriched in Sp1-binding
sites in BE(2)-C cells. Fold enrichment was calculated by dividing PCR products from DNA samples immunoprecipitated with the anti-N-Myc Ab by
PCR products from DNA samples immunoprecipitated with the control Ab, relative to input. Fold enrichment at the negative control region was
artificially set as 1.0. (G) BE(2)-C cells were transfected with control siRNA or N-Myc siRNA-1, followed by co-transfection with Cypridina TK control
construct plus empty vector or linc00467 gene promoter pLightSwitch_Prom construct. Luciferase activities were measured with a LightSwitch Dual
Assay System kit, and expressed as a percentage change relative to control siRNA transfected samples. Error bars represented standard error. *, ** and
*** indicated P,0.05, 0.01 and 0.001 respectively. doi:10 1371/journal pone 0088112 g001 PCR analysis of RD3 mRNA expression was performed in BE(2)-
C and Kelly cells 48 hours after transfection with control siRNA,
N-Myc siRNA-1 or N-Myc siRNA-2. Surprisingly, opposite to our
expectation, knocking-down N-Myc expression with N-Myc siRNA-1 or N-Myc siRNA-2 up-regulated RD3 mRNA expres-
sion (Figure 3A). N-Myc represses RD3 gene expression by direct binding
to the RD3 gene promoter p53 has been shown to indirectly repress the expression of a
subset of genes by inducing the expression of lincRNA-p21 [11]. We therefore examined whether N-Myc increased RD3 mRNA
expression indirectly by suppressing linc00467 expression. RT- Table 1. Modulation of lncRNA expression by N-Myc siRNA-1 by more than 2 fold thirty hours after siRNA transfection, as
identified by lncRNA microarray. Probe name Target ID
Probe Sequence
Fold change
h13721
AL122062
TACTTCTAAAAAAAGTATTTTGTATCTACTTTTGTAACTTCGTCAGAATAAAATATATTG;
TTCGTCAGAATAAAATATATTG
0.33
h03377
AK002005
GTTATCCAGGAAACAATATATATACACTTGTGAACTGTTGTTTGTGATTTAAGCATATAT
0.36
h31926
uc002oww
TTGTAGATTGGTTGTGTTTACACAGTTGTATATATTGACACCCTTGAGTGTTATGACTTC
0.37
h33198
uc002vpg
AATATTCATTTCTGAAATACTTTAGTATGATAGATAAATTTGGTTAAGTTCTTGTTCATT
0.46
h17938
BC039246
ATGTACTAATAATTTTATCTGACTTCTGTTTATATCATTTGTACAATTACATGGTTGTAA
0.47
h26159
linc00467
GAAACAACCACATATGTCACCTTTCCAAGAGGGACTGAAACTGGGCTGACCCTTTTGATT
2.17
doi:10.1371/journal.pone.0088112.t001
PLOS ONE | www.plosone.org
2
February 2014 | Volume 9 | Issue 2 | e88112 Table 1. Modulation of lncRNA expression by N-Myc siRNA-1 by more than 2 fold thirty hours after siRNA transfection, as
identified by lncRNA microarray. Probe name Target ID
Probe Sequence
Fold change
h13721
AL122062
TACTTCTAAAAAAAGTATTTTGTATCTACTTTTGTAACTTCGTCAGAATAAAATATATTG;
TTCGTCAGAATAAAATATATTG
0.33
h03377
AK002005
GTTATCCAGGAAACAATATATATACACTTGTGAACTGTTGTTTGTGATTTAAGCATATAT
0.36
h31926
uc002oww
TTGTAGATTGGTTGTGTTTACACAGTTGTATATATTGACACCCTTGAGTGTTATGACTTC
0.37
h33198
uc002vpg
AATATTCATTTCTGAAATACTTTAGTATGATAGATAAATTTGGTTAAGTTCTTGTTCATT
0.46
h17938
BC039246
ATGTACTAATAATTTTATCTGACTTCTGTTTATATCATTTGTACAATTACATGGTTGTAA
0.47
h26159
linc00467
GAAACAACCACATATGTCACCTTTCCAAGAGGGACTGAAACTGGGCTGACCCTTTTGATT
2.17
doi:10.1371/journal.pone.0088112.t001 February 2014 | Volume 9 | Issue 2 | e88112 2 linc00467 Promotes Cell Survival PCR analysis of RD3 mRNA expression was performed in BE(2)-
siRNA-1 or N-Myc siRNA-2 up-regulated RD3 mRNA expres-
Figure 1. N-Myc represses linc00467 gene expression by direct binding to the linc00467 gene promoter. (A–B). BE(2)-C and Kelly cells
were transfected with scrambled control (Cont) siRNA, N-Myc siRNA-1 or N-Myc siRNA-2 for 48 hours, followed by RNA and protein extraction, real-
time RT-PCR and immunoblot analyses of N-Myc mRNA, protein expression (A) or linc00467 RNA expression (B). (C) SHEP-21N cells were incubated
with or without tetracycline for 48 hours, followed by RNA extraction and RT-PCR analysis of N-Myc and linc00467 RNA expression. (D) Schematic
representation of the linc00467 gene promoter. TSS represented transcription start site, and | represented Sp1-binding sites. (E) ChIP-Seq data from
Dr. Michael Snyder’s group at Yale University for the ENCODE/SYDH project generated from K562 cells. (F) ChIP assays were performed with a control
or anti-N-Myc antibody (Ab) and primers targeting a negative control region or the linc00467 gene core promoter region enriched in Sp1-binding
sites in BE(2)-C cells. Fold enrichment was calculated by dividing PCR products from DNA samples immunoprecipitated with the anti-N-Myc Ab by
PCR products from DNA samples immunoprecipitated with the control Ab, relative to input. Fold enrichment at the negative control region was
artificially set as 1.0. Reduction in DKK1 expression contributes to linc00467-
mediated neuroblastoma cell survival doi:10.1371/journal.pone.0088112.g002 doi:10.1371/journal.pone.0088112.g002 To understand the mechanism through which linc00467
promotes neuroblastoma cell survival, we performed differential
gene expression study of linc00467 target genes in BE(2)-C cells
48 hours after transfection with control siRNA or linc00467
siRNA-1. As shown in Table S1, one of the genes significantly up-
regulated by linc00467 siRNA-1 was DKK1, a Wnt antagonist
tumour suppressor gene known to induce cancer cell apoptosis
[24,25]. RT-PCR analysis confirmed that transfection with
linc00467 siRNA-1 or linc00467 siRNA-2 considerably up-
regulated the expression of DKK1 in BE(2)-C and Kelly cells
(Figure 5A). To understand whether N-Myc directly repressed RD3 gene
transcription, we firstly analysed transcription factor binding sites
at the RD3 gene promoter with Gene-Regulation software (http://
www.gene-regulation.com/pub/programs/alibaba2/index.html.)
Results showed that Sp1-binding sites were enriched at +475 bp to
+731 bp of RD3 gene intron 1, relative to intron 1 start site
(Figure 3C). We then examined a c-Myc ChIP-Seq dataset, which
was generated by Dr. Michael Snyder’s group at Yale University
for the ENCODE/SYDH project. As shown in Figure 3D, the
ChIP-seq data showed that the c-Myc oncoprotein bound to the
RD3 gene intron 1 region matching the Sp1-binding site-enriched
fragment in K562 leukemia cells. Consistently, our own ChIP
assays with primers targeting RD3 intron 1 showed that an anti-N-
Myc antibody efficiently immunoprecipitated the RD3 gene intron
1 region enriched in Sp1-binding sites in BE(2)-C neuroblastoma
cells (Figure 3E). To further understand whether the binding of N-
Myc to the RD3 intron 1 region repressed RD3 gene transcription,
the Sp1-binding site-enriched RD3 intron 1 region was cloned into
a pLightSwitch_Prom construct. Luciferase assays were performed
in BE(2)-C cells after transfection with control siRNA or N-Myc
siRNA-1, followed by transfection with a pLightSwitch_Prom
construct expressing empty vector or the RD3 intron 1 region. Results showed that knocking-down N-Myc expression signifi-
cantly up-regulated luciferase activity of the pLightSwitch_Prom
construct expressing the RD3 intron 1 region (Figure 3F). To
understand whether N-Myc and linc00467 co-operatively reduce
RD3 expression, we transfected BE(2)-C cells with control siRNA, To examine whether up-regulation of the tumour suppressor
gene DKK1 contributed to linc00467 siRNA-mediated apoptosis,
we transfected BE(2)-C cells with control siRNA, linc00467
siRNA-1, DKK1 siRNA, or combination of linc00467 siRNA-1
and DKK1 siRNA. RT-PCR analysis showed that DKK1 siRNA
reduced DKK1 gene expression, and blocked linc00467 siRNA-
mediated DKK1 gene up-regulation (Figure 5B). N-Myc represses RD3 gene expression by direct binding
to the RD3 gene promoter Consistent with this finding, withdrawal of
tetracycline from SHEP-21N neuroblastoma cells, which induced
N-Myc expression, reduced linc00467 RNA expression (Figure 3B). February 2014 | Volume 9 | Issue 2 | e88112 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 3 linc00467 Promotes Cell Survival Figure 2. linc00467 reduces mRNA expression of its neigh-
bouring protein-coding RD3. BE(2)-C and Kelly cells were transfect-
ed with scrambled control siRNA, linc00467 siRNA-1 or linc00467 siRNA-
2 for 48 hours, followed by RNA extraction and and real-time RT-PCR
analysis of the expression of linc00467 (A) or RD3 (B). Error bars
represented standard error. * indicated P,0. 05, and *** indicated
P,0.001. doi:10.1371/journal.pone.0088112.g002 N-Myc siRNA, linc00467 siRNA, or combination of N-Myc
siRNA and linc00467 siRNA. RT-PCR analysis showed that N-
Myc siRNA and linc00467 siRNA did not have co-operative effect
in modulating RD3 expression (Figure S2). Taken together, the
data suggest that N-Myc represses RD3 gene transcription by
direct binding to the Sp1-binding site-enriched region of the RD3
gene promoter and reducing RD3 promoter activity. N-Myc siRNA, linc00467 siRNA, or combination of N-Myc
siRNA and linc00467 siRNA. RT-PCR analysis showed that N-
Myc siRNA and linc00467 siRNA did not have co-operative effect
in modulating RD3 expression (Figure S2). Taken together, the
data suggest that N-Myc represses RD3 gene transcription by
direct binding to the Sp1-binding site-enriched region of the RD3
gene promoter and reducing RD3 promoter activity. Knocking-down linc00467 expression reduces
neuroblastoma cell survival To understand whether repression of linc00467 expression by
N-Myc contributed to an N-Myc-induced cancer phenotype, we
transfected BE(2)-C and Kelly cells with control siRNA or
linc00467 siRNA for 48 hours, followed by Alamar blue assays. As shown in Figure 4A, knocking-down linc00467 expression with
siRNA reduced the number of viable BE(2)-C and Kelly cells. Alamar blue assays in BE(2)-C and Kelly cells 0, 24, 72 and
96 hours after transfection with control siRNA or linc00467
siRNA showed that linc00467 siRNA considerably reduced the
number of viable cells 72 and 96 hours after siRNA transfection
(Figure 4B). To examine whether the effect was due to cell death,
we transfected BE(2)-C and Kelly cells with control siRNA or
linc00467 siRNA, followed by staining with propidium iodide (PI)
and cell cycle study with flow cytometry. We also transfected
BE(2)-C and Kelly cells with control siRNA or linc00467 siRNA,
followed by staining with the apoptosis marker fluorescein
isothiocyanate (FITC)-conjugated Annexin V and analyses with
flow cytometry. Data analyses showed that knocking-down
linc00467 expression with siRNA increased the proportion of
cells at sub-G1 phase of the cell cycle (Figure 4C) and the
proportion of apoptotic cells (Figure 4D). Taken together, the data
suggest that linc00467 promotes neuroblastoma cell survival. Figure 2. linc00467 reduces mRNA expression of its neigh-
bouring protein-coding RD3. BE(2)-C and Kelly cells were transfect-
ed with scrambled control siRNA, linc00467 siRNA-1 or linc00467 siRNA-
2 for 48 hours, followed by RNA extraction and and real-time RT-PCR
analysis of the expression of linc00467 (A) or RD3 (B). Error bars
represented standard error. * indicated P,0. 05, and *** indicated
P,0.001. doi:10 1371/journal pone 0088112 g002 Reduction in DKK1 expression contributes to linc00467-
mediated neuroblastoma cell survival Luciferase activities
were measured with a LightSwitch Dual Assay System kit, and
expressed as a percentage change relative to control siRNA transfected
samples. Error bars represented standard error. * indicated P,0.05. Figure 4. linc00467 enhances neuroblastoma cell survival. (A)
BE(2)-C and Kelly cells were transfected with scrambled control siRNA or
linc00467 siRNA-1 for 48 hours, followed by Alamar blue assays. The
effect of linc00467 siRNA-1 was expressed as a percentage change in
the number of viable cells after transfection with linc00467 siRNA-1,
compared with control siRNA-transfected samples. (B) BE(2)-C and Kelly
cells were transfected with scrambled control siRNA or linc00467 siRNA-
1 for 0, 72 or 96 hours, followed by Alamar blue assays. The effects of
time and siRNAs were expressed as percentages of the number of
viable cells after transfection with control siRNA for 0 hour. (C) After
transfection with control siRNA or linc00467 siRNA-1 for 72 hours, BE(2)-
C and Kelly cells were stained with propodium iodide, and subjected to
flow cytometry analyses of the cell cycle. The percentage of cells at sub-
G1 phase was calculated. (D) After transfection with control siRNA or
linc00467 siRNA-1 for 72 hours, BE(2)-C and Kelly cells were stained with
FITC-conjugated Annexin V, and subjected to flow cytometry analyses. The percentage of cells positively stained by Annexin V was calculated. Error bars represented standard error. * indicated p,0.05, and **
p,0.01. Figure 3. N-Myc represses RD3 gene transcription by direct
binding to the RD3 gene promoter. (A) BE(2)-C and Kelly cells were
transfected with scrambled control (Cont) siRNA, N-Myc siRNA-1 or N-
Myc siRNA-2 for 48 hours, followed by RNA extraction and real-time RT-
PCR analyses of RD3 mRNA expression. (B) SHEP-21N cells were
incubated with or without tetracycline for 48 hours, followed by RNA
extraction and RT-PCR analysis of RD3 RNA expression. (C) Schematic
representation of the RD3 gene promoter. TSS represented transcription
start site, and | represented Sp1-binding sites. (D) ChIP-Seq data from
Dr. Michael Snyder’s group at Yale University for the ENCODE/SYDH
project generated from K562 cells. (E) ChIP assays were performed with
a control or anti-N-Myc antibody (Ab) and primers targeting a negative
control region or the RD3 gene core promoter region enriched in Sp1-
binding sites in BE(2)-C cells. Reduction in DKK1 expression contributes to linc00467-
mediated neuroblastoma cell survival The
effect of linc00467 siRNA-1 was expressed as a percentage change in
the number of viable cells after transfection with linc00467 siRNA-1,
compared with control siRNA-transfected samples. (B) BE(2)-C and Kelly
cells were transfected with scrambled control siRNA or linc00467 siRNA-
1 for 0, 72 or 96 hours, followed by Alamar blue assays. The effects of
time and siRNAs were expressed as percentages of the number of
viable cells after transfection with control siRNA for 0 hour. (C) After
transfection with control siRNA or linc00467 siRNA-1 for 72 hours, BE(2)-
C and Kelly cells were stained with propodium iodide, and subjected to
flow cytometry analyses of the cell cycle. The percentage of cells at sub-
G1 phase was calculated. (D) After transfection with control siRNA or
linc00467 siRNA-1 for 72 hours, BE(2)-C and Kelly cells were stained with
FITC-conjugated Annexin V, and subjected to flow cytometry analyses. The percentage of cells positively stained by Annexin V was calculated. Error bars represented standard error. * indicated p,0.05, and **
p,0.01. doi:10.1371/journal.pone.0088112.g004 Figure 3. N-Myc represses RD3 gene transcription by direct
binding to the RD3 gene promoter. (A) BE(2)-C and Kelly cells were
transfected with scrambled control (Cont) siRNA, N-Myc siRNA-1 or N-
Myc siRNA-2 for 48 hours, followed by RNA extraction and real-time RT-
PCR analyses of RD3 mRNA expression. (B) SHEP-21N cells were
incubated with or without tetracycline for 48 hours, followed by RNA
extraction and RT-PCR analysis of RD3 RNA expression. (C) Schematic
representation of the RD3 gene promoter. TSS represented transcription
start site, and | represented Sp1-binding sites. (D) ChIP-Seq data from
Dr. Michael Snyder’s group at Yale University for the ENCODE/SYDH
project generated from K562 cells. (E) ChIP assays were performed with
a control or anti-N-Myc antibody (Ab) and primers targeting a negative
control region or the RD3 gene core promoter region enriched in Sp1-
binding sites in BE(2)-C cells. Fold enrichment was calculated by
dividing PCR products from DNA samples immunoprecipitated with the
anti-N-Myc Ab by PCR products from DNA samples immunoprecipitated
with the control Ab, relative to input. Fold enrichment at the negative
control region was artificially set as 1.0. (F) BE(2)-C cells were
transfected with control siRNA or N-Myc siRNA-1, followed by co-
transfection with Cypridina TK control construct plus empty vector or
RD3 gene promoter pLightSwitch_Prom construct. Reduction in DKK1 expression contributes to linc00467-
mediated neuroblastoma cell survival Alamar blue
assays (Figure 5C) and flow cytometry analyses of Annexin V
positively stained cells (Figure 5D) showed that linc00467 siRNA-1
reduced the number of viable cells and increased the proportion of
cells positively stained by Annexin V, and that DKK1 siRNA
blocked linc00467 siRNA-1-mediated reduction in the number of
viable neuroblastoma cells and induction of Annexin V positively
stained cells. Taken together, the data suggest that reduction in
DKK1 expression contributes to linc00467-mediated neuroblas-
toma cell survival. February 2014 | Volume 9 | Issue 2 | e88112 PLOS ONE | www.plosone.org 4 linc00467 Promotes Cell Survival Figure 3. N-Myc represses RD3 gene transcription by direct
binding to the RD3 gene promoter. (A) BE(2)-C and Kelly cells were
transfected with scrambled control (Cont) siRNA, N-Myc siRNA-1 or N-
Myc siRNA-2 for 48 hours, followed by RNA extraction and real-time RT-
PCR analyses of RD3 mRNA expression. (B) SHEP-21N cells were
incubated with or without tetracycline for 48 hours, followed by RNA
extraction and RT-PCR analysis of RD3 RNA expression. (C) Schematic
representation of the RD3 gene promoter. TSS represented transcription
start site, and | represented Sp1-binding sites. (D) ChIP-Seq data from
Dr. Michael Snyder’s group at Yale University for the ENCODE/SYDH
project generated from K562 cells. (E) ChIP assays were performed with
a control or anti-N-Myc antibody (Ab) and primers targeting a negative
control region or the RD3 gene core promoter region enriched in Sp1-
binding sites in BE(2)-C cells. Fold enrichment was calculated by
dividing PCR products from DNA samples immunoprecipitated with the
anti-N-Myc Ab by PCR products from DNA samples immunoprecipitated
with the control Ab, relative to input. Fold enrichment at the negative
control region was artificially set as 1.0. (F) BE(2)-C cells were
transfected with control siRNA or N-Myc siRNA-1, followed by co-
transfection with Cypridina TK control construct plus empty vector or
RD3 gene promoter pLightSwitch_Prom construct. Luciferase activities
were measured with a LightSwitch Dual Assay System kit, and
expressed as a percentage change relative to control siRNA transfected
samples. Error bars represented standard error. * indicated P,0.05. doi:10 1371/journal pone 0088112 g003
Figure 4. linc00467 enhances neuroblastoma cell survival. (A)
BE(2)-C and Kelly cells were transfected with scrambled control siRNA or
linc00467 siRNA-1 for 48 hours, followed by Alamar blue assays. Discussion lncRNAs are emerging as critical regulators of gene transcrip-
tion, tumour initiation, progression and metastasis [9,10,26]. Myc
oncoproteins, including N-Myc and c-Myc, are well-known to
exert biological effects by modulating the expression of protein-
coding genes and microRNAs [3,4,15]. However, little is known
about whether Myc modulates the expression of lncRNAs, and
whether regulation of lncRNA expression by Myc plays a role in
Myc oncogenesis. In this study, we have performed genome-wide
differential expression study with lncRNA microarray in neuro-
blastoma
cells
30 hours
after
knocking-down
N-Myc
gene
expression. Data analysis reveals that knocking-down N-Myc gene
expression for 30 hours alters the expression of 6 lncRNAs by
more than 2 fold. One of the lncRNAs most significantly up-
regulated by N-Myc siRNA is linc00467. lncRNAs exert biological effects through in cis and in trans
regulation of RNA expression at both transcriptional and post-
transcriptional levels. For examples, a number of lncRNAs have
been shown to up- or down-regulate the expression of their
neighboring protein-coding genes through modulating chromatin
structure and gene transcription [14,22,27,28]. The lncRNAs
DLEU1 and DLEU2 at 13q14.3 are often deleted in multiple
types of cancers, and DLEU1 and DLEU2 modulate nuclear
factor B function by down-regulating the transcription of their
neighboring protein-coding KPNA3 and the microRNAs miR-15
and miR-16 [23]. Additionally, the lncRNA MALAT1 controls
cell cycle progression by regulating the expression of the oncogenic
transcription factor B-MYB through altering the binding of
splicing factors on B-MYB pre-mRNA and causing aberrant
alternative splicing [29], and the PTEN pseudogene expressed
noncoding RNA antisense RNA (PTENpg1 asRNA) regulates
both PTEN gene transcription and PTEN mRNA stability [30]. In
this study, we have confirmed that knocking-down linc00467 up-
regulates the expression of its neighbouring protein-coding gene
RD3, which encodes a retinal protein responsible for the retinal
degeneration disorder Leber congenital amaurosis type 12 [31,32]. linc00467 was identified by Human Genome Organisation
Gene Nomenclature Committee (HGNC) based on published
DNA and cDNA sequencing data [16,17,18,19,20]. Till today, the
biological function of linc00467 is completely unknown. We have
found that the linc00467 gene core promoter is enriched in Sp1-
binding sites, and that c-Myc binds to the Sp1-binding site-
enriched region of the lin00467 gene core promoter in K562
leukemia cells according to a publically available ChIP-Seq
dataset. Reduction in DKK1 expression contributes to linc00467-
mediated neuroblastoma cell survival Fold enrichment was calculated by
dividing PCR products from DNA samples immunoprecipitated with the
anti-N-Myc Ab by PCR products from DNA samples immunoprecipitated
with the control Ab, relative to input. Fold enrichment at the negative
control region was artificially set as 1.0. (F) BE(2)-C cells were
transfected with control siRNA or N-Myc siRNA-1, followed by co-
transfection with Cypridina TK control construct plus empty vector or
RD3 gene promoter pLightSwitch_Prom construct. Luciferase activities
were measured with a LightSwitch Dual Assay System kit, and
expressed as a percentage change relative to control siRNA transfected
samples. Error bars represented standard error. * indicated P,0.05. doi:10.1371/journal.pone.0088112.g003 Figure 3. N-Myc represses RD3 gene transcription by direct
binding to the RD3 gene promoter. (A) BE(2)-C and Kelly cells were Figure 4. linc00467 enhances neuroblastoma cell survival. (A)
BE(2)-C and Kelly cells were transfected with scrambled control siRNA or
linc00467 siRNA-1 for 48 hours, followed by Alamar blue assays. The
effect of linc00467 siRNA-1 was expressed as a percentage change in
the number of viable cells after transfection with linc00467 siRNA-1,
compared with control siRNA-transfected samples. (B) BE(2)-C and Kelly
cells were transfected with scrambled control siRNA or linc00467 siRNA-
1 for 0, 72 or 96 hours, followed by Alamar blue assays. The effects of
time and siRNAs were expressed as percentages of the number of
viable cells after transfection with control siRNA for 0 hour. (C) After
transfection with control siRNA or linc00467 siRNA-1 for 72 hours, BE(2)-
C and Kelly cells were stained with propodium iodide, and subjected to
flow cytometry analyses of the cell cycle. The percentage of cells at sub-
G1 phase was calculated. (D) After transfection with control siRNA or
linc00467 siRNA-1 for 72 hours, BE(2)-C and Kelly cells were stained with
FITC-conjugated Annexin V, and subjected to flow cytometry analyses. The percentage of cells positively stained by Annexin V was calculated. Error bars represented standard error. * indicated p,0.05, and **
p,0.01. doi:10.1371/journal.pone.0088112.g004 February 2014 | Volume 9 | Issue 2 | e88112 PLOS ONE | www.plosone.org 5 linc00467 Promotes Cell Survival Figure 5. Reduction in DKK1 expression contributes to linc00467-mediated neuroblastoma cell survival. (A) BE(2)-C and Kelly cells
were transfected with scrambled control siRNA, linc00467 siRNA-1 or linc00467 siRNA-2 for 48 hours, followed by RNA extraction and RT-PCR analysis
of DKK1 gene expression. Reduction in DKK1 expression contributes to linc00467-
mediated neuroblastoma cell survival In
this study, we have confirmed that knocking-down linc00467 up-
regulates the expression of its neighbouring protein-coding gene
RD3, which encodes a retinal protein responsible for the retinal
degeneration disorder Leber congenital amaurosis type 12 [31,32]. data demonstrate that linc00467 gene expression is reduced by N-
Myc and up-regulated by N-Myc siRNAs. As N-Myc is well-
known to repress gene transcription by direct binding to target
gene promoter regions enriched in Sp1-binding sites [5,6,7,8], our
data suggest that N-Myc represses linc00467 gene expression by
direct binding to the linc00467 gene promoter region enriched in
Sp1-binidng sites and suppresses linc00467 gene promoter activity. Reduction in DKK1 expression contributes to linc00467-
mediated neuroblastoma cell survival (B) BE(2)-C cells were transfected with scrambled control siRNA, linc00467 siRNA-1, DKK1 siRNA, or combination of
linc00467 siRNA-1 and DKK1 siRNA for 48 hours, followed by RNA extraction and RT-PCR analysis of DKK1 gene expression. (C) BE(2)-C cells were
transfected with scrambled control siRNA, linc00467 siRNA-1, DKK1 siRNA, or combination of linc00467 siRNA-1 and DKK1 siRNA for 72 hours,
followed by Alamar blue assays. The effect of linc00467 siRNA-1 alone, DKK1 siRNA alone, or combination of linc00467 siRNA-1 and DKK1 siRNA was
expressed as a percentage change, compared with control siRNA-transfected samples. (D) BE(2)-C cells were transfected with scrambled control
siRNA, linc00467 siRNA-1, DKK1 siRNA, or combination of linc00467 siRNA-1 and DKK1 siRNA for 72 hours, followed by staining with FITC-conjugated
Annexin V, and subjected to flow cytometry analyses. The percentage of cells positively stained by Annexin V was calculated. Error bars represented
standard error. *, ** and *** indicated p,0.05, 0.01 and 0.001 respectively. doi:10.1371/journal.pone.0088112.g005 Figure 5. Reduction in DKK1 expression contributes to linc00467-mediated neuroblastoma cell survival. (A) BE(2)-C and Kelly cells
were transfected with scrambled control siRNA, linc00467 siRNA-1 or linc00467 siRNA-2 for 48 hours, followed by RNA extraction and RT-PCR analysis
of DKK1 gene expression. (B) BE(2)-C cells were transfected with scrambled control siRNA, linc00467 siRNA-1, DKK1 siRNA, or combination of
linc00467 siRNA-1 and DKK1 siRNA for 48 hours, followed by RNA extraction and RT-PCR analysis of DKK1 gene expression. (C) BE(2)-C cells were
transfected with scrambled control siRNA, linc00467 siRNA-1, DKK1 siRNA, or combination of linc00467 siRNA-1 and DKK1 siRNA for 72 hours,
followed by Alamar blue assays. The effect of linc00467 siRNA-1 alone, DKK1 siRNA alone, or combination of linc00467 siRNA-1 and DKK1 siRNA was
expressed as a percentage change, compared with control siRNA-transfected samples. (D) BE(2)-C cells were transfected with scrambled control
siRNA, linc00467 siRNA-1, DKK1 siRNA, or combination of linc00467 siRNA-1 and DKK1 siRNA for 72 hours, followed by staining with FITC-conjugated
Annexin V, and subjected to flow cytometry analyses. The percentage of cells positively stained by Annexin V was calculated. Error bars represented
standard error. *, ** and *** indicated p,0.05, 0.01 and 0.001 respectively. doi:10.1371/journal.pone.0088112.g005 Figure 5. Reduction in DKK1 expression contributes to linc00467-mediated neuroblastoma cell surv Figure 5. Reduction in DKK1 expression contributes to linc00467-mediated neuroblastoma cell survival. Reduction in DKK1 expression contributes to linc00467-
mediated neuroblastoma cell survival (A) BE(2)-C and Kelly cells
were transfected with scrambled control siRNA, linc00467 siRNA-1 or linc00467 siRNA-2 for 48 hours, followed by RNA extraction and RT-PCR analysis
of DKK1 gene expression. (B) BE(2)-C cells were transfected with scrambled control siRNA, linc00467 siRNA-1, DKK1 siRNA, or combination of
linc00467 siRNA-1 and DKK1 siRNA for 48 hours, followed by RNA extraction and RT-PCR analysis of DKK1 gene expression. (C) BE(2)-C cells were
transfected with scrambled control siRNA, linc00467 siRNA-1, DKK1 siRNA, or combination of linc00467 siRNA-1 and DKK1 siRNA for 72 hours,
followed by Alamar blue assays. The effect of linc00467 siRNA-1 alone, DKK1 siRNA alone, or combination of linc00467 siRNA-1 and DKK1 siRNA was
expressed as a percentage change, compared with control siRNA-transfected samples. (D) BE(2)-C cells were transfected with scrambled control
siRNA, linc00467 siRNA-1, DKK1 siRNA, or combination of linc00467 siRNA-1 and DKK1 siRNA for 72 hours, followed by staining with FITC-conjugated
Annexin V, and subjected to flow cytometry analyses. The percentage of cells positively stained by Annexin V was calculated. Error bars represented
standard error. *, ** and *** indicated p,0.05, 0.01 and 0.001 respectively. doi:10.1371/journal.pone.0088112.g005 data demonstrate that linc00467 gene expression is reduced by N-
Myc and up-regulated by N-Myc siRNAs. As N-Myc is well-
known to repress gene transcription by direct binding to target
gene promoter regions enriched in Sp1-binding sites [5,6,7,8], our
data suggest that N-Myc represses linc00467 gene expression by
direct binding to the linc00467 gene promoter region enriched in
Sp1-binidng sites and suppresses linc00467 gene promoter activity. lncRNAs exert biological effects through in cis and in trans
regulation of RNA expression at both transcriptional and post-
transcriptional levels. For examples, a number of lncRNAs have
been shown to up- or down-regulate the expression of their
neighboring protein-coding genes through modulating chromatin
structure and gene transcription [14,22,27,28]. The lncRNAs
DLEU1 and DLEU2 at 13q14.3 are often deleted in multiple
types of cancers, and DLEU1 and DLEU2 modulate nuclear
factor B function by down-regulating the transcription of their
neighboring protein-coding KPNA3 and the microRNAs miR-15
and miR-16 [23]. Additionally, the lncRNA MALAT1 controls
cell cycle progression by regulating the expression of the oncogenic
transcription factor B-MYB through altering the binding of
splicing factors on B-MYB pre-mRNA and causing aberrant
alternative splicing [29], and the PTEN pseudogene expressed
noncoding RNA antisense RNA (PTENpg1 asRNA) regulates
both PTEN gene transcription and PTEN mRNA stability [30]. Real-time RT-PCR Following siRNA transfections, RNA was extracted from cells
using PureLink RNA Mini kit (Life Technologies) according to the
manufacturer’s instructions. RNA samples were then quantified
using Nanodrop spectrophotometer and treated with DNAse 1
(Life Technologies) to remove remaining genomic DNA. Synthesis
of cDNA from RNA samples was carried out using M-MLV
Reverse Transcriptase (Invitrogen). Real-time RT PCR was
performed in Applied Biosystems 7900 using SYBR green PCR
Master Mix (Life Technologies) as previously described [5,6,7,8]. Discussion Moreover, our own ChIP assays have confirmed that N-
Myc indeed binds to the Sp1-binding site-enriched region of the
lin00467 gene core promoter, luciferase assays show that N-Myc
siRNA enhances linc00467 gene promoter activity, and RT-PCR February 2014 | Volume 9 | Issue 2 | e88112 PLOS ONE | www.plosone.org 6 linc00467 Promotes Cell Survival Surprisingly, we have also confirmed that N-Myc suppresses RD3
gene expression through direct binding to the RD3 gene promoter
region enriched in Sp1-binding sites and reducing RD3 gene
promoter activity. These data indicate that linc00467 reduces
RD3 mRNA expression most likely through an in cis mechanism,
and that N-Myc-mediated suppression of linc00467 gene tran-
scription counterintuitively blocks N-Myc-mediated reduction in
RD3 mRNA expression. In addition, our differential gene
expression study with Affymetrix microarray has identified
DKK1 as one of the genes significantly up-regulated by linc00467
siRNA, suggesting that linc00467 is also likely to modulate gene
expression through in trans mechanisms. lncRNA microarray Neuroblastoma BE(2)-C cells were transfected with scrambled
control siRNA or N-Myc siRNA-1. Thirty hours after siRNA
transfection, RNA was extracted from the cells with RNeasy mini
kit and treated with DNAse 1. Differential gene expression was
examined with NCodeTM Human Non-coding RNA Microarray
(Invitrogene), according to the manufacturer’s instructions. Results
from the microarray hybridization were analysed with GeneSpring
software
(GeneSpring),
and
deposited
at
Gene
Expression
Omnibus website (accession number: GSE52984) While the biological function of linc00467 is completely
unknown in the literature, the Wnt antagonist DKK1 is well-
known to induce cancer cell apoptosis and function as a tumour
suppressor gene [24,25]. This study reveals that knocking-down
linc00467 gene expression reduces the number of viable neuro-
blastoma cells, increases the percentage of cells at sub-G1 phase of
the cell cycle and induces apoptosis in neuroblastoma cells. Importantly,
simultaneous
knocking-down DKK1
expression
blocks linc00467 siRNA-regulated neuroblastoma cell death. The data suggest that linc00467 may play a role in tumourigenesis
through reducing DKK1 expression, leading to enhanced tumour
cell viability, and that N-Myc-mediated suppression of linc00467
gene transcription counterintuitively blocks N-Myc-mediated cell
survival. Chromatin imunoprecipitation (ChIP) assays ChIP assays were performed with an anti-N-Myc antibody
(Merck Millipore) or a control antibody and PCR with primers
targeting negative control region or Sp1-binding site-enriched
region of the linc00467 or RD3 gene core promoter. Fold
enrichment of the linc00467 and RD3 gene core promoter region
by the anti-N-Myc antibody was calculated by dividing the PCR
product from the linc00467 or RD3 gene core promoter region by
the PCR product from the negative control region, relative to
input. siRNA transfection Cells were transfected with AllStars negative control siRNA, N-
Myc siRNA, linc00467 siRNA or DKK1 siRNA (Qiagen) using
Lipofectamine 2000 (Life Technologies) as previously described
[5,6,7,8]. Cell culture Neuroblastoma BE(2)-C cells were cultured in Dulbecco’s
modified Eagle’s medium supplemented with 10% fetal calf serum
[5,6,7,8]. Kelly cells were grown in RPMI 1640 supplemented
with 10% fetal calf serum and 1% L-glutamine. Immunoblot For the analysis of protein expression by immunoblot, cells were
lysed, protein extracted and separated by gel electrophoresis. After
western transfer, membranes were probed with mouse anti-N-Myc
antibody (1:1000) (Santa Cruz Biotech), followed by horseradish
peroxidase-conjugated anti-mouse (1:10000) antiserum (Santa
Cruz Biotech). Protein bands were visualized with SuperSignal
(Pierce). The membranes were lastly re-probed with an anti-actin
antibody (Sigma) as loading controls. Luciferase assays Sp1-binding site enriched linc00467 gene promoter region
(2248 bp upstream of linc00467 gene transcription start site to
+567 bp of intron 1) was custom-cloned into the pLightSwitch_
Prom construct by SwitchGear Genomics. Sp1-binding site
enriched intron 1 region of RD3 (0 bp to +1043 bp) was also
custom-cloned into the pLightSwitch_Prom construct by Switch-
Gear Genomics. BE(2)-C neuroblastoma cells were transfected
with control siRNA or N-Myc siRNA-1, followed by co-
transfection with Cypridina TK control construct plus empty
vector, linc00467 or RD3 gene promoter pLightSwitch_Prom
construct. Luciferase activities were measured with a LightSwitch
Dual Assay System kit (SwitchGear Genomics) according to the
manufacturer’s instructions, and expressed as percentage changes
relative to control siRNA transfected samples. mRNA microarray Neuroblastoma BE(2)-C cells were transfected with scrambled
control siRNA or linc00467 siRNA-1. Fourty-eight hours after
siRNA transfection, RNA was extracted from the cells with
RNeasy mini kit. Differential gene expression was examined with
Affymetrix HuGene-2_0-st Arrays (Affymetrix), according to the
manufacturer’s instructions. Results from the microarray hybrid-
ization were analysed in R (http://www.r-project.org/) with
bioconductor package (http://www.bioconductor.org/), and de-
posited at Gene Expression Omnibus website (accession number:
GSE52985). In summary, this study identifies lncRNAs as targets of N-Myc
in neuroblastoma cells through genome-wide differential gene
expression study, and demonstrates that N-Myc suppresses
linc00467 gene transcription through direct binding to the
linc00467 gene promoter. linc00467 reduces the expression of its
neighbouring protein-coding gene RD3, while N-Myc suppresses
RD3 gene transcription through direct binding to the RD3 gene
promoter. Importantly, linc00467 enhances neuroblastoma cell
survival through reducing DKK1 expression. These findings
therefore
demonstrate
that
N-Myc-mediated
suppression
of
linc00467 gene transcription counterintuitively blocks N-Myc-
mediated reduction in RD3 mRNA expression, and reduces
neuroblastoma cell survival by inducing DKK1 expression. References 1. Maris JM, Hogarty MD, Bagatell R, Cohn SL (2007) Neuroblastoma. Lancet
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N-Myc and linc00467 do not have a co-operative
effect on RD3 expression in neuroblastoma cells. BE(2)-C cells
were transfected with scrambled control siRNA, N-Myc siRNA-1,
N-Myc
siRNA-2,
linc00467
siRNA-1,
linc00467
siRNA-2,
combination of N-Myc siRNA-1 and linc00467 siRNA-1, or
combination of N-Myc siRNA-2 and linc00467 siRNA-2 for
48 hours, followed by RT-PCR analysis of RD3 expression. Error
bars represented standard error. * indicated P,0. 01, compared
with control siRNA-transfected samples. For the analysis of cells at sub-G1 phase, seventy-two hours after
siRNA transfection, cells were harvested, fixed in 80% ethanol,
washed and then stained with 50 mg/ml propidium iodide (Sigma)
in solution containing 2 mg/ml RNase (Roche). Flow cytometric
analysis of the cells was performed using FACS Calibur machine
and FACS Diva software (BD Biosciences). The percentage of cells
at sub-G1 phase of the cell cycle was analyzed. For the analysis of apoptosis, seventy-two hours after siRNA
transfection, cells were incubated with FITC-conjugated Annexin
V (BD Biosciences), and then subjected to flow cytometric analysis
of FITC-positive cells using FACS Calibur machine and FACS
Diva software. Table S1
Modulation of target gene expression by linc00467
siRNA-1 by more than 1.8 fold, as identified by Affymetrix
microarray, in BE(2)-C cells 48 hours after transfection with
control siRNA or linc00467 siRNA-1. The cut-off was set at 1.80
fold, as linc00467 siRNA-1 reduced the expression of linc00467 by
1.841 fold. Alamar blue assays Alamar blue assays were performed as previously described
[33]. Briefly, cells were transfected with various siRNAs in 96 well February 2014 | Volume 9 | Issue 2 | e88112 PLOS ONE | www.plosone.org 7 linc00467 Promotes Cell Survival immunoprecipitated with the anti-N-Myc Ab by PCR products
from DNA samples immunoprecipitated with the control Ab,
relative to input. Fold enrichment at the negative control region
was artificially set as 1.0. Error bars represented standard error. **
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blue (Invitrogen), and the plates were read on a microplate reader
at 570/595 nm. Results were calculated according to the optical
density absorbance units and expressed as percentage change in
viable cell number. Author Contributions Figure S1
N-Myc directly binds to the ODC1 gene promoter. ChIP assays were performed with a control or anti-N-Myc
antibody (Ab) and primers targeting a negative control region or
the ODC1 gene core promoter region. Fold enrichment was
calculated
by
dividing
PCR
products
from
DNA
samples Conceived and designed the experiments: TL. Performed the experiments:
BA PYL NS LZ MW AET. Analyzed the data: BA PYL NS LZ MW AET
GMM TL. Contributed reagents/materials/analysis tools: GMM. Wrote
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noncoding RNA as modular scaffold of histone modification complexes. Science
329: 689–693. 31. Friedman JS, Chang B, Kannabiran C, Chakarova C, Singh HP, et al. (2006)
Premature truncation of a novel protein, RD3, exhibiting subnuclear
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1059–1070. 28. Lai F, Orom UA, Cesaroni M, Beringer M, Taatjes DJ, et al. (2013) Activating
RNAs associate with Mediator to enhance chromatin architecture and
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Retinal degeneration 3 (RD3) protein inhibits catalytic activity of retinal
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30. Johnsson P, Ackley A, Vidarsdottir L, Lui WO, Corcoran M, et al. (2013) A
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translation in human cells. Nat Struct Mol Biol 20: 440–446. February 2014 | Volume 9 | Issue 2 | e88112 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 9
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https://openalex.org/W2182324063
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The Effect of Emotional Intelligence Group Training on Human and Social Capital in Isfahan University of Technology
|
International letters of social and humanistic sciences
| 2,015
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www.ssoar.info www.ssoar.info The effect of emotional intelligence group training
on human and social capital in Isfahan University of
technology
Abedi, Mohammad Reza; Nilforoushan, Parisa; Charsoughi, Batoul Tadayon
Veröffentlichungsversion / Published Version
Zeitschriftenartikel / journal article
Empfohlene Zitierung / Suggested Citation:
Abedi, M. R., Nilforoushan, P., & Charsoughi, B. T. (2015). The effect of emotional intelligence group training on human
and social capital in Isfahan University of technology. International Letters of Social and Humanistic Sciences, 50,
91-99. https://doi.org/10.18052/www.scipress.com/ILSHS.50.91 Empfohlene Zitierung / Suggested Citation:
Abedi, M. R., Nilforoushan, P., & Charsoughi, B. T. (2015). The effect of emotional intelligence group training on human
and social capital in Isfahan University of technology. International Letters of Social and Humanistic Sciences, 50,
91-99. https://doi.org/10.18052/www.scipress.com/ILSHS.50.91 Empfohlene Zitierung / Suggested Citation:
Abedi, M. R., Nilforoushan, P., & Charsoughi, B. T. (2015). The effect of emotional intelligence group training on human
and social capital in Isfahan University of technology. International Letters of Social and Humanistic Sciences, 50,
91-99. https://doi.org/10.18052/www.scipress.com/ILSHS.50.91 Nutzungsbedingungen:
Dieser Text wird unter einer CC BY Lizenz (Namensnennung) zur
Verfügung gestellt. Nähere Auskünfte zu den CC-Lizenzen finden
Sie hier: https://creativecommons.org/licenses/by/4.0/deed.de Online: 2015-03-23 International Letters of Social and Humanistic Sciences
ISSN: 2300-2697, Vol. 50, pp 91-99
doi:10.18052/www.scipress.com/ILSHS.50.91
© 2015 SciPress Ltd., Switzerland ABSTRACT The aim of this study was to investigate the effect of emotional intelligence group training on
human and social capital in the students of Isfahan University of Technology. This research was a
quasi-experimental study with pretest-posttest design and control group. The research population
consists of all the students of Isfahan University of Technology who have been studying in 2012-
2013. For sampling, voluntary sampling method was used that 36 subjects were selected as the
sample and were randomly divided into two groups of experiment and control. The subjects of
experimental group participated in 8 sessions of 90-minute classes while the control group received
no intervention. The study's tool was Mousavi Employability Questionnaire (2013). Analysis of
covariance was used to analyze the data. According to the results, emotional intelligence training
have had impact upon human capital (P<0.004). However, emotional intelligence training had no
effect on social capital. SciPress applies the CC-BY 4.0 license to works we publish: https://creativecommons.org/licenses/by/4.0/ The Effect of Emotional Intelligence Group Training on Human and
Social Capital
in Isfahan University of Technology
Batoul Tadayon Charsoughi1, Mohammad Reza Abedi2, Parisa Nilforoushan3
1 Islamic Azad University, Khomeini Shar Branch, Isfahan, Iran
2 Isfahan University and Islamic Azad University, Khomeini Shar Branch, Isfahan, Iran
3 Psychology Department, Isfahan University, Isfahan, Iran
E-mail address: 1bt.td20@yahoo.com
2dr.mr.abedi@gmail.com
3panilfo@yahoo.com Keywords: emotional intelligence; human capital; social capital 1. INTRODUCTION In each community, employment and having a job is important for the people at working age. Given the outcomes of having a job, a large part of the population seeks employment. In this regard,
the employed people are more healthy and satisfied with their lives. The importance of employment
and its consequent effects have prompted the researchers to make effort for enhancing people
employability. Many definitions have been offered for the concept of employability. Employability is defined
as one's ability to gain initial employment, job retention, job changes within the same organization,
and achieve a new job when needed. This definition includes both unemployed people who are
looking for a job and the employed people who are looking for a new job or want to promote their
position (Hillage& Pollard, 1998). Fugate et al (2004) describe employability as a psychosocial
structure that embodies those personal characteristics that enhances adaptive cognition and
behavior, and reinforces one's working relations. Since it involves those characteristics that cover
the gaps between the individual and the environment, employability is considered a psychosocial
structure. They define employability as one's ability in identifying and specifying career
opportunities within and between organizations. According to them, employability includes the four
aspects of career adaptability, personal adaptability, human capital, and social capital. Individuals and organizations invest in the field of human and social capitals and predict its
future returns in the workplace (Dess& Shaw, 2001). One's ability to identify and realize career
opportunities depends on the extent to which one is influenced by such capitals. These factors are
often included in a broader concept of knowledge, skills and abilities. However, the discussion here
is limited to human and social capitals since these concepts have a specific relationship with iPress applies the CC-BY 4.0 license to works we publish: https://creativecommons.org/licenses/by/4.0/ Volume 50 92 employability. Moreover, human and social capitals are tied to the people's career identities and are
embedded in sociability structure (Fugate et al, 2004). employability. Moreover, human and social capitals are tied to the people's career identities and are
embedded in sociability structure (Fugate et al, 2004). Social capital emanates from the social networks. It provides an interpersonal and social
element for employability, transfers information, and through social networks, provides people with
influence, power and information. 1. INTRODUCTION This influence, power and information not only provide the
individuals with the possibility to have access to career opportunities but also, they are important
components in achieving career aspirations (Fugate, 2006). In this regard, the network's size and
power are two important features of the network that determine the potentiality of the created
influence and information. The breadth and diversity of an individual network is probably
proportional to the amount of information and influence in the network, as well as to the usefulness
and influence of the information (Seibret et al, 2001). The benefits of social capital and its impact on the employability are demonstrated in
employment-related behaviors of individuals. Individuals with a well-developed social capital, not
only benefit from more formal job searching networks (like a company that supports tender
services) but also from informal job searching networks as well (e.g a friend of friend). In this
regard, Boxman, Graaf and Flap (1991) found out that top managers often search for jobs through
informal networks. They also found that social capital has a positive and independent impact on
wages that is higher and beyond human capital. Social networks can increase one's ability in
identifying and understanding career opportunities between organizations and industries and during
the career (Fugate, 2008). An employee's ability to understand job opportunities in the labor market is heavily
influenced by his human capital. Human capital refers to a set of factors that affect the variables
associated with one's career advancements. These factors include age and education, experience and
job training, job performance and organizational tenure, emotional intelligence, cognitive abilities
and knowledge, and skills and abilities that are created by such factors. Among human capital
factors, education and experience are the strongest predictors of career advancement (Fugate et al,
2004). In many types of employments, educational levels, even if they are not necessary, are
preferred. These educational levels include compulsory education, associate degree, bachelor
degree, certificate for a specific job and professional degrees. In fact, some researchers believe that
the value and rate of return for education is measurable (Goleman, 1988). Experience is also very important. Experience often leads to different levels of skills and tacit
knowledge which make the experienced person seem more attractive and prospective in the eyes of
employers. In modern workplaces, experience is shown through portable skills and not through
industry or exposure to a specific occupation. 1. INTRODUCTION eness of one's emotions (self-evaluation and self-esteem). •
Management of the emotions (self-control, trustworthiness, conscientiousness, innovation
and adaptability). •
Self-stimulation (progress motivation, optimism and commitment). •
Self-stimulation (progress motivation, optimism and commitment). •
Identification of others' emotions (understanding others, developing others, variety in the
influence styles, and political awareness). •
Identification of others' emotions (understanding others, developing others, variety in the
influence styles, and political awareness). •
Regulating the relations with others (influence, communication, conflict management,
leadership, bonding and participation, collective capabilities). •
Regulating the relations with others (influence, communication, conflict management,
leadership, bonding and participation, collective capabilities). There have been many studies about the effects of emotional intelligence training upon the
constitutive dimensions of emotional intelligence some of which are mentioned here: William et al
(2008), Soleimani and Ali-beigi (2009), Pourfaraj (2008), Karademas (2006), RaghibiabdRabbani
(2012), Seifi and Pour-mousavi (2010), and Yaryari, Moradi and Yahyazadeh (2007). However, no
research was found emphasizing the impact of emotional intelligence training on social capital or
the relationship between these two. In addition, the content of emotional intelligence deals with the
issues which are important factors in human and social capital. It seems that through training
emotional intelligence, human and social capitals can be influenced. In this regard, doing a research
on the effects of emotional intelligence training upon the social and human capitals seems
necessary. In other words, the aim of this study is to evaluate how emotional intelligence training
influences human and social capitals. Based on the intended variables this study aims to examine
two following hypotheses: •
Emotional intelligence training has impact upon human capital. otional intelligence training has impact upon •
Emotional intelligence training has impact upon social capita 1. INTRODUCTION Portable skills include knowledge, abilities and skills
which are obtained from very different areas and are transferable to other areas. Employees often
choose individuals according to their experiences and experience is associated with productivity and
rewards. In short, human capital refers to one's ability in meeting performance expectations in a
particular profession (Protess, 1998). Therefore, one's level of education and experience are powerful predictors for career
advancement. Human capital will help both individual and organizational adaptability. Furthermore,
investment in human capital throughout a person's career is an adaptive orientation and a
commitment to continuous learning that will lead to further strengthening of employability (Fugate,
2004). Researchers have shown that according to some factors, people with high IQ are not
necessarily successful in their lives, while those with an average IQ may be placed on the road to
success. These factors include another dimension of intelligence called as emotional intelligence by
Goleman. The concept of emotional intelligence was first proposed by Salovay and Mayer as a form
of social intelligence. Salovay used this concept for describing and understanding of people's
feeling, sympathy with the feelings of others, and the ability in optimal management of
temperament. In their definition, they introduced emotional intelligence as the ability to process
emotional information including the proper assessment of one's and others' emotions, appropriate International Letters of Social and Humanistic Sciences Vol. 50 93 expression of emotions, and adaptive regulation of emotions so that it leads to the improvement of
flow of life (Nouri-Emamzadeh, 2009). expression of emotions, and adaptive regulation of emotions so that it leads to the improvement of
flow of life (Nouri-Emamzadeh, 2009). In 1990, Goleman argued that emotional intelligence can be used to identify employees who
might have the best performance and since then, a practical movement was started to increase
research on emotional intelligence. According to Goleman, emotional intelligence refers to
individuals' ability in controlling and governing themselves and their relationships. He also believes
that 80 to 90 percent of individuals' successes can be attributed to emotional intelligence. In
defining emotional intelligence for public, Goleman considers it as a form of social behavior
(Whitte, 2004). (
)
According to Goleman's definition (1995, trans. By Parsa, 2004), emotional intelligence is
classified into five areas of: (
)
According to Goleman's definition (1995, trans. By Parsa, 2004), emotional intelligence is
classified into five areas of: •
Awareness of one's emotions (self-evaluation and self-esteem). Volume 50 Volume 50 94 descriptive statistics including the mean, standard deviation, and inferential statistics including
ANCOVA. The format of the sessions is summarized as follows: ession: explaining the objectives of the sessions, familiarity with the concept, the positio
cessity of employability. The second session: familiarizing the members with the concept of emotional intellig
components of emotional intelligence. The third session: informing the members of self-awareness and the factors that p
awareness of his emotions. The fourth session: familiarizing the members with 5 barriers preventing one's aware
emotions and using some alternatives for these barriers. The fifth session: familiarizing the members with the concept of emotional permeab
how we can reduce permeability. The fifth session: familiarizing the members with the concept of emotional permeability and that
how we can reduce permeability. p
y
ession: members' familiarity with the concept of perception and perception barriers. The sixth session: members' familiarity with the concept of perception and perception barriers. The seventh session: members' familiarity with the concept of accepting and expressing emotions,
and the barriers of it. The sixth session: members familiarity with the concept of perception and perception barriers. The seventh session: members' familiarity with the concept of accepting and expressing emotions,
and the barriers of it. The eighth session: getting feedback from the members on the use and quality of the di The eighth session: getting feedback from the members on the use and quality of the discussions. 1.3. RESEARCH TOOLS To assess the concept of employability, Mousavi Employability Questionnaire (2013) was
used in this study. This questionnaire contains 96 questions that the questions 1 to 6 are related to
age, sex, education, job and training experiences, and have been coded by alphabets. Social-capital-
related questions have been designed in one question that is question 58. The range of scores in this
questionnaire is between 57 and 441. Using Likert scale, scoring process was done as follows:
1 C
l t l
2 A
3 M di
4 Di
5 C
l t l di p
y g
;
g
;
;
g
;
p
y
g
It should be noted that for the questions 3, 8, 32, 16, 11, 33, 34, 49, 50, 51, and 53 scoring
was done reversely: 1- Completely disagree; 2- Disagree; 3- Medium; 4- Agree; 5- Completely
agree. It should be noted that for the questions 3, 8, 32, 16, 11, 33, 34, 49, 50, 51, and 53 scoring
was done reversely: 1- Completely disagree; 2- Disagree; 3- Medium; 4- Agree; 5- Completely
agree. Mousavi has evaluated the content validity of the questionnaire using the opinions of
specialists, and to assess the convergent construct validity, he has reported the correlation of this
questionnaire with employment self-efficacy questionnaire (Salehi, 2005). Using a sample of 20
students of Isfahan University of Technology who have been studying in the academic year of
2012-2013, and using Cronbach alpha, Mousavi, has calculated the reliability of the questionnaire
as 0.94. Cronback alpha reliability of the questionnaire in this study was estimated to be 0.90
through using a sample of 36 students who were studying in Isfahan University of Technology. 1.2. METHODOLOGY This research is a quasi-experimental study with pretest-posttest design and control group. The study's variables include the independent variable of emotional intelligence training and
dependent variables of human and social capitals. The research population consists of all the
students of Isfahan University of Technology who have been studying in 2012-2013. For sampling,
in this study voluntary sampling method with random assignment was used. In this regard, some
declarations were designed declaring: emotional intelligence workshops along with the instructor's
name, and the time and place of registration. Then, the interested students were invited to
participate in the workshop. The sample size was 36 students. They were randomly divided into two
groups of the experimental group (n=18) and the control group (n=18). Criteria for being included in the experimental and control group: a) being a student in Isfahan
University of Technology; b) being a student in the academic year of 2012-2013. After sampling,
the subjects of both groups were assessed using employability questionnaire. Then, the
experimental group received 8 sessions emotional intelligence group training while the control
group remained in the waiting list. After the end of the sessions, both groups responded again to the
questionnaire. Then, the data obtained from both experimental and control groups were analyzed. For data analyzing, SPSS16 software was used. The statistical method used in this study has been 2. DISCUSSION Given the purpose and method of the research that is based on comparing the two groups of
experiment and control in their scores of employability and its dimensions and considering that it
was necessary to control pretest, analysis of covariance (ANCOVA) was used. However, to use this
test, its pre-assumptions/defaults have to be examined. In continue, at first, these pre-assumptions
will be discussed and then the results of analysis of covariance for the scores of both human and
social capitals are provided separately. Table 1 summarizes the results of Kolmogorov-Smirnov and
Shapiro-Willkie tests to verify the default normal distribution of scores on the dependent variable
(posttest score) and the scores of auxiliary random variable (pretest score). Table 1. The summary results of the default normal distribution of the dependent variable (posttest score)
and the auxiliary random variables (pretest score)
The variable
Pretest
Posttest
Kolmogorov-Smirnov
Shapiro-Willkie
Kolmogorov-Smirnov
Shapiro-Willkie
statistic df Significance statistic df Significance statistic df Significance statistic df Significance
Human capital
0.12
36
0.18
0.98
36
0.58
0.09
36
0.20
0.98
36
0.80
Social capital
0.10
36
0.20
0.98
36
0.59
0.09
36
0.20
0.97
36
0.52 The summary results of the default normal distribution of the dependent variable (posttest score)
and the auxiliary random variables (pretest score) International Letters of Social and Humanistic Sciences Vol. 50 95 As it is shown in table 1, the default normal distribution in each of the dependent variables (posttest
scores) and the auxiliary random variables (pretest scores) cannot be rejected. Table 2 summarizes
the results of the Levine test to examine the default equality of variances of the dependent variable
scores (posttest score) and the scores of auxiliary random variable (pretest score). Table 2. The summary result of Levine test in examining the default equality of variances of the dependent
variables (posttest scores) and the auxiliary random variables (pretest score) summary result of Levine test in examining the default equality of variances of the dependent
variables (posttest scores) and the auxiliary random variables (pretest score) Table 2. 2. DISCUSSION The summary result of Levine test in examining the default equality of variances of the dependent
variables (posttest scores) and the auxiliary random variables (pretest score)
The variables
Pretest
Posttest
F
df1
df2
Significance
F
df1 df2
Significance
Human capital
3.63
3
32
0.02
1.36
3
32
0.27
Social capital
5.82
3
32
0.000
2.77
3
32
0.06 The variables
Pretest
Posttest
F
df1
df2
Significance
F
df1 df2
Significance
Human capital
3.63
3
32
0.02
1.36
3
32
0.27
Social capital
5.82
3
32
0.000
2.77
3
32
0.06 According to the Levine test (Table 2), the default equality of variances cannot be rejected. Now,
given the observance of the default, analysis of covariance can be used. 2.1. THE RESULTS OF ANALYSIS OF COVARIANCE FOR HUMAN CAPITAL SCORES 2.1. THE RESULTS OF ANALYSIS OF COVARIANCE FOR HUMAN CAPITAL SCORES
Descriptive statistic results for the pretest and posttest scores of human capital are 2.1. THE RESULTS OF ANALYSIS OF COVARIANCE FOR HUMAN CAPITAL SCORES
Descriptive statistic results for the pretest and posttest scores of human capital are
summarized in table 3. In the posttest phase, the averages are calculated after controlling the effects 2.1. THE RESULTS OF ANALYSIS OF COVARIANCE FOR HUMAN CAPITAL SCORES
Descriptive statistic results for the pretest and posttest scores of human capital are
summarized in table 3. In the posttest phase, the averages are calculated after controlling the effects
of auxiliary random variable. Descriptive statistic results for the pretest and posttest scores of human capital are
summarized in table 3. In the posttest phase, the averages are calculated after controlling the effects
of auxiliary random variable. Table 3. Descriptive statistics of human capital both in pretest and posttest stages
Group
belonging of
therapy
Group
belongings
of sex
Sample
size
Pretest
Posttest
Mean
Standard
deviation
Modified mean
Standard
deviation
Experimental
Male
13
22.23
3.83
24.25
4.98
Female
5
20.40
1.52
23.76
3.91
Control
Male
13
22.46
4.94
21.88
4.35
Female
5
19.00
5.29
21.11
5.03
Total
Experimental
18
20.31
3.41
24.01
4.71
Control
18
21.73
5.14
21.49
4.78 Table 3. Descriptive statistics of human capital both in pretest and posttest stages As it can be observed in table 3, both in the experimental group and the control group, there have
been differences between the pretest and posttest means, but the differences have been more in the
experimental group. 2. DISCUSSION Good criteria for the means and determining the significance and non-
significance of the differences are given in figure Volume 50 96 1:
Figure 1. Comparison of human capital means in both experimental and control groups in pretest posttest
stages and at the confidence level of 95%. The between-line points represent the averages and the lines'
extents show the confidence intervals of the means. 12Posttest Control
11.Posttest Exp
10.Pretest Control
9.Pretest Exp
8.Posttest F
Control
7.Posttest M
Control
6.Posttese F
Exp
5.Posttest M
Exp
4.Pretest F Control
3.Pretest M
Control
2.Pretest female, Exp
1.Pretest male
Exp
96
Volume 50 Figure 1. Comparison of human capital means in both experimental and control groups in pretest posttest
stages and at the confidence level of 95%. The between-line points represent the averages and the lines'
extents show the confidence intervals of the means. In terms of intergroup comparisons, significance of the differences is shown in figure 1. Combined with the results of the table 4, these differences are discussed in continue: Table 4. The summary results of covariance analysis to determine intergroup differences in posttest scores of
human capital
Effect source
Degree of
freedom (df)
Mean
squares
F
Statistical
significance
Effect size
Statistical
power
Pretest
1
544.42
118.49
0.0005
0.79
1.00
Intervention
1
45.52
9.91
0.004
0.24
0.86
According to the results of table 4 human capital posttest significant difference between the two Table 4. The summary results of covariance analysis to determine intergroup differences in po
human capital ummary results of covariance analysis to determine intergroup differences in posttest scores of
human capital According to the results of table 4, human capital posttest significant difference between the two
groups of experiment and control (P=0.004; F=9.91; R2=0.24) indicates the effectiveness of
emotional intelligence on the human capital (the comparison of lines 11 and 12 of figure 1 also
suggest the same significant difference). Therefore, the first research hypothesis is confirmed. Emotional intelligence training has been able to predict 24 percent increase in human capital,
suggesting the effectiveness of the intervention (R2>0.10). 2.2.ANALYSIS OF COVARIANCE FOR SOCIAL CAPITAL SCORES Descriptive statistic results for the pretest and posttest scores of social capital are summarized
in table 5. In the posttest phase, the averages are calculated after controlling the effects of auxiliary
random variable. Table 5. Descriptive statistics of social capital both in pretest and posttest stages
Group
belonging of
therapy
Group
belongings
of sex
Sample
size
Pretest
Posttest
Mean
Standard
deviation
Modified mean
Standard
deviation
Experimental
Male
13
63.77
24.87
66.36
26.09
Female
5
79.00
9.41
61.13
11.59 International Letters of Social and Humanistic Sciences Vol. 50 International Letters of Social and Humanistic Sciences Vol. 50 Control
Male
13
72.54
29.28
67.87
29.93
Female
5
57.60
25.88
65.47
27.62
Total
Experimental
18
71.39
22.51
63.75
23.03
Control
18
65.07
28.46
66.67
29.50
International Letters of Social and Humanistic Sciences Vol. 50
97 97 Control
Male
13
72.54
29.28
67.87
29.93
Female
5
57.60
25.88
65.47
27.62
Total
Experimental
18
71.39
22.51
63.75
23.03
Control
18
65.07
28.46
66.67
29.50 As it can be observed in table 5, there have been differences between the pretest and posttest
means both in the experimental group and the control group, but the differences have been more in
the experimental group. Good criteria for the means and determining the significance and non-
significance of the differences are given in figure 2: Figure 2. Comparison of social capital means in both experimental and control groups in pretest posttest
stages and at the confidence level of 95%. The between-line points represent the averages and the lines'
extents show the confidence intervals of the means. 12Posttest
Control
11.Posttest
Exp
10.Pretest
Control
9.Pretest Exp
8.Posttest F
Control
7.Posttest M
Control
6.Posttese F
Exp
5.Posttest M
Exp
4.Pretest F
Control
3.Pretest M
Control
2.Pretest
female, Exp
1.Pretest male
Exp Figure 2. Comparison of social capital means in both experimental and control groups in pretest posttest
stages and at the confidence level of 95%. The between-line points represent the averages and the lines'
extents show the confidence intervals of the means. T bl 6 Th
lt
f
i
l
i t d t
i
i t
diff
i
tt
t
f
12Posttest
Control
11.Posttest
Exp
10.Pretest
Control
9.Pretest Exp
8.Posttest F
Control
7.Posttest M
Control
6.Posttese F
Exp
5.Posttest M
Exp
4.Pretest F
Control
3.Pretest M
Control
2.Pretest
female, Exp
1.Pretest male
Exp Figure 2. 2.2.ANALYSIS OF COVARIANCE FOR SOCIAL CAPITAL SCORES Comparison of social capital means in both experimental and control groups in pretest posttest
stages and at the confidence level of 95%. The between-line points represent the averages and the lines'
extents show the confidence intervals of the means. Table 6. The summary results of covariance analysis to determine intergroup differences in posttest scores of
human capital
Effect source
Degree of
freedom (df)
Mean
squares
F
Statistical
significance
Effect size
Statistical
power
Pretest
1
18733.17
153.76
0.0005
0.83
1.00
Intervention
1
60.91
0.50
0.48
0.02
0.11 Table 6. The summary results of covariance analysis to determine intergroup differences in posttest scores of
human capital
Effect source
Degree of
freedom (df)
Mean
squares
F
Statistical
significance
Effect size
Statistical
power
Pretest
1
18733.17
153.76
0.0005
0.83
1.00
Intervention
1
60.91
0.50
0.48
0.02
0.11 ummary results of covariance analysis to determine intergroup differences in posttest scores of
human capital According to the results of table 6, human capital posttest non-significant difference between
the two groups of experiment and control (P=0.48; F=0.50; R2=0.02) suggests the non-effectiveness
of emotional intelligence on the social capital (the comparison of lines 11 and 12 of figure 2 also
suggests this significant difference). Therefore, this hypothesis is rejected. Emotional intelligence
training has been not able to predict the increase in social capital, suggesting the practical non-
effectiveness of the intervention (R2<0.10). 3. CONCLUSION The results obtained from the analysis of covariance showed that there is a significant
difference between the posttest of the experimental group and the control group in terms of human
capital. Therefore, it can be concluded that emotional intelligence training has had impact on the
human capital of students in the Isfahan University of Technology. Moreover, the results obtained
from the analysis of covariance showed that there is no significant difference between the posttests
of the experimental and control groups in the social capital. Hence, it can be concluded that
emotional intelligence training has had no impact on the social capital of students in the Isfahan
University of Technology. Volume 50 98 To explain the first hypothesis, it can be said that regardless of the conducted researches, the
effect of emotional intelligence training is evident on the human capital. As mentioned in the
methodology part, training pattern of increasing emotional intelligence in this intervention has been
based on increasing the dimensions of emotional intelligence. Thus, it is clear that this intervention
has had effect on the various aspects of human capital (such as self-awareness, self-regulation,
motivation, empathy and social skills), and finally has led to the increase of human capital in this
study. It is also noteworthy that the effects of an intervention are measured by its measuring tools. Regarding this study's tools, to measure human capital some questions have been used about
emotions control, emotions expression and emotions management. Hence, it is obvious that this
intervention has been more effective on human capital. In the course of this study, as a result of interaction with each other, the members gained new
learning during these training sessions. At these sessions, the members defined their experiences
with regard to the controlling and expressing their emotions. In this process, members, through
observing each other's experiences and integration of assigned duties, gain the ability to manage,
know and express their emotions. To explain the second hypothesis, it can be said that although no research was found on the
effect of emotional intelligence training on social capital and the relationship between these two,
this non-effectiveness can be explained in this way that by social capital in the Fugate et al model
(2004), it means the networks that give influence and information to the individuals and help them
to identify and realize career opportunities. 3. CONCLUSION A group of these capitals such as parents, siblings, relatives and so on are with a person at
his/her birth, and another group such as school, university, friends and so forth are created over the
time. The second group of capitals can be created under the influence of the first group. For
example, educated parents can make their children familiar with books or enroll them in high level
schools. In this regard, the first group of capitals is to a large extent out of one's control and is
controlled by the deterministic powers. The second category of capitals, as it is formed by the first
group, is also, to some extent, out of one's control but it cannot be claimed definitely that they are
completely out of one's control. Since these capitals have been established in the individual from years ago and out of the
individual's control, they cannot be increased in the process of the intervention. For example, some
parts of these capitals are created by media and their contents cannot be changed, or they are
established by the family, relatives and friends networks which cannot be influenced directly by the
intervention. Hence, this intervention cannot in a short time change or increase the capitals whose
processes of creation have been from many years ago. However, perhaps if the intervention followed up, we could see the effect of the intervention
on the increase of such capitals; since, on the one hand, this intervention was itself a social capital
and, on the other hand, according to the dimension of social skills in emotional intelligence, the
individual sought information, trainings and measures to increase such capitals in oneself, but, due
to the limited scope of the study it was impossible for us to do it. In conclusion, it can be said that the efficacy of an intervention can be measured by its
measuring tools. In the employability questionnaire, social capital is measured by such questions as
"how much have your parents contributed to the creation of employment opportunities in you?"
Since for increasing the scores of these questions, such factors as trainings, family counseling and
social capitals should be considered and due to the fact that such trainings were impossible in our
intervention, the intervention could not increase the members' social capital. Acknowledgement At the end we express our endless gratitude and indebtedness to all people who help us in this
project. In addition, we would like to thank Mr. Roohollah Datli Beigi, M.A. in English Language
and Literature, University of Isfahan, whose linguistic and grammatical comments were helpful and
constructive in preparation of this paper. International Letters of Social and Humanistic Sciences Vol. 50 International Letters of Social and Humanistic Sciences Vol. 50 99 References [1] Dess, G.G., & Shaw, S.D.(2001). “Voluntary turnover, Social capital, and organizational
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Intelligence of the Boys in Keman Juvenile Institution".The Journal of Educational-
Psychological Studies.9 (15): 107-124. [12] Seibert, S. E., Kraimer, M. L., &Crant, J. M. (2001). “What do proactive people do? A
longitudinal model linking proactive personality and career success”, Personnel Psychology,
54(4), 845-874. [13] Seifi, M. Y. and Pour-mousavi, M (2009). Investigation of the Relationship between Emotional
Intelligence and Locus of Control (Internal-External).BA Thesis, Psychology,Payam Noor
University of Boroujen. [14] Soleimani, N and Ali-beigi, F (2009)."Relationship betweenEmotional Intelligence and Self-
efficacy of the Managers in the Branches of Islamic Azad University".Journal of Educational
Leadership and Management.(10): 137-154. [15] Whitte, R. (2004). Emotional intelligence and career success among the highly intelligent. Journal of career Assessment. [16] Yaryari, F. Moradi, A. R. and Yahyazadeh, S (2007)."Relationship of Emotional Intelligence
and Control Locus with Mental Health of the Students in the University of
Mazandaran".Journal of Psychological Studies.(1): 120-128.
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English
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Zerumbone, a cyclic sesquiterpene, exerts antimitotic activity in HeLa cells through tubulin binding and exhibits synergistic activity with vinblastine and paclitaxel
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Cell proliferation
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cc-by
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O R I G I N A L A R T I C L E O R I G I N A L A R T I C L E Funding information g
This work is partly supported by the grant
from the DST/SERB, Government of India
(SR/SO/BB‐0013/2010), to Dr Rathinasamy
K and partly by TEQIP‐II, NIT Calicut, for the
Student Project to SMA (Ref. NITC/TEQIP‐
II/R&D/2014). Results: Zerumbone selectively inhibited the proliferation of HeLa cells with an IC50
of 14.2 ± 0.5 μmol/L through enhanced cellular uptake compared to the normal cell
line L929. It induced a strong mitotic block with cells exhibiting bipolar spindles at the
IC50 and monopolar spindles at 30 μmol/L. Docking analysis indicated that tubulin is
the principal target of zerumbone. In vitro studies indicated that it bound to goat
brain tubulin with a Kd of 4 μmol/L and disrupted the assembly of tubulin into micro‐
tubules. Zerumbone and colchicine had partially overlapping binding site on tubulin. Zerumbone synergistically enhanced the anti‐proliferative activity of vinblastine and
paclitaxel through augmented mitotic block. Conclusion: Our data suggest that disruption of microtubule assembly dynamics is
one of the mechanisms of the anti‐cancer activity of zerumbone and it can be used in
combination therapy targeting cell division. Shabeeba M. Ashraf | Jomon Sebastian | Krishnan Rathinasamy Shabeeba M. Ashraf | Jomon Sebastian | Krishnan Rathinasamy School of Biotechnology, National Institute
of Technology Calicut, Calicut, Kerala, India Abstract
Objectives: The aim of this study was to elucidate the antimitotic mechanism of
zerumbone and to investigate its effect on the HeLa cells in combination with other
mitotic blockers. Correspondence
Krishnan Rathinasamy, School of
Biotechnology, National Institute of
Technology Calicut, Calicut, Kerala, India. Email: rathin@nitc.ac.in
Funding information
This work is partly supported by the grant
from the DST/SERB, Government of India
(SR/SO/BB‐0013/2010), to Dr Rathinasamy
K and partly by TEQIP‐II, NIT Calicut, for the
Student Project to SMA (Ref. NITC/TEQIP‐
II/R&D/2014). Materials and methods: HeLa cells and fluorescence microscopy were used to ana‐
lyse the effect of zerumbone on cancer cell lines. Cellular internalization of zerum‐
bone was investigated using FITC‐labelled zerumbone. The interaction of zerumbone
with tubulin was characterized using fluorescence spectroscopy. The Chou and
Talalay equation was used to calculate the combination index. Received: 28 July 2018 | Revised: 19 September 2018 | Accepted: 10 October 2018
DOI: 10.1111/cpr.12558 Received: 28 July 2018 | Revised: 19 September 2018 | Accepted: 10 October 2018
DOI: 10.1111/cpr.12558 y 2018 | Revised: 19 September 2018 | Accepted: 10 October 2018 Received: 28 July 2018 | Revised: 19 September 2018 | Accepted: 10 October 2018 DOI: 10.1111/cpr.12558 Cell Proliferation. 2019;52:e12558.
https://doi.org/10.1111/cpr.12558 Cell Proliferation. 2019;52:e12558.
| 1 of 17
wileyonlinelibrary.com/journal/cpr
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited
© 2018 The Authors. Cell Proliferation Published by John Wiley & Sons Ltd 2.1 | Materials Paclitaxel, vinblastine sulphate, podophyllotoxin, colchicine, 5,5′‐
dithiobis‐2‐nitrobenzoic acid (DTNB), sulforhodamine B (SRB),
Hoechst 33342, guanosine 5′‐triphosphate (GTP), propidium iodide,
EGTA, MgCl2, piperazine‐N,N′‐bis (2‐ethanesulphonic acid) (PIPES),
mouse monoclonal anti‐α‐tubulin IgG and FITC‐conjugated anti‐
mouse IgG, fluorescein isothiocyanate isomer (FITC) and dimeth‐
ylformamide were purchased from Sigma‐Aldrich (St. Louis, MO,
USA). Foetal bovine serum (FBS) and Alexa Fluor 568‐conjugated
anti‐mouse IgG were purchased from Invitrogen (Thermo Scientific,
Massachusetts, USA). Acridine orange (AO), hydroxysuccinimide,
minimal essential medium (MEM), cell culture tested antibiotic so‐
lution, and phosphate‐buffered saline (PBS) were purchased from
HiMedia (Mumbai, India). Dichloromethane, n‐hexane, hydroxy‐
lamine hydrochloride and triethylamine were purchased from Merck,
India. 1‐Ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide hydrochlo‐
ride was purchased from SRL chemicals. All other reagents used in
the study were of analytical grade. The clinically successful antitubulin agents such as the pacli‐
taxel and the vinblastine are obtained from plants. Natural product
research is gaining a huge attention because many of the phyto‐
chemicals exhibit excellent chemopreventive and chemotherapeutic
potential in addition to their selectivity against cancer cells and low
cost of production.10 Natural products such as genistein, apigenin,
quercetin, curcumin, berberine, limonene, coumarin, indirubin, bras‐
sinin, indole‐3‐carbinol, lycopene and resveratrol are in clinical/
preclinical trials either alone or in combination therapy for the treat‐
ment of cancer.11-13 In the present study, we have investigated the
anti‐proliferative mechanism of the natural product zerumbone iso‐
lated from the plant Zingiber zerumbet belonging to the ginger family
of flowering plants (Zingiberaceae). Zerumbone is a sesquiterpene
and is reported to exhibit anti‐cancer potential and other pharma‐
cological activities such as anti‐inflammatory, antibacterial, antima‐
larial and antioxidant properties.14-17 Zerumbone was found to be
effective in preventing tumour angiogenesis by inhibiting the VEGF
expression and NF‐κB activity.18 It was reported to induce apoptosis
in various cancer cell lines by modulating the FAS and TRAIL signal‐
ling pathways, through enhanced expression of TNF and modulating
Bax/Bcl‐2 ratio .19-21 Recently, zerumbone was reported to block the
cell cycle at mitosis17,22 and induce apoptosis in cancer cells through
inhibition of microtubule assembly.23 However, the mechanism be‐
hind the mitotic block was not clearly established, and hence, we
have performed this study to elaborate its anti‐cancer mechanism
through biophysical, biochemical and cell culture studies. kinases such as the Aurora family of proteins; the Polo‐like kinase;
and the mitotic checkpoint proteins.5,7 its inhibitory effects on tubulin and induction of mitotic block, and
combination of zerumbone and other anti‐cancer drugs might pro‐
vide a therapeutic advantage in controlling the growth of cancer cells. Microtubules, the key components of the cytoskeleton, are com‐
posed of alpha and beta tubulin heterodimer. They are highly dy‐
namic polymers that undergo polymerization and depolymerization
in a short span of time and play essential role in the maintenance
of cell shape, intracellular trafficking, cell motility and cell signalling
apart from cell division and mitosis.8 Tubulin has two nucleotide
(GTP) binding sites and three well‐characterized drug binding sites
such as the colchicine binding site, the paclitaxel binding site and
the Vinca alkaloid binding site. The GTP binding site is located at
the N‐terminal region of the α and the β subunits, and the colchicine
binding site is present at the interface of the α‐ β subunit.8,9 The
paclitaxel binding site is located at the β‐tubulin, and the Vinca alka‐
loids binding site is located in the N‐terminal region of the β‐tubulin
subunit close to the GTP binding site.9 2.2 | Isolation, purification and
characterization of zerumbone Fresh rhizomes of Zingiber zerumbet were collected from the farms
of the Indian Institute of Spice Research (IISR), Calicut, Kerala (India),
and it was authenticated by Dr D Prasath, Principal Scientist, IISR,
Calicut. Zerumbone was extracted and isolated from the rhizomes
of Zingiber zerumbet as described earlier.24 Briefly, 1 kg of fresh rhi‐
zomes was washed under running tap water and cut into slices. The
slices were then shade‐dried at 37°C for 5 days. The dried samples
were then soaked in methanol for 3 days, and the methanolic ex‐
tract was concentrated by using the rotary evaporator (Heidolph
Instruments, GmbH & CO. KG, Schwabach, Germany). The extract
was then fractionated by silica gel (mesh size 200) column chroma‐
tography using organic solution mixture of hexane: ethyl acetate
(8:2; v/v). Zerumbone thus obtained was further purified by crystal‐
lization. The purity of zerumbone was analysed and confirmed using
the Shimadzu liquid chromatography‐mass spectrometry (LC‐MS)
and Bruker Avance III Nuclear Magnetic Resonance spectroscopy
(NMR) using the standard procedure. Cell culture studies showed an excellent observation that zerum‐
bone exhibited selective toxicity against HeLa cells through enhanced
internalization of the compound and inhibited their migration. The
anti‐proliferative effect of zerumbone in HeLa cells correlated well
with its ability to inhibit the cell cycle at mitosis through tubulin bind‐
ing. Zerumbone bound to tubulin at the colchicine binding site and
inhibited the polymerization of tubulin into microtubules. Zerumbone
exhibited excellent synergistic antimitotic and anti‐proliferative ac‐
tivity in HeLa cells when combined with clinically established drugs
such as paclitaxel and vinblastine. Together, the results suggest that
the anti‐proliferative effects of zerumbone could be partly through 2 of 17 ASHRAF et al. 1 | INTRODUCTION 1 and invasion. Hence, chemotherapeutic agents that target cell di‐
vision are highly valuable in cancer chemotherapy. Mammalian cell
division requires the coordinated actions of the cytoskeleton, the
membrane proteins, the motor proteins and the cell cycle regulatory
proteins which are precisely controlled in space and time.4,5 Among
the various players in cell division, tubulin is indispensable for mito‐
sis and chromosome segregation, and antimitotic agents targeting
tubulin are the most successful in the treatment of various types of
tumours.5,6 The other essential antimitotic targets include the mi‐
totic kinesins such as Eg5, CENP‐E, MCAK, and MKLP1; the mitotic Cancer is one of the leading causes of death all over the world, and
cancer treatment remains one of the greatest challenges in front of
the healthcare professionals.1,2 Most of the anti‐cancer agents cur‐
rently available in the market are non‐selective and could destroy
the normal healthy cells. In addition, they could pose greater risk of
developing drug resistance in the tumour cells and are ineffective
in inhibiting metastasis.3 Cancer is a complex disease characterized
by an overactive cell cycle that promotes increased cell proliferation | 1 of 17
wileyonlinelibrary.com/journal/cpr 1 of 17 wileyonlinelibrary.com/journal/cpr (EDF) to characterize the binding site of zerumbone on tubulin. Zerumbone oxime was synthesized as described earlier.25 In brief,
zerumbone (0.3 g) was dissolved in 10 mL of ethanol containing 0.9 g
of hydroxylamine hydrochloride and 1.8 g potassium carbonate. The
mixture was stirred for 5 hours at room temperature. The reaction
mixture was then filtered, and the residue was washed with methanol. The filtrate was concentrated under reduced pressure and was then
mixed with dichloromethane (10 mL). The organic layer was collected
and washed with water. The resultant mixture was concentrated
and dried to get crystalline zerumbone oxime, which was subjected
to FTIR analysis. Fluoresceinthiocarbamyl ethylenediamine (EDF)
was synthesized as described earlier.26 Zerumbone oxime (20 mg),
1‐ethyl‐3‐(3‐dimethylaminopropyl)
carbodiimide
hydrochloride
(20 mg) and hydroxysuccinimide (6 mg) were dissolved in 1 mL
of dimethylformamide. The mixture was stirred continuously for
1 hour; and to this mixture, 5 mL of dimethylformamide containing
25 mg of EDF was added dropwise over a period 30 minutes. The
reaction mixture was allowed to resolve on a preparative silica gel
using ethyl acetate/methanol/acetic acid (90/8/2 v/v/v) as mobile
phase. The spots on the preparative TLC plate were identified in
a UV chamber, and the spot corresponding to FITC‐conjugated
zerumbone (Rf 0.49) was eluted using methanol.26 estimated as mmol/cell.29 Standard curve of fluorozerumbone was
obtained using the standard solution in the range of 1‐100 µmol/L. Spectral scan was analysed using Systronics AU‐2701 UV‐visible
double beam spectrophotometer at 200‐800 nm. estimated as mmol/cell.29 Standard curve of fluorozerumbone was
obtained using the standard solution in the range of 1‐100 µmol/L. Spectral scan was analysed using Systronics AU‐2701 UV‐visible
double beam spectrophotometer at 200‐800 nm. 2.8 | Mitotic index assay and
Immunofluorescence microscopy For determination of mitotic index (MI), HeLa cells were grown on
poly‐l‐lysine‐coated glass coverslips (12 mm) in 24‐well tissue culture
plates and were subsequently treated with different concentrations
of zerumbone for 24 hours. The cells were then fixed with 3.7% (v/v)
formaldehyde solution in PBS for 30 minutes at 37°C. The cells were
permeabilized with cold methanol at −20°C for 30 minutes. Cells were
then stained with Hoechst 33342 (1.5 µg/mL). The coverslips were
washed twice with PBS and were mounted on clean glass slides with the
mounting medium containing 1,4‐diazabicyclo [2.2.2] octane (DABCO)
as anti‐quenching agent. The number of mitotic and interphase
cells was counted using a Nikon ECLIPSE Ti‐E inverted fluorescent
microscope (Tokyo, Japan). The MI was calculated as the percentage
of cells blocked at mitosis.31 At least 1000 cells were counted for each
concentrations of zerumbone. The HeLa cells that were treated with
different concentrations of zerumbone for 24 hours were fixed with
formaldehyde and processed to visualize the interphase and mitotic
microtubules using mouse monoclonal alpha‐tubulin antibody and goat %Cell migration %Cell migration
=
[
1−
(width of scratch at specific time point t
width of the scratch at zero time
)]
×100 %Cell migration
=
[
1−
(width of scratch at specific time point t
width of the scratch at zero time
)]
×100 2.7 | Cell migration assay HeLa cells (1 × 106 cells/mL) were grown in minimum essential medium
supplemented with 10% FBS in 35 mm cell culture dishes. At 90%
confluence, a wound was made using a sterile micropipette tip.30,31
The floating cells were removed immediately after wounding, and the
media were changed with fresh one containing different concentra‐
tions of zerumbone (0, 5, 10 and 15 μmol/L). Cells were observed at 24,
48 and 72 hours of intervals, and the bright‐field images of the wound
closure were recorded using the Nikon ECLIPSE Ti inverted micro‐
scope. Percentage wound healing was calculated by using the formula: 2.6 | Calculating the percentage of apoptotic cell
death using AO staining HeLa cells (0.5 × 105 cells/mL) grown on poly‐l‐lysine‐coated glass
coverslips (12 mm) in 24‐well tissue culture plates were treated with
either 0.1% DMSO or different concentrations of zerumbone (10, 20
and 30 μmol/L) for 24 hours. The live cells were immediately viewed
under an inverted Nikon ECLIPSE Ti (Tokyo, Japan) fluorescent mi‐
croscope after adding AO (2 μg/mL), and the images were captured
using the CoolSNAP digital camera. 2.3 | Fluorescent labelling of zerumbone Zerumbone does not have any characteristic fluorescence; hence,
we labelled it with fluorescein isothiocyanate (FITC) by conjugating
zerumbone oxime with fluoresceinthiocarbamyl ethylenediamine ASHRAF et al. 3 of 17 3 of 17 2.4 | Cell culture and cell proliferation assay Human cervical cancer cell line (HeLa) and mouse fibroblast cell line
(L929) were obtained from the National Centre for Cell Science,
Pune, India. The cells were grown in 25 cm2 tissue culture flasks in
a humidified atmosphere containing 5% CO2 and 95% air at 37°C. HeLa cells were grown and maintained in minimal essential me‐
dium (MEM) supplemented with 10% (v/v) FBS, sodium bicarbonate
and antibiotic solution containing 100 units of penicillin, 100 µg
of streptomycin and 0.25 µg of amphotericin B per mL. L929 cells
were grown and maintained in Dulbecco’s modified Eagle’s medium
(DMEM) supplemented with 10% FBS, sodium bicarbonate and anti‐
biotic solution. The cytotoxic effect of zerumbone on HeLa cells and
L929 cells was determined in 96‐well tissue culture plates using the
standard SRB assay.27,28 2.13 | Determination of Kd For calculating the dissociation constant, tubulin (1 µmol/L) was in‐
cubated with varying concentrations of zerumbone in PEM buffer at
37°C for 30 minutes. The samples were excited at 295 nm to spe‐
cifically excite the tryptophan residues of tubulin, and the emission
spectrum was recorded. The fraction of binding sites (X) occupied
by zerumbone was evaluated using the equation X = (Fo − Fc)/ΔFmax,
where Fo and Fc represent the fluorescence intensity of tubulin in the
absence and presence of varying concentrations of zerumbone. The
maximum change in the fluorescence intensity, ΔFmax, was calculated
from the Y‐intercept of the graph 1/ΔF vs 1/[zerumbone]. Assuming
a single binding site of zerumbone per tubulin dimer, the dissociation
constant (Kd) was estimated using the relationship, 1/X = 1 + (Kd/Lf),
where Lf is the concentration of free zerumbone.30,39 The experi‐
ment was repeated three times. 2.14 | Sedimentation assay The in vitro microtubule sedimentation assay was performed to
detect the effect of zerumbone on the polymerization of tubulin. Different concentrations of zerumbone were incubated with tubu‐
lin (12 μmol/L) in PEM buffer containing 0.8 mol/L glutamate and
1 mmol/L GTP at 37°C for 1 hour. The reaction mixture was then
subjected to centrifugation at 50 000 × g for 1 hour. The superna‐
tant and pellet were collected separately, and the protein concentra‐
tion in the supernatant was measured using Bradford assay.30 protein was stored in aliquots at −80°C until further use. All the
experiments with tubulin were performed in PEM buffer (25 mmol/L
PIPES, 1 mmol/L EGTA, 3 mmol/L MgCl2, pH 6.8). anti‐mouse IgG conjugated to Alexa Fluor 568. The DNA was stained
with Hoechst 33342 to visualize the DNA. Gamma tubulin staining
was performed using rabbit monoclonal anti‐gamma tubulin antibody
at 1:1000 dilutions as described earlier.27,32,33 Immunofluorescence
images were acquired using the CoolSNAP digital camera and were
processed by using ImageJ (NIH, USA). 2.10 | Prime/MM‐GBSA scoring The binding energy (ΔG binding) of zerumbone to each protein
was calculated using Prime/MM‐GBSA method (Schrodinger,
LLC) using the equation ΔGbind = ΔEMM + ΔGSolv + ΔGSA, where
ΔEMM is the difference in the minimized energies between pro‐
tein‐zerumbone complex and the sum of the minimized energies
of unbound protein and zerumbone, ΔGSolv is the difference in the
GBSA solvation energy of protein‐zerumbone complex and the
sum of the solvation energies of unbound protein and zerumbone,
and ΔGSA is the difference in the surface area energies of complex
and the sum of the surface area energies of unbound protein and
zerumbone. The Prime/MM‐GBSA calculations were performed
based on the protein‐zerumbone complexes obtained from Glide
docking using OPLS‐2005 forcefield and VSGB2.0 solvent model. 2.9 | Molecular docking study The interaction of zerumbone with tubulin dimer and other cell divi‐
sion proteins such as Eg5, Aurora A, Plk1, Kif2 and Nek2 was analysed
through molecular docking. The 3D crystal coordinates of tubulin heter‐
odimer (5J2U), Eg5 motor domain (1X88), Aurora A (5LXM), Polo‐box do‐
main of Plk1(4WHK), Kif2 motor domain (2GRY) and Nek2 (2W5A) were
obtained from Protein Data Bank (http://www.rcsb.org/pdb/home/
home.do). The protein preparation wizard of Glide, Schrodinger Maestro
v11.1 (Schrodinger, LLC, New York, NY, USA) was used to prepare the
protein structures. The structures were refined by adding missing side
chains and removing water molecules, ions, cofactors and inhibitors. Then, they were energy‐minimized until the average RMSD of the non‐
hydrogen atoms reached 0.3 Å. The 3D structure of zerumbone was
obtained from PubChem (https://pubchem.ncbi.nlm.nih.gov, PubChem
CID: 5470187). The low energy conformations of zerumbone were pre‐
pared using LigPrep module of Schrodinger. The molecular docking of
zerumbone with different proteins was performed using Grid‐Based
Ligand Docking with Energetics (Glide) module of Schrodinger. A grid
box covering the entire protein was generated to perform blind docking
of zerumbone using extra precision (XP) mode of Glide script. The best
docking pose was selected based on Glide scoring function, Glide en‐
ergy, Emodel energy, EvdW and Ecoul using Glide XP Visualizer. 2.12 | Spectral measurements All the absorbance measurements were carried out in Systronics
AU‐2701 UV‐visible double beam spectrophotometer using a
cuvette of 1 cm path length. FTIR spectrometer (PerkinElmer,
PerkinElmer Inc., Waltham, MA, USA) was used to record the FTIR
spectra. The fluorescence measurements are performed in JASCO
FP‐8300 spectrofluorometer (Tokyo, Japan) equipped with a ther‐
mostatted cell holder directly connected to a circulating water
bath for maintaining constant temperature. For all the fluorescence
measurements, the inner filter correction was done according to the
equation F = Fobs × antilog [(Aex + Aem)/2], where Aex is the absorb‐
ance of ligand at the excitation wavelength, and Aem is the absorb‐
ance of ligand at the emission wavelength.37,38 The background
fluorescence exhibited by buffer and free ligands was routinely sub‐
tracted from all the samples. 2.11 | Purification of tubulin Goat brain tubulin was isolated by two cycles of polymerization
and depolymerization in the presence of glutamate as described
earlier.34,35 Bradford assay was used to estimate the tubulin
concentration using bovine serum albumin as the standard.36 The 4 of 17 4 of 17 ASHRAF et al. 2.5 | Cellular uptake of fluorescein zerumbone
(fluorozerumbone) HeLa cells and L929 cells (∼1 × 106 cells/mL) were incubated with
0.1% DMSO or 40 µmol/L zerumbone or 40 µmol/L fluorozerumbone
for 4 hours. After the incubation period, the cells were collected
by trypsinization and counted. Cells were then centrifuged at 800
x g for 10 minutes and washed three times with cold PBS. The cell
pellet was then dried and suspended in 800 µL of methanol and
sonicated till fluorozerumbone is completely extracted into the
methanol fraction. The cell lysate was centrifuged at 2000 x g for
5 minutes. The absorbance and fluorescence spectra (excitation
at 494; emission at 500‐600) of the supernatant containing
flourozerumbone were recorded. The total cellular uptake was 2.17 | Determination of combination index HeLa cells were incubated with zerumbone or vinblastine or pacli‐
taxel alone or zerumbone (5, 10 and 12 µmol/L) and paclitaxel (5 and
10 nmol/L) in combination or zerumbone (5, 10 and 12 µmol/L) and
vinblastine (6 and 1.2 nmol/L) in combination for 24 hours. The CI
was calculated to understand the effect of zerumbone on the cy‐
totoxic activity of paclitaxel and vinblastine. The CI was calculated
using the Chou and Talalay41 equation: 3.1 | Isolation and characterization Colchicine has a very weak fluorescence in aqueous buffers but ex‐
hibits a strong fluorescence after binding to tubulin.40 This fluores‐
cence property of colchicine is exploited in binding site competition
assays to predict the binding site of unknown compounds. Tubulin
(1 μmol/L) was incubated with colchicine (10 μmol/L) for 1 hour at
37°C to form a stable tubulin‐colchicine (T‐C) complex which has sev‐
eral fold higher fluorescence than unbound colchicine.40 Different
concentrations of zerumbone were then added to the T‐C complex
and incubated for further 30 minutes at 37°C. The samples were
excited at 360 nm, and the emission spectra were recorded.30,39
Alternatively, competition assay was also done using the fluores‐
cence of the tubulin‐fluorozerumbone complex. Tubulin (2 µmol/L)
was incubated with 10 µmol/L fluorozerumbone and incubated
for 20 minutes at 37°C. After the incubation period, different con‐
centrations of zerumbone (10 µmol/L or 20 µmol/L), or 20 µmol/L
colchicine, or 5 µmol/L vinblastine were added to the tubulin‐
fluorozerumbone complex and incubated under dark at 37°C for fur‐
ther 30 minutes. The samples were excited at 494 nm, and emission
spectra were recorded. The competition assay was repeated using
EDF in place of fluorozerumbone with different concentrations of
zerumbone (10 and 20 µmol/L) and 20 µmol/L colchicine. The methanolic extract of 1 kg of fresh Zingiber zerumbet extract
yielded 1.8 g of crystalline zerumbone. The isolated product showed
M+ ion at 219 when analysed by LC‐MS (Figure 1A). The compound
was further analysed and confirmed by 1H‐NMR (500 MHz, CDCl3)
δ = 6‐6.03 (1H,d), 5.95 (1H,s), 5.88‐5.84(1H,s), 5.23‐5.27(1H,m),
2.47‐2.28 (3H,m), 2.19‐2.25(2H,d), 1.88‐1.91(3H,d), 1.54 (3H, s),
1.79(3H, s), 1.2(3H,s), 1.07(3H,s) (Figure 1B). 3.2 | Selective toxicity of zerumbone on the
proliferation of cancer cells Zerumbone inhibited the proliferation of human cervical cancer cell
line (HeLa) in a concentration‐dependent manner. After 24 hours
of incubation, the half‐maximal inhibitory concentration (IC50)
of zerumbone on HeLa cells was found to be 14.2 ± 0.5 μmol/L
(Figure 2A). Cells treated with 20, 40, 60 and 80 μmol/L zerum‐
bone inhibited the cell proliferation by 63%, 86%, 98% and 100%,
respectively. We analysed the effect of zerumbone on L929 cells
since it is generally used as an ideal in vitro model to test the chemi‐
cal toxicity, drug cytotoxicity and material biocompatibility.43,44
Interestingly, zerumbone exhibited less cytotoxic effect on nor‐
mal mouse fibroblast cells (L929) with an IC50 of 30.5 ± 1.5 μmol/L
(Figure 2B). Cells treated with 10, 20, 40 and 80 μmol/L zerumbone
inhibited the growth of L929 cells by 20%, 33%, 70% and 83%,
respectively. Different concentrations of zerumbone were added to 12 μmol/L
tubulin in the polymerization buffer containing 25 mmol/L PIPES,
1 mmol/L EGTA, 3 mmol/L MgCl2 and 0.8 mol/L glutamate. The
assembly reaction was initiated by adding 1 mmol/L GTP and
incubated at 37°C.38 The polymerization of tubulin was monitored
by light scattering at 550 nm for 15 minutes using JASCO FP‐8300
spectrofluorometer (Tokyo, Japan) connected with circulating water
bath maintained at 37°C. where Dm is the median dose, fa is the fraction affected, and fu is
the fraction unaffected (fu = 1 − fa). The median dose (Dm) was cal‐
culated as described earlier.42 A CI < 1 indicates synergism, CI = 1
shows additivity, and CI > 1 specifies antagonism. HeLa cells grown
on coverslips in 24‐well tissue culture plate were treated with zerum‐
bone in combination with paclitaxel or vinblastine and processed to
visualize microtubules and DNA. 2.15 The effect of zerumbone on the assembly of microtubule was also
analysed by monitoring the kinetics of tubulin polymerization. 5 of 17 ASHRAF et al. 3.3 | Internalization of fluorozerumbone by
HeLa and L929 cells The fluorozerumbone internalized by HeLa and L929 cells treated
with 40 µmol/L fluorozerumbone for 4 hours was extracted
using methanol and quantified based on the absorption spectra
of the standard fluorozerumbone. We found that the uptake of
fluorozerumbone by HeLa cells was 26 nmol/cell and that by L929
cells was 14.8 nmol/cell. The methanolic extracts were further
subjected to fluorometric analysis by exciting them at 494 nm as
explained in Materials and methods. As shown in Figure 2C, the
fluorescence intensity of the extract obtained from HeLa cells was
16% higher than that of the extract obtained from L929 cells. These
results indicate that the cellular uptake of fluorozerumbone is much
higher in tumour cells. CI=[(D) 1∕(Dx) 1]+[(D) 2∕(Dx) 2] CI=[(D) 1∕(Dx) 1]+[(D) 2∕(Dx) 2] Where, (D)1 and (D)2 are the concentrations of drug 1 (zerum‐
bone) and drug 2 (vinblastine or taxol) in combination that produces
a given effect, (Dx)1 and (Dx)2 are the concentrations of drug 1 and
drug 2 that also produces the same effect when used alone. (Dx),
the concentration of the drug which produces any particular effect,
was calculated from the median effect equation of the Chou and
Talalay41: (Dx)=Dm
[fa∕fu
]1∕m (Dx)=Dm
[fa∕fu
]1∕m 6 of 17 |
ASHRAF et al
3.4 | Zerumbone‐induced apoptosis in HeLa cells
Acridine orange staining is a common method used to detect
apoptotic cell death. After 24 hours, the control cells remained
which indicated the characteristic of apoptosis (Figure 3A)
Approximately 20% and 36% apoptotic cells were detected in
the cells treated with 10 and 20 μmol/L zerumbone for 24 hours
(Figure 3B). At higher concentration of zerumbone (30 μmol/L)
FI G U R E 1 Characterization of zerumbone isolated from Zingiber zerumbet. A, LC‐MS analysis of zerumbone. Zerumbone exhibited an M+
ion at 219.17. The inset shows the chemical structure of zerumbone [(2E,6E,10E)‐2,6,9,9‐tetramethylcycloundeca‐2,6,10‐trien‐1‐one]. B, 1H‐
NMR spectrum of zerumbone (500 MHz, CDCl3) 6 of 17 |
ASHRAF et al. ASHRAF et al. 6 of 17 FI G U R E 1 Characterization of zerumbone isolated from Zingiber zerumbet. A, LC‐MS analysis of zerumbone. Zerumbone exhibited an M
ion at 219.17. The inset shows the chemical structure of zerumbone [(2E,6E,10E)‐2,6,9,9‐tetramethylcycloundeca‐2,6,10‐trien‐1‐one]. B, 1H FI G U R E 1 Characterization of zerumbone isolated from Zingiber zerumbet. A, LC‐MS analysis of zerumbone. Zerumbone exhibited an M+
ion at 219.17. The inset shows the chemical structure of zerumbone [(2E,6E,10E)‐2,6,9,9‐tetramethylcycloundeca‐2,6,10‐trien‐1‐one]. 3.3 | Internalization of fluorozerumbone by
HeLa and L929 cells B, 1H‐
NMR spectrum of zerumbone (500 MHz, CDCl3) which indicated the characteristic of apoptosis (Figure 3A). Approximately 20% and 36% apoptotic cells were detected in
the cells treated with 10 and 20 μmol/L zerumbone for 24 hours
(Figure 3B). At higher concentration of zerumbone (30 μmol/L),
~50% cells were found to be in the later stages of apoptotic cell
death with the characteristic features such as membrane blebbing migration of cancer cells even at concentrations lower than the
IC50. After 24 hours, the control cells have shown 30% migration;
during the same time, 5, 10 and 15 μmol/L zerumbone exhibited
20%, 11% and 9% migration, respectively. After 48 hours,
the control cells displayed ~56% wound healing, and in the
cells treated with zerumbone 5, 10 and 15 μmol/L, the wound
healing was found to be 41%, 36% and 15%, respectively. Similar
concentration‐dependent inhibition of wound healing was
observed after 72 hours of treatment, and the complete wound
healing was observed in control cells after 96 hours, while 63%,
56% and 35% wound healing were observed in the cells treated
with 5, 10 and 15 μmol/L zerumbone (Figure 4B). The results
suggest that zerumbone can effectively prevent the migration of
cancer cells in a concentration‐ and time‐dependent manner even
at concentrations lower than the IC50. 3.6 | Zerumbone caused depolymerization of
interphase and mitotic microtubules in HeLa cells and
blocked the cells at mitosis Zerumbone induced significant depolymerization of interphase
and mitotic microtubules in HeLa cells. The control cells treated
with vehicle (0.1% DMSO) exhibited typical microtubule or‐
ganization with the microtubules spread over the entire cell. Zerumbone at the IC50 (15 μmol/L) and lower concentrations
such as 10 μmol/L did not alter the interphase microtubule net‐
work (Figure 5A). However, higher concentrations such as 30
and 100 μmol/L caused significant disruption of the interphase
microtubules with shrunken cells. Similarly, zerumbone did not
cause significant disruption of the mitotic spindles at the IC50
(15 μmol/L) and lower concentrations but perturbed the organi‐
zation of the chromosomes at the metaphase plate (Figure 5B). Surprisingly, we found that significant number of the mitotic cells
had monopolar spindles. Hence, the mitotic cells were further
analysed by observing the centrosome using gamma tubulin stain‐
ing. As shown in Figure 5C, zerumbone inhibited the centrosome
separation and caused the formation of monopolar spindles with
rosette‐like chromosomes around it. The estimation of HeLa cells
blocked at mitosis 24 hours of post‐treatment with zerumbone
revealed a strong mitotic block with both bipolar and monopolar
spindles. In the cells treated with 10, 15, and 30 μmol/L zerum‐
bone, the number of cells with monopolar spindles was calculated
to be 3%, 7% and 27%, respectively, and the MI, which is the ratio
of the total number of the mitotic cells to the total cells, was found
to be 15%, 20% and 34%, respectively. Under similar conditions,
the MI of the control cells was 3.5% (Figure 5D). FI G U R E 2 Zerumbone differentially inhibited the proliferation
of HeLa (A) and L929 (B) cells. The inhibition of proliferation was
determined by the standard SRB assay after treating the cells with
different concentration of zerumbone. C, Fluorescence spectra of
fluorozerumbone extracted from (▲) L929 and (∆) HeLa cells treated
with 40 µmol/L fluorozerumbone. Control L929 (●) and HeLa (○)
cells were treated with 0.1% DMSO and cell shrinkage. Under similar conditions, the number of
apoptotic cells in the control was 8%. 3.4 | Zerumbone‐induced apoptosis in HeLa cells Acridine orange staining is a common method used to detect
apoptotic cell death. After 24 hours, the control cells remained
viable and healthy and the zerumbone‐treated cells displayed
brightly stained hypercondensed nucleus and membrane blebbing, ASHRAF et al. FI G U R E 2 Zerumbone differentially inhibited the proliferatio
of HeLa (A) and L929 (B) cells. The inhibition of proliferation was
determined by the standard SRB assay after treating the cells wi
different concentration of zerumbone. C, Fluorescence spectra o
fluorozerumbone extracted from (▲) L929 and (∆) HeLa cells trea
with 40 µmol/L fluorozerumbone. Control L929 (●) and HeLa (○)
cells were treated with 0.1% DMSO 7 of 17 ASHRAF et al. 3.5 | Zerumbone inhibited the migration of HeLa
cells in a concentration‐dependent manner Since zerumbone treatment produced cells with mitotic
abnormalities, we investigated the interaction of the zerumbone
with cell division proteins such as tubulin, Eg5, Aurora A, Plk1, Wound healing assay was used to check the migration of HeLa
cells upon treatment with different concentrations of zerumbone. As shown in Figure 4A, zerumbone effectively inhibited the 8 of 17 |
ASHRAF et al
FI G U R E 3 Zerumbone‐induced apoptosis in HeLa cells in a dose‐dependent manner. A, HeLa cells (0.5 × 105 cells/mL) were incubated
with different concentrations of the zerumbone for 24 h. After the incubation period, AO was added and the live cells were viewed under
the fluorescent microscope using FITC filter. Apoptotic cells appeared brightly stained with hypercondensed nucleus. Scale bar represents
20 μm. B, Graph represents percentage of apoptotic cells observed after 24 h of treatment with different concentrations of zerumbone. At
least, 600 cells were counted for each concentration. The experiment was repeated thrice, and data represent mean ± SD
(A)
(B) ASHRAF et al. 8 of 17 (B) (B) FI G U R E 3 Zerumbone‐induced apoptosis in HeLa cells in a dose‐dependent manner. A, HeLa cells (0.5 × 105 cells/mL) were incubated
with different concentrations of the zerumbone for 24 h. After the incubation period, AO was added and the live cells were viewed under
the fluorescent microscope using FITC filter. Apoptotic cells appeared brightly stained with hypercondensed nucleus. Scale bar represents
20 μm. B, Graph represents percentage of apoptotic cells observed after 24 h of treatment with different concentrations of zerumbone. At
least, 600 cells were counted for each concentration. The experiment was repeated thrice, and data represent mean ± SD spectrofluorometer by measuring the intrinsic tryptophan
fluorescence of tubulin. Zerumbone quenched the intrinsic
fluorescence of tubulin in a concentration manner (Figure 7A). Figure 7B shows the change in the fluorescence intensity of
tubulin incubated with different concentrations of zerumbone
and the analysis of the reduction in the fluorescence of tubulin
as a function of zerumbone concentration yielded a dissociation
constant (Kd) of 4 μmol/L (Figure 7B inset). Kif2 and Nek2 that play role in the organization of mitotic
spindle and organization of chromosomes at the metaphase plate
through computational docking. Results of the docking analysis
with tubulin implied that zerumbone bound at the interface of
the α/β‐tubulin dimer with a Glide docking score of −3.608 kcal/
mol (Figure 6). 3.5 | Zerumbone inhibited the migration of HeLa
cells in a concentration‐dependent manner The MM‐GBSA scoring has shown that zerumbone
has bound to tubulin dimer at this position with a strong affinity
(ΔG = −50.638 kcal/mol). The number of hydrogen bonds and
the list of protein residues interacting with zerumbone are
given in the Table 1. The interaction between tubulin dimer and
zerumbone was stabilized by one hydrogen bond with Valβ355
and many hydrophobic interactions with residues Valα177,
Proα222, Tyrα224, Leuβ248 and Metβ325. Comparative analysis
of the binding sites of the ligands such as paclitaxel,45 colchicine46
and vinblastine47 on the tubulin heterodimer gave an inference
that zerumbone binding site partially overlaps with the DAMA
colchicine binding site. The residues that both zerumbone and
DAMA colchicine interact in the tubulin heterodimer are Serα178,
Thrα179, Leuβ248 and Alaβ354 (Table 1). The docking results of
other cell division proteins such as Eg5, Aurora kinase A, Polo‐
like kinase 1 (Plk1), Kif2A and NIMA‐related kinase 2 (Nek2) are
shown in Figure 6B‐F. Based on the Glide docking score (Table 1)
and MM‐GBSA scoring, it is possible to speculate that Eg5 and
Aurora kinase A could also be the potential target for zerumbone
in addition to tubulin. 3.9 | Zerumbone inhibited the polymerization of
tubulin in vitro The effect of zerumbone on tubulin assembly was analysed
by using the sedimentation assay and the light scattering
assay. Tubulin (12 µmol/L) was allowed to polymerize in the
presence or absence of different concentrations of zerumbone
as explained in Materials and methods. Zerumbone inhibited the
polymerization of tubulin in a concentration‐dependent manner
(Figure 8A). The polymer mass of tubulin treated with 20, 40 and
80 µmol/L zerumbone was found to be 82.6%, 78.5% and 74.3%,
respectively, compared to the control, which is considered as
100%. The kinetics of tubulin polymerization upon treatment
with zerumbone was analysed using light scattering assay. Similar
to the results obtained in the sedimentation assay, light scattering
assay also indicated inhibition of tubulin polymerization by
zerumbone (Figure 8B), in a concentration‐dependent manner. At 20 μmol/L zerumbone, the polymer mass was inhibited by
12%, and at 40 μmol/L, the polymer mass was decreased by 20%
compared to control. The steady‐state reading at the saturation
point of polymerization was taken for the calculation of inhibition
of polymerization. FI G U R E 4 Effect of zerumbone on the migration of HeLa cells. A, Migration of HeLa cells in the absence and presence of the
indicated concentrations of zerumbone at different time intervals
(24, 48 72 and 96 h) was monitored as mentioned in Section 2. The scale bar represents 200 µm. B, Percentage of cell migration
at specific time intervals in the absence and presence of 5, 10 and
15 µmol/L zerumbone was calculated as described in Section 2. The data shown are mean ± SD of three independent experiments
(*P < 0.001; ‡ P < 0.01; †P < 0.05)
(A)
(B) (A) Addition of 20 µmol/L colchicine to the tubulin‐fluorozerumbone
reduced the fluorescence by 44%, confirming that colchicine and
zerumbone might share their binding site. Since zerumbone induced
depolymerization of microtubules, we also analysed its competition
with vinblastine and found that vinblastine (5 µmol/L) enhanced
the fluorescence of fluorozerumbone by 44%. The results suggest
that binding of vinblastine to tubulin might stabilize the tubulin‐
fluorozerumbone complex and both the ligands could bind simulta‐
neously to tubulin. To confirm that the observed effects are not due
to the fluorescence tag (EDF), we repeated the same competition
assay with EDF in place of fluorozerumbone and found that both
zerumbone and colchicine could not reduce the EDF fluorescence
(Figure 9C). (B) 3.11 | Zerumbone inhibited the proliferation of
HeLa cells synergistically in combination with
vinblastine and paclitaxel The scale bar represents 200 µm. B, Percentage of cell migration
at specific time intervals in the absence and presence of 5, 10 and
15 µmol/L zerumbone was calculated as described in Section 2. The data shown are mean ± SD of three independent experiments
(*P < 0.001; ‡ P < 0.01; †P < 0.05) 9 of 17 9 of 17 ASHRAF et al. 3.11 | Zerumbone inhibited the proliferation of
HeLa cells synergistically in combination with
vinblastine and paclitaxel Vinblastine and paclitaxel are FDA‐approved clinically used drugs
for the treatment of various types of tumours.48 The effect of
zerumbone was similar to other classical antimitotic drugs; hence,
to further explore its potential cancer therapeutics we performed
the combination studies with vinblastine and paclitaxel. Vinblastine
and paclitaxel inhibited the HeLa cell proliferation with an IC50 of
1.2 and 10 nmol/L and a median dose of 1.10 and 9.21 nmol/L,
respectively (Figure 10A‐D). The logarithmic plot of the cytotoxic
data yielded a median dose of 13.68 µmol/L for zerumbone
(Figure 10E). Zerumbone synergistically inhibited the proliferation
of HeLa cells when combined with vinblastine and paclitaxel. When 0.6 nmol/L vinblastine was combined with zerumbone of
5, 10 and 12 µmol/L, the inhibition of proliferation of HeLa cell
was determined to be 65%, 82% and 86%, respectively. When
5, 10 and 12 µmol/L zerumbone was combined with 1.2 nmol/L
vinblastine, the inhibition of proliferation was found to be 84%,
98% and 100%, respectively (Figure 10F). The combination
index was calculated based on the Chou and Talalay equation as
explained in Materials and methods to demonstrate quantitatively
the relationship between the combination of zerumbone
with vinblastine and paclitaxel. The CI for the combination of
0.6 nmol/L vinblastine and 5, 10 and 12 µmol/L zerumbone was
calculated to be 0.55, 0.48 and 0.45, respectively (Figure 10G)
and CI for the combination of 1.2 nmol/L vinblastine with 5 and
10 µmol/L zerumbone was found to be 0.39 and 0.10, respectively
(Figure 10G). Combination of 5 nmol/L paclitaxel with 5, 10 and
12 µmol/L of zerumbone inhibited the proliferation of HeLa cells
by 82%, 85% and 92%, respectively (Figure 10H). When paclitaxel
10 nmol/L was combined with 5 µmol/L zerumbone, the inhibition
of proliferation of HeLa cell was found to be 98%. The CI of 5, 10,
and 12 µmol/L zerumbone with 5 nmol/L paclitaxel was calculated
to be 0.18, 0.25 and 0.15, respectively. When 5 µmol/L zerumbone
was combined with 10 nmol/L paclitaxel, the CI was calculated
to be 0.04 (Figure 10I). All the calculated combination indices
were found to be lesser than 1, signifying that the combination FI G U R E 4 Effect of zerumbone on the migration of HeLa cells. A, Migration of HeLa cells in the absence and presence of the
indicated concentrations of zerumbone at different time intervals
(24, 48 72 and 96 h) was monitored as mentioned in Section 2. 3.8 | Binding of zerumbone to tubulin Results from the cell culture studies and docking analysis indicated
that tubulin could be one of the primary targets for Zerumbone. Hence, binding of zerumbone on tubulin was analysed using 3.10 | Zerumbone competes with colchicine for the
binding site on tubulin The data represent mean ± SD FI G U R E 5 Effect of different concentrations of zerumbone on interphase and mitotic microtubules in HeLa cells. A, HeLa cells were
incubated with the indicated concentrations of the zerumbone for 24 h. Microtubules (red) and DNA (blue) were visualized as mentioned
in Section 2. Scale bar represents 20 μm. B, Effect of zerumbone on the spindle microtubules and chromosome organization in HeLa
cells. Zerumbone induced abnormal spindles and misalignment of chromosomes at the metaphase plate. Scale bar represents 10 μm. C, Zerumbone induced the formation of monopolar spindles in HeLa cells. HeLa cells were incubated with different concentrations of
zerumbone for 24 h, and the cells were then fixed and processed to visualize centrosomes, DNA and microtubules. Scale bar represents
5 μm. D, Zerumbone treatment increased the number of mitotic cells with monopolar spindles in HeLa cells. Percentage of cells with bipola
(black) and monopolar (grey) spindles post‐treatment with zerumbone for 24 h are shown in the graph. Inset shows the percentage of total
mitotic cells. All the experiments were performed three times. The data represent mean ± SD with 10 and 12 µmol/L zerumbone, the MI was found to be 32%
and 37%, respectively (Figure 11B), and when 10 nmol/L paclitaxel
was combined with 10 and 12 µmol/L zerumbone, the MI was in‐
creased to 54% and 60%, respectively, while paclitaxel alone at 5
and 10 nmol/L induced 15% and 31% mitotic block (Figure 11B). In
addition to the enhanced mitotic arrest, the combined addition of
two drugs induced drastic mitotic abnormalities in the organiza‐
tion of the mitotic spindle and alignment of chromosomes (Figures
11C,D). of zerumbone‐vinblastine and zerumbone‐paclitaxel is strongly
synergistic in inhibiting the proliferation of HeLa cells. The effect of the combination of zerumbone with vinblastine
and paclitaxel on mitotic cells was analysed by calculating the MI. Zerumbone synergistically increased the MI in combination with
vinblastine and paclitaxel. As shown in Figure 11A, vinblastine
when used alone induced a mitotic block of 9.4%; however, when
combined with zerumbone 5, 10 and 12 µmol/L, the mitotic block
was increased to 20%, 25% and 31%, respectively. Similarly, vin‐
blastine 1.2 nmol/L when used alone induced a mitotic block of
19%, and when combined with zerumbone 5, 10 and 12 µmol/L,
the mitotic block was found to be increased 28%, 38% and 46%,
respectively. 3.10 | Zerumbone competes with colchicine for the
binding site on tubulin The effect of zerumbone on the binding of colchicine to tubulin
was analysed by monitoring the T‐C fluorescence in the presence
of increasing concentrations of zerumbone. As shown in Figure 9A,
zerumbone quenched the T‐C fluorescence in a concentration‐de‐
pendent manner. Zerumbone at 5 µmol/L quenched the T‐C fluores‐
cence by 29%, and it is clear from the figure that at concentrations
more than 5 µmol/L zerumbone, the fluorescence quenching was
nearly equal to 30%. This suggests that 5 µmol/L zerumbone is the
saturating concentration. Under similar conditions, 40 µmol/L po‐
dophyllotoxin, which is reported to bind on the colchicine site of tu‐
bulin, quenched the fluorescence of T‐C complex by 32%. To further
confirm the binding site of zerumbone on tubulin, we have carried
out the competition assay using fluorozerumbone, zerumbone and
colchicine by analysing the fluorescence exhibited by fluorozerum‐
bone. As shown in Figure 9B, zerumbone 10 µmol/L quenched
the fluorescence of fluorozerumbone by 33%. Approximately 60%
quenching of fluorescence was observed with 20 µmol/L zerum‐
bone, indicating that fluorozerumbone binds to the zerumbone
binding site and labelling with FITC has not altered its binding site. ASHRAF et al. 10 of 17 10 of 17 |
ASHRAF et a
FI G U R E 5 Effect of different concentrations of zerumbone on interphase and mitotic microtubules in HeLa cells. A, HeLa cells were
incubated with the indicated concentrations of the zerumbone for 24 h. Microtubules (red) and DNA (blue) were visualized as mentioned
in Section 2. Scale bar represents 20 μm. B, Effect of zerumbone on the spindle microtubules and chromosome organization in HeLa
cells. Zerumbone induced abnormal spindles and misalignment of chromosomes at the metaphase plate. Scale bar represents 10 μm. C, Zerumbone induced the formation of monopolar spindles in HeLa cells. HeLa cells were incubated with different concentrations of
zerumbone for 24 h, and the cells were then fixed and processed to visualize centrosomes, DNA and microtubules. Scale bar represents
5 μm. D, Zerumbone treatment increased the number of mitotic cells with monopolar spindles in HeLa cells. Percentage of cells with bipolar
(black) and monopolar (grey) spindles post‐treatment with zerumbone for 24 h are shown in the graph. Inset shows the percentage of total
mitotic cells. All the experiments were performed three times. 3.10 | Zerumbone competes with colchicine for the
binding site on tubulin Zerumbone induced a significant hike in the mitotic
cells when combined with paclitaxel, similar to its synergistic ac‐
tivity with vinblastine. When 5 nmol/L paclitaxel was combined 4 | DISCUSSION Several secondary metabolites from plants have successful
application as chemotherapeutic agents either in their unmodified | 11 of 17
ASHRAF et al. FI G U R E 6 Computational docking
analysis of zerumbone with different
cell division proteins. Interaction of
zerumbone with A, tubulin heterodimer
(5J2U), B, Eg5 motor domain (1X88), C,
Aurora A (5LXM), D, Polo‐box domain of
Plk1(4WHK), E, Kif2 motor domain (2GRY)
and F, Nek2 (2W5A)
TA B LE 1 Computational docking analysis of zerumbone with tubulin, Eg5 motor domain, Aurora A, Polo‐box domain of Plk1, Kif2 motor
domain and Nek2 ASHRAF et al. | 11 of 17 TA B LE 1 Computational docking analysis of zerumbone with tubulin, Eg5 motor domain, Aurora A, Polo‐box domain of Plk1, Kif2 motor
domain and Nek2 TA B LE 1 Computational docking analysis of zerumbone with tubulin, Eg5 motor domain, Aurora A, Polo‐box domain of Plk1, Kif2 motor
d
i
d N k2 TA B LE 1 Computational docking analysis of zerumbone with tubulin, Eg5 motor domain, Aurora A, Polo‐box domain of Plk1, Kif2 motor
domain and Nek2 Protein
Docking score
(kcal/mol)
ΔG binding
(kcal/mol)
No. of H‐ bonds
Interacting residues
Tubulin
heterodimer
−3.608
−50.638
1 (Valβ355)
Glnα176, Valα177, Serα178, Thrα179, Argα221, Proα222, Thrα223,
Tyrα224, Glnβ247, Leuβ248, Metβ325, Thrβ353, Alaβ354, Valβ355
Eg5
−4.672
−42.675
1 (Val194)
Ser159, Leu161, Asp187, Arg189, Asn190, Gly193, Val194, Ile195,
Ile196, Leu199, Glu201, Val238, Ser240, Thr242, Lys260, Asn262,
Ile319
Aurora A
−3.657
−50.326
‐
Leu139, Gly140, Lys141, Lys143, Val147, Lys162, Leu210, Gly216,
Thr217, Arg220, Glu260, Asn261, Leu263, Ala273, Asp274
Plk1
−2.636
−30.562
1 (Trp514)
Ser418, Leu435, Phe436, Asn437, Ser439, Thr513, Trp514
Kif2a
−2.590
−30.574
1 (Asn207)
Arg204, Pro205, Asn207, Gly289, Ser290, Gly291, Hie294
Nek2
−2.445
−38.151
‐
Val97, Lys100, Gly101, Glu104, Gln106, Tyr107, Leu108, Asp109,
Phe112
Glide docking score, ΔG binding, number of hydrogen bonds and interacting residues are shown. Glide docking score, ΔG binding, number of hydrogen bonds and interacting residues are shown. form such as paclitaxel, vinblastine, vincristine, camptothecin and
podophyllotoxin or in the synthetically modified form such as
docetaxel, vinorelbine, vinflunine, topotecan and etoposide.49,50 cell culture studies indicated that zerumbone exerted selective
toxicity against HeLa cells compared to L929 cells. The half‐maxi‐
mal inhibitory concentration for L929 (30 μmol/L) cells was 2‐fold
higher than that for the HeLa cells (15 μmol/L). 4 | DISCUSSION To further under‐
stand the preferential killing of HeLa cells, we made an attempt
to measure the quantitative uptake of zerumbone using the flu‐
orescently labelled compound fluorozerumbone. Although there
are several reports on the anti‐cancer activity of zerumbone,14,15
the internalization of zerumbone was not studied as it does not
have a characteristic absorbance or fluorescence. Results from
the internalization studies indicated that the uptake of fluo‐
rozerumbone was higher in the HeLa cells compared to the L929
cells. Thus, this property is highly favourable for zerumbone to Results from LC‐MS and NMR were in conformity with the mo‐
lecular weight (218.34) and the structure of the compound. The
data were in agreement with the earlier published reports.25 Our 12 of 17 |
FI G U R E 7 Zerumbone bound to tubulin and quenched the
intrinsic tryptophan fluorescence in a concentration‐dependent
manner. A, Tubulin (1 µmol/L) was incubated with zerumbone
0 (●), 5 (○), 10 (▼), 20 (∆), 40 (■) and 80 (□) µmol/L for 30 min at
37°C. The samples were then excited at 295 nm, and the emission
spectrum was recorded. B, The change in the intrinsic tryptophan
fluorescence intensity was plotted against different concentrations
of zerumbone. Inset shows the double reciprocal plot, which
yielded a Kd of 4 μmol/L 12 of 17 | ASHRAF et al. 12 of 17 Microtubules play a very important role in cell migration,54 and
most of the potent tubulin‐targeted drugs inhibit the migration of
the cell at concentrations lower than their IC50.55,56 Since zerumbone showed excellent mitotic block and inhibited
the migration of cancer cells, we analysed the effect of zerumbone
on interphase and mitotic microtubules using immunofluorescence
microscopy. In our study, we observed that zerumbone at IC50 in‐
duced moderate depolymerization of interphase microtubules in
HeLa cells, while at 30 μmol/L (2 × IC50) and higher concentrations,
it strongly depolymerized the interphase microtubules. Its effect on
mitotic cells was more visible and dramatic as both bipolar and mo‐
nopolar cells were observed depending on the concentration used. At the IC50, most of the mitotic cells had bipolar spindles, while at
30 μmol/L, most of the mitotic cells exhibited monopolar spindles,
indicating that at the IC50, microtubules are the preferential target;
however, at higher concentrations, it is quite possible that it might
have additional targets. 4 | DISCUSSION At the IC50, most of the mitotic cells had bipolar spindles, while at
30 μmol/L, most of the mitotic cells exhibited monopolar spindles,
indicating that at the IC50, microtubules are the preferential target;
however, at higher concentrations, it is quite possible that it might
have additional targets. Our results are in agreement with previous
report in which zerumbone inhibited the assembly of microtubule
and induced apoptosis in PC‐3 and DU‐145 cells.23 But the capabil‐
ity of zerumbone to induce monopolar spindles was not explored
earlier. The mitotic cells with monopolar spindles had condensed
chromosomes in a rosette‐like configuration similar to that of the
monastrol‐treated cells. Monopolar spindles are generally induced
by drugs, which target the mitotic kinesins and mitotic kinases that
are involved in the organization of mitotic spindle.57 Hence, we
performed the docking analysis with tubulin, Eg5, Aurora kinase,
Plk1, Nek2 and Kif2A. Our molecular docking results indicated that
zerumbone has strong affinity towards Eg5 and Aurora A in addi‐
tion to tubulin. It has been well documented that Eg5 and Aurora A
play important role in the separation of centrosomes and inhibition
of them could result in the formation monopolar mitotic cells.58-60 FI G U R E 7 Zerumbone bound to tubulin and quenched the
intrinsic tryptophan fluorescence in a concentration‐dependent
manner. A, Tubulin (1 µmol/L) was incubated with zerumbone
0 (●), 5 (○), 10 (▼), 20 (∆), 40 (■) and 80 (□) µmol/L for 30 min at
37°C. The samples were then excited at 295 nm, and the emission
spectrum was recorded. B, The change in the intrinsic tryptophan
fluorescence intensity was plotted against different concentrations
of zerumbone. Inset shows the double reciprocal plot, which
yielded a Kd of 4 μmol/L be used as a relatively less toxic, safe and effective chemothera‐
peutic agent. Curcumin, the dihydroxy polyphenol from Curcuma
longa, was also reported to induce selective toxicity in cancer cells
due to preferential uptake by the cancer cells compared to nor‐
mal cells.29 AO staining was used to analyse whether the cytotoxic
effect of zerumbone was due to necrotic cell death or apoptosis. 4 | DISCUSSION Our results are in agreement with previous
report in which zerumbone inhibited the assembly of microtubule
and induced apoptosis in PC‐3 and DU‐145 cells.23 But the capabil‐
ity of zerumbone to induce monopolar spindles was not explored
earlier. The mitotic cells with monopolar spindles had condensed
chromosomes in a rosette‐like configuration similar to that of the
monastrol‐treated cells. Monopolar spindles are generally induced
by drugs, which target the mitotic kinesins and mitotic kinases that
are involved in the organization of mitotic spindle.57 Hence, we
performed the docking analysis with tubulin, Eg5, Aurora kinase,
Plk1, Nek2 and Kif2A. Our molecular docking results indicated that
zerumbone has strong affinity towards Eg5 and Aurora A in addi‐
tion to tubulin. It has been well documented that Eg5 and Aurora A
play important role in the separation of centrosomes and inhibition
of them could result in the formation monopolar mitotic cells.58-60
The nearly equal affinity of zerumbone towards Eg5 and Aurora A
obtained by docking analysis indicated that it might target both the
proteins or any one which can be confirmed only through in vitro
analysis. It is important to note that zerumbone at its IC50 had more
bipolar mitotic cells and at higher concentration such as 30 μmol/L
had more of monopolar mitotic cells. The results suggest that tubulin
could be the primary target of tubulin at the IC50, and it might target
Eg5 or Aurora A or both at 30 μmol/L. At concentrations higher than
30 μmol/L, the number of mitotic cells decreased with simultaneous
increase in the number of apoptotic cells. This behaviour is similar to
other antimitotic drugs, which at higher concentration will activate
the apoptotic pathways much earlier in the cell cycle.32,61,62 Since zerumbone showed excellent mitotic block and inhibited
the migration of cancer cells, we analysed the effect of zerumbone
on interphase and mitotic microtubules using immunofluorescence
microscopy. In our study, we observed that zerumbone at IC50 in‐
duced moderate depolymerization of interphase microtubules in
HeLa cells, while at 30 μmol/L (2 × IC50) and higher concentrations,
it strongly depolymerized the interphase microtubules. Its effect on
mitotic cells was more visible and dramatic as both bipolar and mo‐
nopolar cells were observed depending on the concentration used. 4 | DISCUSSION B, Tubulin (2 μmol/L) was
incubated with 10 μmol/L flourozerumbone for 20 min at 37°C. This was followed by the addition of 0 (●), 10 (○) and 20 (▼)
µmol/L zerumbone to the tubulin‐fluorozerumbone complex. Colchicine 20 (∆) µmol/L and vinblastine 5 (■) µmol/L were used
as positive controls. All the samples were excited at 494 nm, and
emission spectra were recorded. (C) The competition assay was
repeated using EDF in place of fluorozerumbone with different
concentrations of zerumbone 0 (●), 10 (○) 20 (▼) and 20 (∆)
µmol/L colchicine FI G U R E 8 Effect zerumbone on the assembly of tubulin. A, A FI G U R E 8 Effect zerumbone on the assembly of tubulin. A, A
sedimentation assay was carried out with different concentrations
of zerumbone (20, 40 and 80 µmol/L) to determine the percentage
of polymer mass of tubulin. The experiment was done three times,
and the data represent mean ± SD. B, Zerumbone inhibited the
glutamate‐induced polymerization of tubulin. The assembly kinetics
of tubulin in the presence and absence of zerumbone 0 (●), 20 (○)
and 40 (▼) µmol/L was monitored by recording the light scattering
at 550 nm for 15 min as described in Section 2 FI G U R E 8 Effect zerumbone on the assembly of tubulin. A, A
sedimentation assay was carried out with different concentrations
of zerumbone (20, 40 and 80 µmol/L) to determine the percentage
of polymer mass of tubulin. The experiment was done three times,
and the data represent mean ± SD. B, Zerumbone inhibited the
glutamate‐induced polymerization of tubulin. The assembly kinetics
of tubulin in the presence and absence of zerumbone 0 (●), 20 (○)
and 40 (▼) µmol/L was monitored by recording the light scattering
at 550 nm for 15 min as described in Section 2 of tubulin are also analysed in a cell‐free system using goat/bovine
brain tubulin isolated in vitro .37,38 The results from the sedimenta‐
tion assay and the light scattering assay of tubulin polymerization
in the presence of glutamate are in excellent agreement with the
results obtained from the cell culture studies, where zerumbone
caused strong depolymerization of the interphase and mitotic micro‐
tubules in HeLa cells. 4 | DISCUSSION FI G U R E 8 Effect zerumbone on the assembly of tubulin. A, A
sedimentation assay was carried out with different concentrations
of zerumbone (20, 40 and 80 µmol/L) to determine the percentage
of polymer mass of tubulin. The experiment was done three times,
and the data represent mean ± SD. B, Zerumbone inhibited the
glutamate‐induced polymerization of tubulin. The assembly kinetics
of tubulin in the presence and absence of zerumbone 0 (●), 20 (○)
and 40 (▼) µmol/L was monitored by recording the light scattering
at 550 nm for 15 min as described in Section 2 | 13 o
G U R E 9 Competition experiment of zerumbone and
orozerumbone with colchicine, podophyllotoxin and
blastine for the localization of binding site on tubulin. A,
lchicine (10 μmol/L) was allowed to form complex with
bulin (2 μmol/L) for 1 h at 37°C. Different concentrations of
umbone 0 (●), 5 (○), 10 (▼) and 20 (∆) µmol/L were added
the complex. Podophyllotoxin 40 (■) µmol/L was used as a
sitive control. The samples were excited at 360 nm, and the
ission spectrum was recorded. B, Tubulin (2 μmol/L) was
ubated with 10 μmol/L flourozerumbone for 20 min at 37°C ASHRAF et al. 13 of 17 | FI G U R E 8 Effect zerumbone on the assembly of tubulin. A, A
sedimentation assay was carried out with different concentrations
of zerumbone (20, 40 and 80 µmol/L) to determine the percentage
of polymer mass of tubulin. The experiment was done three times,
and the data represent mean ± SD. B, Zerumbone inhibited the
glutamate‐induced polymerization of tubulin. The assembly kinetics
of tubulin in the presence and absence of zerumbone 0 (●), 20 (○)
and 40 (▼) µmol/L was monitored by recording the light scattering
at 550 nm for 15 min as described in Section 2 F I G U R E 9 Competition experiment of zerumbone and
fluorozerumbone with colchicine, podophyllotoxin and
vinblastine for the localization of binding site on tubulin. A,
Colchicine (10 μmol/L) was allowed to form complex with
tubulin (2 μmol/L) for 1 h at 37°C. Different concentrations of
zerumbone 0 (●), 5 (○), 10 (▼) and 20 (∆) µmol/L were added
to the complex. Podophyllotoxin 40 (■) µmol/L was used as a
positive control. The samples were excited at 360 nm, and the
emission spectrum was recorded. 4 | DISCUSSION Since zerumbone induced depolymerization
of microtubules, and the docking analysis predicted colchicine bind‐
ing domain as the binding site of zerumbone, we decided to confirm
the binding site using competition binding assay using two different
approaches. In one method, the change in the fluorescence of col‐
chicine‐tubulin complex upon addition of increasing concentrations
of zerumbone was monitored. In the other approach, the change in
the fluorescence of tubulin‐fluorozerumbone upon addition of dif‐
ferent compounds was monitored. Fluorescently labelled molecules
are widely used to characterize the binding interactions and binding
site of the ligand on the protein.63 Unlabelled zerumbone effectively
quenched the fluorescence of the T‐C complex; similarly, colchicine
could quench the fluorescence of fluorozerumbone through com‐
petitive displacement, indicating that fluorozerumbone, zerumbone
and colchicine have overlapping binding sites. To further confirm F I G U R E 9 Competition experiment of zerumbone and F I G U R E 9 Competition experiment of zerumbone and
fluorozerumbone with colchicine, podophyllotoxin and
vinblastine for the localization of binding site on tubulin. A,
Colchicine (10 μmol/L) was allowed to form complex with
tubulin (2 μmol/L) for 1 h at 37°C. Different concentrations of
zerumbone 0 (●), 5 (○), 10 (▼) and 20 (∆) µmol/L were added
to the complex. Podophyllotoxin 40 (■) µmol/L was used as a
positive control. The samples were excited at 360 nm, and the
emission spectrum was recorded. B, Tubulin (2 μmol/L) was
incubated with 10 μmol/L flourozerumbone for 20 min at 37°C. This was followed by the addition of 0 (●), 10 (○) and 20 (▼)
µmol/L zerumbone to the tubulin‐fluorozerumbone complex. Colchicine 20 (∆) µmol/L and vinblastine 5 (■) µmol/L were used
as positive controls. All the samples were excited at 494 nm, and
emission spectra were recorded. (C) The competition assay was
repeated using EDF in place of fluorozerumbone with different
concentrations of zerumbone 0 (●), 10 (○) 20 (▼) and 20 (∆)
µmol/L colchicine F I G U R E 9 Competition experiment of zerumbone and
fluorozerumbone with colchicine, podophyllotoxin and
vinblastine for the localization of binding site on tubulin. A,
Colchicine (10 μmol/L) was allowed to form complex with
tubulin (2 μmol/L) for 1 h at 37°C. Different concentrations of
zerumbone 0 (●), 5 (○), 10 (▼) and 20 (∆) µmol/L were added
to the complex. Podophyllotoxin 40 (■) µmol/L was used as a
positive control. 4 | DISCUSSION Apoptotic cells will appear brightly stained, hypercondensed and
often fragmented chromatin in spherical or irregular shapes under
fluorescent microscope.52 It was clearly evident that by the end
of one cell cycle, most of the zerumbone‐treated cells underwent
apoptosis since their characteristics were similar to those of the
apoptotic cells.52 Molecules preventing metastasis are highly
valuable in cancer chemotherapy as they can prevent the cancer
cells spreading to other tissues. Results from the cell migration
assays indicate that zerumbone strongly inhibited the migration
of cancer cells at 5 and 10 μmol/L, which are much lower concen‐
trations than its IC50. This result is in agreement with the previous
report suggesting the anti‐metastatic property of zerumbone.53 be used as a relatively less toxic, safe and effective chemothera‐
peutic agent. Curcumin, the dihydroxy polyphenol from Curcuma
longa, was also reported to induce selective toxicity in cancer cells
due to preferential uptake by the cancer cells compared to nor‐
mal cells.29 AO staining was used to analyse whether the cytotoxic
effect of zerumbone was due to necrotic cell death or apoptosis. Apoptotic cells will appear brightly stained, hypercondensed and
often fragmented chromatin in spherical or irregular shapes under
fluorescent microscope.52 It was clearly evident that by the end
of one cell cycle, most of the zerumbone‐treated cells underwent
apoptosis since their characteristics were similar to those of the
apoptotic cells.52 Molecules preventing metastasis are highly
valuable in cancer chemotherapy as they can prevent the cancer
cells spreading to other tissues. Results from the cell migration
assays indicate that zerumbone strongly inhibited the migration
of cancer cells at 5 and 10 μmol/L, which are much lower concen‐
trations than its IC50. This result is in agreement with the previous
report suggesting the anti‐metastatic property of zerumbone.53 Results from the docking studies and the cell culture studies in
which zerumbone induced a strong mitotic block and depolymeriza‐
tion of interphase microtubules motivated us to study its effect on
purified tubulin isolated from goat brains using fluorescence spec‐
troscopy. Tubulin heterodimer contains eight tryptophan residues,
and interaction of small molecules will disturb the conformation
of tubulin, leading to change in the intrinsic fluorescence contrib‐
uted by the tryptophan residues.38 Zerumbone binding to tubulin
with a Kd of 4 μmol/L indicates that it has a high affinity for tubulin. The effects of microtubule targeted drugs on the polymerization | 13 of 1
ASHRAF et al. 4 | DISCUSSION All the experiments were
performed three independent times, and
the error bars represent mean ± SD | 15 of 17 | 15 of 17
ASHRAF et al. FI G U R E 11 Zerumbone potentiated the mitotic block in HeLa cells in combination with vinblastine and paclitaxel. Zerumbone increased
the number of mitotic cells in HeLa cells in combination with vinblastine (A) and paclitaxel (B). All the experiments were performed three
times. The data represents mean ± SD. Effect of zerumbone on the spindle microtubule and chromosome alignment in combination with
vinblastine (C) and paclitaxel (D). HeLa cells treated with indicated concentrations of zerumbone, vinblastine and paclitaxel were fixed and
processed for immunofluorescence microscopy as described in Section 2. The scale bar represents 10 μm ASHRAF et al. FI G U R E 11 Zerumbone potentiated the mitotic block in HeLa cells in combination with vinblastine and paclitaxel. Zerumbone increased
the number of mitotic cells in HeLa cells in combination with vinblastine (A) and paclitaxel (B). All the experiments were performed three
times. The data represents mean ± SD. Effect of zerumbone on the spindle microtubule and chromosome alignment in combination with
vinblastine (C) and paclitaxel (D). HeLa cells treated with indicated concentrations of zerumbone, vinblastine and paclitaxel were fixed and
processed for immunofluorescence microscopy as described in Section 2. The scale bar represents 10 μm that the observed quenching of fluorozerumbone is not due to arte‐
fact induced by the labelled molecule, we have used EDF in the place
of fluorozerumbone and found that colchicine could not quench the
FITC fluorescence. microtubule depolymerizing drug colchicine, it exhibited strong
mitotic block when combined with a MT‐stabilizing drug such
as paclitaxel or a MT‐destabilizing drug such as vinblastine at
very low concentrations. It is well documented that microtubule
polymerization inducing agents and microtubule depolymerizing
agents inhibit the cell proliferation at the IC50 only by suppress‐
ing the microtubule dynamics.34,65 It is reasonable to assume
that the synergistic activity of zerumbone with vinblastine and
taxol could be due to the strong suppression of microtubule
dynamics. Combination therapy which uses two or more drugs is excel‐
lent for cancer treatment because it is more effective against
tumour growth and metastasis. 4 | DISCUSSION The samples were excited at 360 nm, and the
emission spectrum was recorded. B, Tubulin (2 μmol/L) was
incubated with 10 μmol/L flourozerumbone for 20 min at 37°C. This was followed by the addition of 0 (●), 10 (○) and 20 (▼)
µmol/L zerumbone to the tubulin‐fluorozerumbone complex. Colchicine 20 (∆) µmol/L and vinblastine 5 (■) µmol/L were used
as positive controls. All the samples were excited at 494 nm, and
emission spectra were recorded. (C) The competition assay was
repeated using EDF in place of fluorozerumbone with different
concentrations of zerumbone 0 (●), 10 (○) 20 (▼) and 20 (∆)
µmol/L colchicine 14 of 17 | 14 of 17 |
ASHRAF et al. 14 of 17 | 14 of 17 |
ASHRAF et
FI G U R E 10 Zerumbone inhibited th
proliferation of HeLa cells in synergism
with vinblastine and paclitaxel. HeLa
cells were treated with different
concentrations of vinblastine (A) and
paclitaxel (B) for 24 h, and the inhibition
of cell proliferation was determined
using SRB assay as described in Section
2. Median effect plot for the inhibition
of cell proliferation by vinblastine
(C), paclitaxel (D) and zerumbone (E). Zerumbone synergistically inhibited the
proliferation of HeLa cells in combinatio
with vinblastine (F) and paclitaxel (G). The combined effect of zerumbone with
vinblastine (H) and paclitaxel (I) on the
proliferation of HeLa cells was calculate
using the Chou and Talalay equation, an
the CI at different drug concentrations
was calculated. All the experiments wer
performed three independent times, and
the error bars represent mean ± SD ASHRAF et al. 14 of 17 FI G U R E 10 Zerumbone inhibited the
proliferation of HeLa cells in synergism
with vinblastine and paclitaxel. HeLa
cells were treated with different
concentrations of vinblastine (A) and
paclitaxel (B) for 24 h, and the inhibition
of cell proliferation was determined
using SRB assay as described in Section
2. Median effect plot for the inhibition
of cell proliferation by vinblastine
(C), paclitaxel (D) and zerumbone (E). Zerumbone synergistically inhibited the
proliferation of HeLa cells in combination
with vinblastine (F) and paclitaxel (G). The combined effect of zerumbone with
vinblastine (H) and paclitaxel (I) on the
proliferation of HeLa cells was calculated
using the Chou and Talalay equation, and
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Wood Waste Turned Into Value Added Products: Thermal Plasticization by Benzylation Process
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ner, Köse, Yürümez, Ümit Yalçın, Akgül: Wood Waste Turned Into Value Added Products ner, Köse, Yürümez, Ümit Yalçın, Akgül: Wood Waste Turned Into Value Added Products Birol Üner1, Gökhan Köse1, Yeşim Yürümez1, Ömer Ümit Yalçın2, Mehmet Akgül3 1 Autori su izvanredni profesor, dodiplomski student i doktorand Sveučilišta Suleyman Demirel, Šumarski fakultet, Zavod za inženjerstvo
proizvoda baziranih na šumi, Isparta, Turska. 2Autor je profesor Sveučilišta Suleyman Demirel, Doktorska škola prirodnih i primijenjenih
znanosti, Isparta, Turska. 3Autor je profesor Sveučilišta Necmettin Erbakan, Fakultet inženjerstva, Zavod za inženjerstvo materijala,
Seydişehir/Konya, Turska. 1 Authors are associate professor, undergraduate student and PhD student at Suleyman Demirel University, Faculty of Forestry Department of
Forest Products Engineering, Isparta, Turkey. 2Author is professor at Suleyman Demirel University, Graduate School of Natural and Applied
Sciences, Isparta, Turkey. 3Author is professor at Necmettin Erbakan University, Engineering Faculty, Material Engineering Department.,
Seydişehir/Konya, Turkey. 1 Authors are associate professor, undergraduate student and PhD student at Suleyman Demirel University, Faculty of Forestry Department of
Forest Products Engineering, Isparta, Turkey. 2Author is professor at Suleyman Demirel University, Graduate School of Natural and Applied
Sciences, Isparta, Turkey. 3Author is professor at Necmettin Erbakan University, Engineering Faculty, Material Engineering Department.,
Seydişehir/Konya, Turkey.
1 Autori su izvanredni profesor, dodiplomski student i doktorand Sveučilišta Suleyman Demirel, Šumarski fakultet, Zavod za inženjerstvo
proizvoda baziranih na šumi, Isparta, Turska. 2Autor je profesor Sveučilišta Suleyman Demirel, Doktorska škola prirodnih i primijenjenih
znanosti, Isparta, Turska. 3Autor je profesor Sveučilišta Necmettin Erbakan, Fakultet inženjerstva, Zavod za inženjerstvo materijala,
Seydişehir/Konya, Turska. Pretvorba drvnog otpada u proizvod dodane
vrijednosti: toplinsko plastificiranje primjenom
procesa benzilacije Original scientifi c paper • Izvorni znanstveni rad
Received – prispjelo: 16. 9. 2015. Accepted – prihvaćeno: 6. 9. 2016. UDK: 630*839.8; 630*812.14; 630*871.1
doi:10.5552/drind.2016.1534 ABSTRACT • Sawdust is usually considered as waste in wood-using industries. These materials can be converted
into value added thermoplastics by means of benzylation. Products can be utilized in different applications where
plastics are used. Thermoplasticization process was carried out with benzyl chloride under different alkaline
conditions, 15, 25 and 35 %, respectively. Alkali ions and concentration affect the substitution reaction. Tailored
material structure was remarkably changed. In order to detect changes, crystallinity index of the materials and
thermal properties were analyzed. In FTIR spectra, the peaks appeared at 698 cm-1 and 740 cm-1, which indicates
the aromatic C-C angular deformation. Multiple peak appears at 3030 cm-1, which indicates benzylation of the ma-
terials. The peak increase can be observed at 1596 cm-1 due to the aromatic deformation. Benzyl groups attached
to hydroxyl to form ether groups increase the peak intensity. As a result of that, hydrogen bond energy changes
and crystallinity of the materials is reduced. This substitution of functional groups changes the decomposition
temperature of modifi ed materials. It reduces the decomposition temperature to between 330 and 350 ºC. Thermo-
gravimetric analysis revealed that modifi ed products were characterized by poorer thermal stability compared to
raw materials. Keywords: thermoplastic resin, hydrogen bond energy, crystallinity, thermal properties SAŽETAK • Piljevina se u drvoprerađivačkoj industriji često smatra otpadom. Međutim, ona se postupkom benzi-
lacije može pretvoriti u termoplastični materijal kao proizvod dodane vrijednosti. Tako dobiveni materijal moguće
je upotrijebiti za različite namjene za koje se obično rabe plastične mase. Toplinski postupak plastifi ciranja pro-
vodi se benzil kloridom pri različitim lužnatim uvjetima (15, 25 ili 35 %). Koncentracija lužnatih iona utječe na
reakciju supstitucije. Struktura materijala značajno se mijenja. Radi otkrivanja promjene, analizirani su indeks
kristaliničnosti materijala i toplinska svojstva. U infracrvenom dijelu spektra pikovi su se pojavili na 698 cm-1 DRVNA INDUSTRIJA 67 (4) 315-322 (2016) 315 ner, Köse, Yürümez, Ümit Yalçın, Akgül: Wood Waste Turned Into Value Added Products i 740 cm-1, što upućuje na aromatske C-C kutne deformacije. Višestruki maksimum pojavljuje se pri 3030 cm-1,
što označava benzilaciju materijala. Zbog aromatske deformacije porast pika može se primijetiti na 1596 cm-1. 1 INTRODUCTİON
1. UVOD plasticity was achieved by benzylation process, and
characterization of the material was accomplished
with crystallinity index and thermogravimetric analy-
sis. Increased utilization of wood results in more
waste due to debarking, cutting, shaving, sanding, etc. How to deal with this waste is an important issue. En-
vironmental concern and defi ciency in natural resourc-
es impose solutions that involve reducing waste and
recovering and recycling this material to conserve nat-
ural resources. Progress has been made in efforts to
reduce waste, but additional works need to be done to
solve this problem. Pretvorba drvnog otpada u proizvod dodane
vrijednosti: toplinsko plastificiranje primjenom
procesa benzilacije Benzilne skupine vezane na hidroksilne skupine tvore eterske skupine i povećavaju intenzitet pika, zbog čega se
mijenja energija vodikove veze i kristaliničnost materijala se smanjuje. Taj postupak supstitucije na funkcionalnim
skupinama mijenja temperaturu razgradnje modifi ciranih materijala. Smanjuje se temperatura dekompozicije na
330 i 350 °C. Termogravimetrijska analiza pokazala je da modifi cirani proizvodi imaju lošiju toplinsku stabilnost
nego sirovina od koje su proizvedeni. Ključne riječi: termoplastične smole, energija vodikove veze, kristaliničnost, toplinska svojstva 2.2.1 Alkali treatment
2.2.1. Lužnata obrada Sawdust was treated with different concentra-
tions of sodium, lithium, potassium hydroxide and
guanidine for 24 hrs at room temperature. After treat-
ment, materials were rinsed and dried at 102±3 ºC and
tested with Fourier transform infrared spectroscopy
(FTIR). 2.1 Materials
2 1
Materijali 2.1 Materials
2.1. Materijali Turkish red pine (TRP) sawdust were obtained
from the Isparta region in Turkey. Materials were dried
under room temperature. Meal samples were prepared
using a Wiley mill and ground to pass various mesh
screens (60-80 mesh). Sawdust and wood shavings are produced by a
number of sectors and this raw material is part of the
municipal waste stream. A tremendous range of prod-
ucts can be obtained from these lignocellulosic waste
materials due to complexity of cell wall structure. Lig-
nocellulosic materials refer to woody and nonwoody
plants that are composed of cellulose, hemicellulose,
and lignin. Chemical properties of these materials
make them suitable for a large number of products. The
utilization of lignocellulosic materials in the produc-
tion of plastic and composites is becoming more and
more attractive (Biswas et al., 2006; Bodirlau et al.,
2008; Çöpür et al., 2007; Ebringerova and Heinze,
2000; Hassan et al., 2001; Hon and Luis, 1989; Hon
and Ou, 1989; Pereira et al.,1997; Rowell, 1990; Su et
al., 2015). 2.2.2 Benzylation process
2.2.2. Proces benzilacije Plastics are being favored for many applications
in place of other materials due to formability, light
weight and strength properties (Hon and Shiraishi,
2001). In order to plasticize, lignocelullosic materials
are chemically modifi ed. Chemical modifi cation of cell
wall polymers is usually carried out either in acidic or
alkali conditions (Chen et al., 2012; Huang et al., 2014;
Qu et al., 2014). Chemical alteration of hydroxyl
groups can reduce the interaction among the chain seg-
ments, creates free volume and changes the thermal
properties of the material (Nakano, 1994). New modi-
fi ed lignocellulosic materials have thermoplasticity,
when large or many side-chains are introduced by
chemical modifi cation. Benzylation process was used similarly to Hon
and Ou process. Dried and ground samples (3 grams)
were pretreated with various alkali concentrations (15,
25, 35 %) for preswelling. The slurry was transferred
to a 250 ml round bottom fl ask containing benzyl chlo-
ride (BC) (27.6 ml). The reaction was conducted at 110
ºC for 6 hours with continuous stirring. The crude ben-
zylated material was collected by fi ltration and exhaus-
tively washed with water to remove any residuals and
fi nally washed with ethanol to remove any residual
benzyl alcohol. The fi nal product was dried overnight
at 40 ºC. 2 RESULTS AND DISCUSSION
2. REZULTATI I RASPRAVA The degree of crystallinity is an important pa-
rameter for plasticization of lignocellulosic materials. Cell wall polymers contain signifi cant amounts of hy-
droxyl groups. Chemical modifi cation of lignocellu-
losic materials usually takes place between these hy-
droxyl groups of the cell wall polymers and chemical
reagent with or without catalyst (Rowell, 1990). Tai-
lored hydroxyl groups can help lignocellulosic materi-
als to exhibit thermoplastic properties due to change in
hydrogen bonding ability. Figure 1 Assignment of hydroxyl bands in FTIR spectra for
untreated Turkish red pine
Slika 1. Raspored hidroksilnih skupina na infracrvenom
dijelu spektra za netretiranu piljevinu od drva turskoga
crvenog bora of the bonded OH groups, and K is a constant (1/ K =
2.625 · 102 kJ). In order to increase reactivity and swell lignocel-
lulosic materials before modifi cation, the material was
treated with alkaline solution. Alkaline treatments con-
tracts microfi brils in the cell wall and changes the di-
mension (Nakano, 2010). The mechanism of microfi -
bril contraction and anisotropic dimensional changes
of cells in wood are treated with aqueous NaOH solu-
tion. As a result, chemical reagent can reach and react
with hydroxyl groups on cell wall polymers. In untreat-
ed materials, there are free, intra and inter hydrogen
bonded hydroxyl groups available (Figure 1). The in-
tramolecular hydrogen bond in lignin can be observed
at 3560-3580 cm-1 (Kondo, 1997; Poletto et al., 2012). Intramolecular hydrogen bond in cellulose appears
around 3430 cm-1 and 3340 cm-1 (Kondo, 1997; Poletto
et al., 2014). After alkaline treatment, these hydroxyl
groups affected crystalline structure changes. Table 1
shows the crystallinity and hydrogen bond energy
change after alkaline treatment (Table 1). Introducing new chemicals to cell wall polymers
causes breaking up of some hydrogen bonds and
changes crystallinity. This change indicates the possi-
bility of plasticization and can be detected with FTIR
spectroscopy. In order to detect the change in crystal-
linity, different peak ratios and hydrogen bond energy
can be used (Poletto et al., 2013; Akgul et al., 2007). The ratio between the heights of the bands at 1370 cm-1
and 2900 cm-1 was used to determine the total crystal-
line index (TCI), and the ratio between the areas of the
bands at 1430 cm-1 and 896 cm-1 was used to determine
lateral order index (LOI) of the materials (Akerholm,
et al., 2004; Ma, 2007). 2.2.4 Thermal properties
2.2.4. Toplinska svojstva 3
2.5
2
1.5
1
0.5
0
3800 3700 3600 3500 3400 3300 3200 3100 3000 2900 2800
Wavenumber / valni broj, cm–1
Absorbance / apsorpcija, a.u
Figure 1 Assignment of hydroxyl bands in FTIR spectra for
untreated Turkish red pine
Slika 1. Raspored hidroksilnih skupina na infracrvenom
dijelu spektra za netretiranu piljevinu od drva turskoga
crvenog bora Perkin Elmer Diamond model thermogravimetric
analyzer (TGA) was used to study the thermal proper-
ties of etherifi ed samples. The heating rate was set at
10 ºC/min and the temperature ranged from 25 to 600
ºC. Measurements on 4 mg samples were carried out
under nitrogen atmosphere (100 ml/min). 2.2.3 Fourier transform infrared spectroscopy
(FTIR) Thermoplastic materials are usually produced
from petrochemicals, which is dependent on limited
source of fossil fuels whose price is fl uctuating and
may be running out. In addition to that, fossil fuels
release gases which cause the greenhouse effect. One
solution is to use tailored renewable resources. There-
fore, the objectives of this paper were to explore the
chemical modifi cation of Turkish red pine sawdust
via etherifi cation to impart thermoplasticity. Thermo- (
)
2.2.3. Fourierova transformacija infracrvene
spektroskopije FTIR spectra were obtained on untreated and
benzylated substrates from KBr pellet using Perkin
Elmer spectrum one model FTIR spectrometer. Each
sample was scanned 10 times between 4000 and 400
cm-1 and changes in the chemical structure were re-
corded. DRVNA INDUSTRIJA 67 (4) 315-322 (2016) 316 ner, Köse, Yürümez, Ümit Yalçın, Akgül: Wood Waste Turned Into Value Added Products DRVNA INDUSTRIJA 67 (4) 315-322 (2016)
317 2 RESULTS AND DISCUSSION
2. REZULTATI I RASPRAVA The energy of the hydrogen
bonds EH for the OH stretching band was calculated
using equation 1: (1) (1) Where νo is the standard frequency correspond-
ing to free OH groups (3650 cm-1), ν is the frequency Where νo is the standard frequency correspond-
ing to free OH groups (3650 cm-1), ν is the frequency There is a small difference in crystallinity index
and hydrogen bond energy (EH) of the materials. TCI There is a small difference in crystallinity index
and hydrogen bond energy (EH) of the materials. TCI Table 1 Crystallinity of alkaline treated materials of Turkish red pine (TRP)
Tablica 1. Kristaliničnost materijala piljevine turskog crvenog bora (TPR) tretiranog lužinama Table 1 Crystallinity of alkaline treated materials of Turkish red pine (TRP)
Tablica 1. Kristaliničnost materijala piljevine turskog crvenog bora (TPR) tretiranog lužinama
Crystallinity of raw materials / Kristaliničnost sirovine
TCI
LOI
EH (kJ)
δʋ (cm-1) 2
δʋ (cm-1) 3
δʋ (cm-1) 6
Untreated Turkish red pine
0.43
0.36
15.75
2.80
2.79
2.77
15 % NaOH TRP sawdust
7.99
2.18
13.59
2.80
2.78
2.77
25 % NaOH TRP sawdust
5.56
2.81
12.87
2.80
2.78
2.77
35 % NaOH TRP sawdust
5.04
21.63
14.38
2.80
2.78
2.77
15 % KOH TRP sawdust
1.50
9.43
16.54
2.80
2.78
2.77
25 % KOH TRP sawdust
2.07
2.73
17.69
2.80
2.78
2.77
35 % KOH TRP sawdust
2.24
10.74
17.04
2.80
2.78
2.77
15 % LiOH TRP sawdust
3.61
1.17
16.54
2.80
2.78
2.77
25 % LiOH TRP sawdust
1.96
1.37
17.98
2.80
2.78
2.77
35 % LiOH TRP sawdust
2.14
1.44
20.35
2.80
2.78
2.77
15 % Guanidine TRP sawdust
3.60
20.20
16.54
2.80
2.78
2.77
25 % Guanidine TRP sawdust
4.70
0.25
17.40
2.80
2.78
2.77
35 % Guanidine TRP sawdust
0.89
4.05
16.54
2.80
2.78
2.77 317 öse, Yürümez, Ümit Yalçın, Akgül: Wood Waste Turned Into Value Added Products... .. transition take place under the alkaline solution. As the
concentration of alkali, in particular NaOH, increases,
TCI decreases. In contrast to that, LOI increased with
the NaOH, KOH and LiOH concentration. It is probably
due to the transition between cellulose polymorph and
the effect of alkali ion (Nakano, 2010; Borysiak and
Doczekalska, 2005). The lowest TCI and LOI values
were observed on untreated materials, which may indi-
cate that the amorphous region is higher in untreated
materials than after alkali treatment when mercerization
takes place. 2 RESULTS AND DISCUSSION
2. REZULTATI I RASPRAVA Hydrogen bond energy indicates the interac-
tion between the adjacent cellulose chains (Poletto et al.,
2012). It increases when cellulose chains approach each
other, and the packing density also increases. There was
a slight difference among bond distances in alkali treat-
ed materials. Hydrogen bond energy increased with al-
kali concentration. 0
0.5
1
1.5
2
2.5
3
400
1400
2400
3400
Untreated TRP
Alkali Treated TRP
Benzylated TRP
*
Wavenumber / valni broj, cm–1
Absorbance / apsorpcija, a.u
Figure 2 FTIR spectra of untreated and chemically treated
TRP sawdust of Turkish red pine (TRP)
Slika 2. FTIR spektar netretirane i kemijski tretirane
piljevine od drva turskoga crvenog bora Figure 2 FTIR spectra of untreated and chemically treated
TRP sawdust of Turkish red pine (TRP)
Slika 2. FTIR spektar netretirane i kemijski tretirane
piljevine od drva turskoga crvenog bora After alkali treatment, the slurry was transferred
to a round bottom fl ask containing benzyl chloride. The benzylation process proceeded with continuous
stirring. Untreated and chemically treated samples
show different FTIR spectra (Figure 2). Benzylation of
the materials caused a reduction of hydroxyl peaks at
3400 cm-1and an increase of the aromatic C-C axial de-
formations around 1595 cm-1 (Figure 3). In addition to
that, aromatic multiple peak appeared at 3030-3100
cm-1. This could be due to monosubstituted benzyl
rings. Guanidine showed spectra similar to those of un-
treated materials. and LOI of the untreated sample were lower than those
of alkaline treated materials. In contrast, hydrogen bond
energy was high considering the alkaline treated materi-
als. Alkaline treated samples change the crystalline
structure and hydrogen bond energy and the degree of
this change increases as NaOH < KOH ≤ Guanidine <
LiOH at the same concentration. Wood powder merceri-
zation under alkaline conditions and the polymorphic and LOI of the untreated sample were lower than those
of alkaline treated materials. In contrast, hydrogen bond
energy was high considering the alkaline treated materi-
als. Alkaline treated samples change the crystalline
structure and hydrogen bond energy and the degree of
this change increases as NaOH < KOH ≤ Guanidine <
LiOH at the same concentration. Wood powder merceri-
zation under alkaline conditions and the polymorphic 318
DRVNA INDUSTRIJA 67 (4) 315-322 (2016)
Figure 3 Aromatic stretch at 3030 - 3050 cm-1 due to monosubstituted benzyl rings
Slika 3. 2 RESULTS AND DISCUSSION
2. REZULTATI I RASPRAVA Aromatski dio na 3030 – 3050 cm-1 zbog monosupstitucije benzilskih prstenova
0.22
0.72
1.22
1.72
2.22
2800
3300
3800
Absorbance / apsorpcija, a.u
Wavenumber / valni broj, cm–1
NaOH
Untreated
15 %
25 %
35 %
+
x
X
0
0.5
1
1.5
2
2800
3300
3800
Absorbance / apsorpcija, a.u
Wavenumber / valni broj, cm–1
KOH
0
0.5
1
1.5
2
2800
3300
3800
Absorbance / apsorpcija, a.u
Wavenumber / valni broj, cm–1
LiOH
-0.1
0,4
0.9
1.4
1.9
2800
3300
3800
Absorbance / apsorpcija, a.u
Wavenumber / valni broj, cm–1
GuOH
2.4 0.22
0.72
1.22
1.72
2.22
2800
3300
3800
Absorbance / apsorpcija, a.u
Wavenumber / valni broj, cm–1
NaOH
Untreated
15 %
25 %
35 %
+
x
X 0
0.5
1
1.5
2
2800
3300
3800
Absorbance / apsorpcija, a.u
Wavenumber / valni broj, cm–1
KOH Absorbance / apsorpcija, a.u Wavenumber / valni broj, cm–1 Wavenumber / valni broj, cm–1 0
0.5
1
1.5
2
2800
3300
3800
Absorbance / apsorpcija, a.u
Wavenumber / valni broj, cm–1
LiOH j,
-0.1
0,4
0.9
1.4
1.9
2800
3300
3800
Absorbance / apsorpcija, a.u
Wavenumber / valni broj, cm–1
GuOH
2.4 Wavenumber / valni broj, cm–1 Figure 3 Aromatic stretch at 3030 - 3050 cm-1 due to monosubstituted benzyl rings
Slika 3. Aromatski dio na 3030 – 3050 cm-1 zbog monosupstitucije benzilskih prstenova DRVNA INDUSTRIJA 67 (4) 315-322 (2016) DRVNA INDUSTRIJA 67 (4) 315-322 (2016) 318 ner, Köse, Yürümez, Ümit Yalçın, Akgül: Wood Waste Turned Into Value Added Products ..Üner, Köse, Yürümez, Ümit Yalçın, Akgül: Wood Waste Turned Into Value Added Produ
0
0.2
0.4
0.6
0.8
1
1.2
1400
1500
1600
1700
Absorbance / apsorpcija, a.u
Wavenumber / valni broj, cm–1
0
0.2
0.4
0.6
0.8
1
1.2
1400
1500
1600
1700
Absorbance / apsorpcija, a.u
Wavenumber / valni broj, cm–1
-0.1
0.1
0.3
0.5
0.7
0.9
1.1
1.3
1400
1500
1600
1700
Absorbance / apsorpcija, a.u
Wavenumber / valni broj, cm–1
0.22
0.42
0.62
0.82
1.02
1.22
1400
1500
1600
1700
Absorbance / apsorpcija, a.u
Wavenumber / valni broj, cm–1
NaOH
KOH
LiOH
GuOH
x Untreated Ƒ 15% + 25% o 35%
Figure 4 Increase of aromatic C-C axial deformations around 1595 cm-1 due to benyzlation
Slika 4. 2 RESULTS AND DISCUSSION
2. REZULTATI I RASPRAVA Porast aromatske C-C aksijalne deformacije oko 1595 cm-1 zbog benzilacije
0.22
0.72
1.22
1.72
2.22
625
725
825
925
Absorbance / apsorpcija, a.u
Wavenumber / valni broj, cm–1
0
0.5
1
1.5
2
625
725
825
925
Absorbance / apsorpcija, a.u
Wavenumber / valni broj, cm–1
0
0.5
1
1.5
2
625
725
825
925
Absorbance / apsorpcija, a.u
Wavenumber / valni broj, cm–1
0
0.5
1
1.5
2
625
725
825
925
Absorbance / apsorpcija, a.u
Wavenumber / valni broj, cm–1
x Untreated Ƒ 15% + 25% o 35%
NaOH
KOH
LiOH
GuOH
Figure 5 Appearance of bands at 698 cm-1, 740 cm-1
Slika 5. Pojava skupina na 698 cm-1, 740 cm-1 0
0.2
0.4
0.6
0.8
1
1.2
1400
1500
1600
1700
Absorbance / apsorpcija, a.u
Wavenumber / valni broj, cm–1
0
0.2
0.4
0.6
0.8
1
1.2
1400
1500
1600
1700
Absorbance / apsorpcija, a.u
Wavenumber / valni broj, cm–1
-0.1
0.1
0.3
0.5
0.7
0.9
1.1
1.3
1400
1500
1600
1700
Absorbance / apsorpcija, a.u
Wavenumber / valni broj, cm–1
0.22
0.42
0.62
0.82
1.02
1.22
1400
1500
1600
1700
Absorbance / apsorpcija, a.u
Wavenumber / valni broj, cm–1
NaOH
KOH
LiOH
GuOH
x Untreated Ƒ 15% + 25% o 35%
Figure 4 Increase of aromatic C-C axial deformations around 1595 cm-1 due to benyzlation
Slika 4. Porast aromatske C-C aksijalne deformacije oko 1595 cm-1 zbog benzilacije 0
0.2
0.4
0.6
0.8
1
1.2
1400
1500
1600
1700
Absorbance / apsorpcija, a.u
Wavenumber / valni broj, cm–1
0
0.2
0.4
0.6
0.8
1
1.2
1400
1500
1600
1700
Absorbance / apsorpcija, a.u
Wavenumber / valni broj, cm–1
-0.1
0.1
0.3
0.5
0.7
0.9
1.1
1.3
1400
1500
1600
1700
Absorbance / apsorpcija, a.u
Wavenumber / valni broj, cm–1
0.22
0.42
0.62
0.82
1.02
1.22
1400
1500
1600
1700
Absorbance / apsorpcija, a.u
Wavenumber / valni broj, cm–1
NaOH
KOH
LiOH
GuOH
x Untreated Ƒ 15% + 25% o 35%
Figure 4 Increase of aromatic C-C axial deformations around 1595 cm-1 due to benyzlation
Slika 4. 2 RESULTS AND DISCUSSION
2. REZULTATI I RASPRAVA Porast aromatske C-C aksijalne deformacije oko 1595 cm-1 zbog benzilacije 0.22
0.42
0.62
0.82
1.02
1.22
1400
1500
1600
1700
Absorbance / apsorpcija, a.u
Wavenumber / valni broj, cm–1
NaOH 0
0.2
0.4
0.6
0.8
1
1.2
1400
1500
1600
1700
Absorbance / apsorpcija, a.u
Wavenumber / valni broj, cm–1
KOH -0.1
0.1
0.3
0.5
0.7
0.9
1.1
1.3
1400
1500
1600
1700
Absorbance / apsorpcija, a.u
Wavenumber / valni broj, cm–1
GuOH Figure 4 Increase of aromatic C-C axial deformations around 1595 cm-1 due to benyzlation
Slika 4. Porast aromatske C-C aksijalne deformacije oko 1595 cm-1 zbog benzilacije 0.22
0.72
1.22
1.72
2.22
625
725
825
925
Absorbance / apsorpcija, a.u
Wavenumber / valni broj, cm–1
0
0.5
1
1.5
2
625
725
825
925
Absorbance / apsorpcija, a.u
Wavenumber / valni broj, cm–1
0
0.5
1
1.5
2
625
725
825
925
Absorbance / apsorpcija, a.u
Wavenumber / valni broj, cm–1
0
0.5
1
1.5
2
625
725
825
925
Absorbance / apsorpcija, a.u
Wavenumber / valni broj, cm–1
x Untreated Ƒ 15% + 25% o 35%
NaOH
KOH
LiOH
GuOH
Figure 5 Appearance of bands at 698 cm-1, 740 cm-1
Slika 5. Pojava skupina na 698 cm-1, 740 cm-1 DRVNA INDUSTRIJA 67 (4) 315-322 (2016)
0.22
0.72
1.22
1.72
2.22
625
725
825
925
Absorbance / apsorpcija, a.u
Wavenumber / valni broj, cm–1
0
0.5
1
1.5
2
625
725
825
925
Absorbance / apsorpcija, a.u
Wavenumber / valni broj, cm–1
0
0.5
1
1.5
2
625
725
825
925
Absorbance / apsorpcija, a.u
Wavenumber / valni broj, cm–1
0
0.5
1
1.5
2
625
725
825
925
Absorbance / apsorpcija, a.u
Wavenumber / valni broj, cm–1
x Untreated Ƒ 15% + 25% o 35%
NaOH
KOH
LiOH
GuOH
Figure 5 Appearance of bands at 698 cm-1, 740 cm-1
Slika 5. 2 RESULTS AND DISCUSSION
2. REZULTATI I RASPRAVA Pojava skupina na 698 cm-1, 740 cm-1 0
0.5
1
1.5
2
625
725
825
925
Absorbance / apsorpcija, a.u
Wavenumber / valni broj, cm–1
KOH 0.22
0.72
1.22
1.72
2.22
625
725
825
925
Absorbance / apsorpcija, a.u
Wavenumber / valni broj cm–1
NaOH Wavenumber / valni broj, cm
0
0.5
1
1.5
2
625
725
825
925
Absorbance / apsorpcija, a.u
Wavenumber / valni broj, cm–1
GuOH Wavenumber / valni broj, cm
0
0.5
1
1.5
2
625
725
825
925
Absorbance / apsorpcija, a.u
Wavenumber / valni broj, cm–1
LiOH Absorbance / apsorpcija, a.u x Untreated Ƒ 15% + 25% o 35% Figure 5 Appearance of bands at 698 cm-1, 740 cm-1
Slika 5. Pojava skupina na 698 cm-1, 740 cm-1 Figure 5 Appearance of bands at 698 cm-1, 740 cm-1
Slika 5. Pojava skupina na 698 cm-1, 740 cm-1 DRVNA INDUSTRIJA 67 (4) 315-322 (2016) DRVNA INDUSTRIJA 67 (4) 315-322 (2016) 319 319 öse, Yürümez, Ümit Yalçın, Akgül: Wood Waste Turned Into Value Added Products... .. 0.22
0.72
1.22
1.72
2.22
900
1000
1100
1200
1300
Absorbance / apsorpcija, a.u
Wavenumber / valni broj, cm–1
0
0.5
1
1.5
2
2.5
900
1000
1100
1200
1300
Absorbance / apsorpcija, a.u
Wavenumber / valni broj, cm–1
0
0.5
1
1.5
2
2.5
900
1000
1100
1200
1300
Absorbance / apsorpcija, a.u
Wavenumber / valni broj, cm–1
0
0.5
1
1.5
2
2.5
900
1000
1100
1200
1300
Absorbance / apsorpcija, a.u
Wavenumber / valni broj, cm–1
x Untreated Ƒ 15% + 25% o 35%
NaOH
KOH
LiOH
GuOH
Figure 6 C-O stretch of the substituted benzyl ether Wavenumber / valni broj, cm
0
0.5
1
1.5
2
2.5
900
1000
1100
1200
1300
Absorbance / apsorpcija, a.u
Wavenumber / valni broj, cm–1
LiOH j,
0
0.5
1
1.5
2
2.5
900
1000
1100
1200
1300
Absorbance / apsorpcija, a.u
Wavenumber / valni broj, cm–1
GuOH x Untreated Ƒ 15% + 25% o 35% Figure 6 C-O stretch of the substituted benzyl ether
Slika 6. C-O dio etera koji je supstituiran benzilom The peak at 670 cm–1gives information on C–OH
out of plane bending (Ma, 2007). Spectra of the sam-
ples showed a reduction of peak intensity at 670 cm-1. In contrast, peak appears at 698 cm-1due to aromatic
C-C angular deformation in treated materials (Figure
5). 2 RESULTS AND DISCUSSION
2. REZULTATI I RASPRAVA In addition to that, benzyl group can be detected at
740 cm-1. The guanidine peak is lower compared to
others. Even though it is strong alkali, the size of the
molecule may restrict the entrance into the cell wall. After the reaction, an increase of aryl ether band at
1265cm-1 and of alkyl ether band at 1155-1060 cm-1
(due to C-O stretch from the substituted benzyl ether)
was observed. (Figure 5). This absorbance band im-
plied the substitution of hydroxyl groups in materials
with benzyl groups. weight loss curve can be used to tell the point at which
weight loss is most apparent (Figure 8). According to
the results obtained, raw materials begin to decompose
at a higher temperature (Ti = 380 °C) when compared
to modifi ed materials (Ti = 330-340 °C). It was seen that there was a signifi cant difference
between modifi ed and untreated materials in relation to
their respective thermal decomposition temperatures,
which may be related to the difference in crystallinity
between untreated and modifi ed materials. Tailored hy-
droxyl groups change the crystalline structure of the
materials, when the intra and intermolecular hydrogen
bonds between chains are broken. Rearrangement of
the bonding ability of the treated materials reduces the
decomposition temperature. As a result, the crystalline
domains in the untreated materials show a higher ther-
mal stability, when compared to modifi ed materials. Cellulose is the main component in wooden ma-
terials and it controls the thermal properties of wood
(Hon and Shiraishi, 2001). Hydrogen bonds between
cellulose chain and in the chain shift to melting tem-
perature over decomposition temperature. Modifi ca-
tion of wood helped to disrupt any hydrogen bonds
between adjacent chains and in the chain. Therefore, it
helps wood to become thermoformable. 4 CONCLUSION
4. ZAKLJUČAK Sawdust is a very promising source of raw materi-
als for commercial products such as cellulose, plastics,
etc. for many countries due to its availability and abun-
dance. However, it is considered as waste. Thermoplas-
ticization can be achieved by benzylation. Alkaline spe-
cies and alkali concentration affect the substitution
reaction and change the material properties. These
changes are signifi cant with NaOH, and it can be de-
tected with crystallinity, hydrogen bond energy change
and thermal analysis. Hydroxyl groups were substituted Figure 7 shows differential thermogravimetric
analysis (DTGA) curves of alkali treated with different
alkali ions, concentration and untreated materials. The
fi rst step of weight loss, from room temperature to 120
°C, was related to the evaporation of absorbed water. The second step ends near 410 °C, and can be described
by an abrupt mass loss stage that was related to the
main thermal decomposition process. A derivative DRVNA INDUSTRIJA 67 (4) 315-322 (2016) 320 r, Köse, Yürümez, Ümit Yalçın, Akgül: Wood Waste Turned Into Value Added Products... ner, Köse, Yürümez, Ümit Yalçın, Akgül: Wood Waste Turned Into Value Added Products -0.04
0.01
0.06
0.11
0.16
0.21
0.26
0.31
0
100
200
300
400
500
600
Deriv. Weight / promjena mase, %/min
Temperature / temperatura, °C
-0.05
0
0.05
0.1
0.15
0.2
0.25
0.3
0
100
200
300
400
500
600
Deriv. Weight / promjena mase, %/min
Temperature / temperatura, °C
x Untreated Ƒ 15% + 25% o 35%
NaOH
KOH
Figure 7 DTGA thermogram of untreated and benzylated wood with different alkali
Slika 7. DTGA termogrami netretiranog drva i drva benziliranoga različitim lužinama -0.04
0.01
0.06
0.11
0.16
0.21
0.26
0.31
0
100
200
300
400
500
600
Deriv. Weight / promjena mase, %/min
Temperature / temperatura, °C
NaOH Temperature / temperatura, °C -0.05
0
0.05
0.1
0.15
0.2
0.25
0.3
0
100
200
300
400
500
600
Deriv. Weight / promjena mase, %/min
Temperature / temperatura, °C
x Untreated Ƒ 15% + 25% o 35%
KOH
Figure 7 DTGA thermogram of untreated and benzylated wood with different alkali
Slika 7. DTGA termogrami netretiranog drva i drva benziliranoga različitim lužinama Figure 7 DTGA thermogram of untreated and benzylated wood with different alkali
Slika 7. 5 REFERENCES
5. LITERATURA 1. Akgul, M; Gumuskaya, E.; Korkut, S., 2007: Crystalline
structure of heat-treated Scots pine [Pinus sylvestris L.]
and Uludag˘ fi r [Abies nordmanniana (Stev.) subsp. born-
muelleriana (Mattf.)] wood. Wood Science and Technol-
ogy, 41: 281-289. http://dx.doi.org/10.1007/s00226-006-0110-9. 18. Pereira, R.; Campana Filho, S. P.; Curvelo, A. A. S.,
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lulose, 4: 21-31. http://dx.doi.org/10.1023/A:1018459016966. http://dx.doi.org/10.1023/A:1018459016966. 19. Poletto, M.; Pistor, V.; Zanetta, A. J., 2013: Structural
Characteristics and Thermal Properties of Native Cellu-
lose, in Cellulose, Fundamental Aspects Edited by Theo
Van de Ven and Louis Godbout published by In Tech
Croatia, 2013. 2. Akerholm, M.; Hinterstoisser, B.; Salmen, L., 2004:
Characterization of the crystalline structure of cellulose
using static and dynamic FT-IR spectroscopy. Carbohy-
drate Research, 339: 569-578. http://dx.doi.org/10.1016/j.carres.2003.11.012. 20. Poletto, M.; Junior, H. L. O.; Zattera, A. J., 2014: Native
Cellulose: Structure Characterization and Thermal Prop-
erties. Materials, 7: 6105-6119. http://dx.doi.org/10.3390/ma7096105. 3. Biswas, A.; Saha, B. C.; Lawton, J. W.; Shogren, R. L.;
Willet, J. L., 2006: Process for obtaining cellulose acetate
from agricultural by-products. Carbohydrate Polymers,
64:134-137. http://dx.doi.org/10.1016/j.carbpol.2005.11.002. 21. Poletto, M.; Zattera, A. J.; Santana, R. M. C., 2012: Stru-
cural differences between wood species: Evidence from
Chemical Composition, FTIR Spectroscopy and Ther-
mogravimetric Analysis. Journal of Applied Polymer
Science, 126, E336-E343. http://dx.doi.org/10.1002/app.36991. http://dx.doi.org/10.1016/j.carbpol.2005.11.002. 4. Bodirlau, R.; Teaca, C. A.; Spiridon, I., 2008: Chemical
Modifi cation Of Beech Wood: Effect On Thermal Stabil-
ity. Bioresources, 3 (3): 789-800. 5. Borysiak, S.; Doczekalska, B., 2005: X-ray Diffraction
Study of Pine Wood Treated with NaOH. Fibres & Tex-
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5 (53), 87-89. 22. Qu, B.; Qin, T.; Chu, F.; 2014: Cyanoethylation of sev-
eral fi ber materials and their utilization as adhesive in
wood-based panels. Wood Science Technology, 48: 519-
531. http://dx.doi.org/10.1007/s00226-014-0623-6. 6. Çöpür, Y.; Güler, C.; Akgül, M.; Taşçıoğlu, C., 2007:
Some chemical properties of hazelnut husk and its suita-
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and
syntheticpoly mers-Biode gradabilityan-
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9-14; Dallas, TX. Washington, DC: American Chemical
Society; 1990. Chapter 21. https://doi.org/10.1021/bk-1990-0433.ch021. 7. 4 CONCLUSION
4. ZAKLJUČAK DTGA termogrami netretiranog drva i drva benziliranoga različitim lužinama DRVNA INDUSTRIJA 67 (4) 315-322 (2016)
321
-0.05
0
0.05
0.1
0.15
0.2
0.25
0
10
20
30
40
50
60
70
80
90
100
0
100
200
300
400
500
600
Weight / masa, %
Temperature / temperatura, °C
-0.05
0
0.05
0.1
0.15
0
10
20
30
40
50
60
70
80
90
100
0
100
200
300
400
500
600
Deriv.Weight, %/min
promjena mase, %/min
Weight / masa, %
Temperature / temperatura, °C
-0.2
-0.1
0
0.1
0.2
0.3
0
10
20
30
40
50
60
70
80
90
100
0
200
400
600
Weight / masa, %
Temperature / temperatura, °C
-0.1
-0.05
0
0.05
0.1
0.15
0.2
0.25
0.3
0
20
40
60
80
100
0
100
200
300
400
500
600
Weight / masa, %
Temperature / temperatura, °C
25 %
LiOH
25 % GuOH
25 % KOH
25% NaOH
Deriv.Weight, %/min
promjena mase, %/min
Deriv.Weight, %/min
promjena mase, %/min
Deriv.Weight, %/min
promjena mase, %/min
Figure 8 DTG thermograms of benzylated materials under different alkali concentrations
Slika 8. DTG termogrami benziliranog materijala pri različitim koncentracijama lužine -0.05
0
0.05
0.1
0.15
0.2
0.25
0
10
20
30
40
50
60
70
80
90
100
0
100
200
300
400
500
600
Weight / masa, %
Temperature / temperatura, °C
25 % KOH
Deriv.Weight, %/min
promjena mase, %/min -0.05
0
0.05
0.1
0.15
0
10
20
30
40
50
60
70
80
90
100
0
100
200
300
400
500
600
Deriv.Weight, %/min
promjena mase, %/min
Weight / masa, %
Temperature / temperatura, °C
25% NaOH -0.1
-0.05
0
0.05
0.1
0.15
0.2
0.25
0.3
0
20
40
60
80
100
0
100
200
300
400
500
600
Weight / masa, %
Temperature / temperatura, °C
25 %
LiOH
Deriv.Weight, %/min
promjena mase, %/min -0.2
-0.1
0
0.1
0.2
0.3
0
10
20
30
40
50
60
70
80
90
100
0
200
400
600
Weight / masa, %
Temperature / temperatura, °C
25 % GuOH
Deriv.Weight, %/min
promjena mase, %/min Figure 8 DTG thermograms of benzylated materials under different alkali concentrations
Slika 8. DTG termogrami benziliranog materijala pri različitim koncentracijama lužine DRVNA INDUSTRIJA 67 (4) 315-322 (2016)
321 DRVNA INDUSTRIJA 67 (4) 315-322 (2016) DRVNA INDUSTRIJA 67 (4) 315-322 (2016) 321 321 öse, Yürümez, Ümit Yalçın, Akgül: Wood Waste Turned Into Value Added Products... .. 5 REFERENCES
5. LITERATURA Chen, C.; Cho, M.; Kim, B.-W.; Namb, J.-D.; Lee, Y.,
2012: Thermo plasticization and characterization of ke-
naf fi ber by benzylation. Journal of Industrial Engineer-
ing and Chemistry, 18: 1107-1111. http://dx.doi.org/10.1016/j.jiec.2011.12.012. http://dx.doi.org/10.1016/j.jiec.2011.12.012. 24. Su, M.; Cheng, J.; Pan, Z.; Li, X.; Xu, A.; Hong, J., 2015:
Study on the Preparation and Mechanical Properties of
Injection-Moulded Wood-Based Plastics. Journal of Ap-
plied Polymer Science, 132 (5): 41376. http://dx.doi. org/10.1002/app.41376. 8. Ebringerova, A.; Heinze, T., 2000: Xylan and xylan de-
rivatives – biopolymers with valuable properties. Macro-
molecular Rapid Communication, 21: 542-556. 9. Hassan, M. L.; El-Wakil, N. A.; Sefain, M. Z., 2001:
Thermoplasticization of Bagasse by Cyanoethylation. Journal of Applied Polymer Science, 79: 1965-1978. 4 CONCLUSION
4. ZAKLJUČAK 12. Hon, D. N.-S.; Shiraishi, N., 2001: Wood and Cellulosic
Chemistry, second edition. Marcel Dekker Inc. New
York, Basel. with benzyl groups and this caused the change of FTIR
spectra. Guanidine shows different trends. This could be
due to the molecule size. It cannot penetrate the pore on
the cell wall, because the pore size could be smaller than
the molecule size. Na Li and K are effective on thermo-
plasticization. Multiple peaks appear at around 3030
cm-1 indicating benzylation of the materials. The appear-
ance of the peak at 698 cm-1 and 740 cm-1 indicates the
aromatic C-C angular deformation. Thermogravimetric
analysis revealed that modifi ed products were character-
ized by poorer thermal stability compared to raw materi-
als. Deformed crystallinity of the materials reduced sig-
nifi cantly the decomposition temperature, and the
reduction of the decomposition temperature ranged be-
tween 40 to 60 oC. 13. Huang, K.; Zhang, M.; Zhang, G.; Jiang, X.; Huang, D.,
2014: Acetylation Modifi cation of Rice Straw Fiber And
Its Thermal Properties. Cellulose Chemistry and Tech-
nology, 48 (3-4): 199-207. 14. Kondo, T., 1997: The assignment of IR absorption bands
due to free hydroxyl groups in cellulose. Cellulose, 4:
281-292. http://dx.doi.org/10.1023/A:1018448109214. 15. Ma, L., 2007: Plasticization of Wood By Benzylation. MSc. Thesis University of Idaho. 16. Nakano, T., 1994: Mechanism Of Thermoplasticity for
Chemically-Modifi ed Wood. Holzforschung-Internation-
al Journal of the Biology, Chemistry, Physics and Tech-
nology of Wood, 48 (4): 318-324. http://dx.doi.org/10.1515/hfsg.1994.48.4.318. 17. Nakano, T., 2010: Mechanism of microfi bril contraction
and anisotropic dimensional changes for cells in wood
treated with aqueous NaOH solution. Cellulose, 17: 711-
719. http://dx.doi.org/10.1007/s10570-010-9414-x. Corresponding address: 10. Hon, D. N-S.; Louis, J. M. S., 1989: Thermoplasticiza-
tion of Wood. II. Cyanoethylation. Journal of Polymer
Science. Part A: Polymer Chemistry, 27: 4143-4160. http://dx.doi.org/.1002/pola.1989.080271221. Assoc. prof. BIROL ÜNER, Ph.D. Assoc. prof. BIROL ÜNER, Ph.D. Suleyman Demirel University, Faculty of Forestry
Department of Forest Products Engineering
Isparta 32260, TURKEY
e-mail: biroluner09@gmail.com Suleyman Demirel University, Faculty of Forestry 11. Hon, D. N.-S.; Ou, N.-H., 1989: Thermoplasticization of
Wood. I. Benyzlation of wood. Journal of Polymer Sci-
ence. Part A: Polymer Chemistry, 27: 2457-2482. http://dx.doi.org/10.1002/pola.1989.080270725. DRVNA INDUSTRIJA 67 (4) 315-322 (2016) DRVNA INDUSTRIJA 67 (4) 315-322 (2016) DRVNA INDUSTRIJA 67 (4) 315-322 (2016) 322
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https://openalex.org/W2891902755
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https://revistas.pucsp.br/index.php/ReCaPe/article/download/37285/26434
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Gamificação e gestão de pessoas: um estudo de caso sobre treinamento e ambiente de diversidade cultural
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Revista de Carreiras e Pessoas
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RESUMO Este artigo tem como objetivo discutir a aplicação de gamificação
na gestão de pessoas, mais especificamente no treinamento
e desenvolvimento de competências de colaboradores de
diferentes níveis culturais dentro de uma grande organização. Para tal estudo, iremos trabalhar, principalmente, com as
teorias de carreira sem fronteiras e de gamificação voltada para
o aprendizado ligados a um estudo de caso do desenvolvimento
de projeto piloto de treinamento corporativo em empresa de
grande porte, com mais de 4 mil colaboradores, ligada ao
setor de infraestrutura, com atuação na região metropolitana
de São Paulo. O estudo de caso utilizou entrevistas não
somente dos profissionais do departamento de treinamento da
empresa, como também coletou dados da empresa terceirizada
responsável pelo processo de gamificação além de entrevistas
com os desenvolvedores do game/ treinamento. Os resultados
da pesquisa mostraram as motivações da escolha desta
dinâmica e como a empresa se preparou para a implementação
do projeto. ABSTRACT ABSTRACT This article aims to discuss the application of gamification in people
management, more specifically in the training and development
of skills of employees of different cultural levels within a large
organization. For this study, we will work mainly with theories
of career without frontiers and gamification focused on learning
linked to a case study of the development of pilot project of
corporate training in large company, with more than 4 thousand
employees, linked to the infrastructure sector, operating in the
metropolitan region of São Paulo. The case study used interviews
not only from the professionals of the training department of the
company, but also collected data from the outsourced company
responsible for the gamification process in addition to interviews
with the developers of the game / training. The results of the
research showed the motivations of choosing this dynamic and
how the company prepared for the implementation of the project. Dados para contato:
Matheus Matsuda Marangoni
Escola Superior de Propaganda e
Marketing. Rua Dr. Álvaro Alvim, 123 - Vila Mariana,
04018-010, São Paulo, SP, Brasil. URL da Homepage:
https://www.espm.br/
Recebido em: 06/02/2017
Aprovado em: 14/12/2017
DOI:
http://dx.doi.org/10.20503/recape. v8i3.37285 Mauro Miguel
Rodrigues Berimbau Professor de Graduação e de
Pós-Graduação Lato Sensu da
Escola Superior de Propaganda e
Marketing. Coordenador do
Game Lab ESPM (Brasil) mauroberimbau@gmail.com Palavras-chave: carreira sem fronteiras; gamificação; gestão
de pessoas; treinamento. ISSN: 2237-1427
Vol. 8 | Nº 3 | Ano 2018
Páginas 431-444 Matheus Matsuda
Marangoni GAMIFICACIÓN Y GESTIÓN DE PERSONAS:
UN ESTUDIO DE CASO SOBRE ENTRENAMIENTO Y
AMBIENTE DE DIVERSIDAD CULTURAL Professor de Graduação e de
Pós-Graduação Lato Sensu da
Escola Superior de Propaganda e
Marketing (Brasil) matheus.marangoni@espm.br RESUMO RESUMEN Este artículo tiene como objetivo discutir la aplicación de gamificación en la gestión de perso-
nas, más específicamente en el entrenamiento y desarrollo de competencias de colaboradores de
diferentes niveles culturales dentro de una gran organización. Para este estudio, vamos a trabajar
principalmente con las teorías de carrera sin fronteras y de gamificación orientada al aprendizaje
vinculado a un estudio de caso del desarrollo de un proyecto piloto de entrenamiento corporativo en
una empresa de gran tamaño, con más de 4 mil colaboradores, vinculada al sector de infraestructura,
con actuación en la región metropolitana de São Paulo. El estudio de caso utilizó entrevistas no sólo
de los profesionales del departamento de entrenamiento de la empresa, como también recolectó
datos de la empresa tercerizada responsable del proceso de gamificación además de entrevistas
con los desarrolladores del juego / entrenamiento. Los resultados de la investigación mostraron las
motivaciones de la elección de esta dinámica y cómo la empresa se preparó para la implementación
del proyecto. Palabras clave: carrera sin fronteras, gamificación, gestión de personas, entrenamiento. Dados para contato: Matheus Matsuda Marangoni
Escola Superior de Propaganda e
Marketing. Rua Dr. Álvaro Alvim, 123 - Vila Mariana,
04018-010, São Paulo, SP, Brasil. URL da Homepage:
https://www espm br/ Recebido em: 06/02/2017
Aprovado em: 14/12/2017 Matheus Matsuda Marangoni, Mauro Miguel Rodrigues Berimbau Keywords: career without borders, gamification, people management, training. Keywords: career without borders, gamification, people management, training. 1 TREINAMENTO, CARREIRA E APRENDIZAGEM A gestão de carreira nas últimas décadas passa por grandes mudanças, conforme Dutra (2010)
nos apresenta em um panorâmica histórico das trajetórias de carreira desde os anos 1980. O papel
da gestão de carreira que antes voltado mais para as preocupações do indivíduo dentro das organi-
zações, passa no final dos anos 1990 e início dos anos 2000, a ser compartilhado entre as motiva-
ções pessoais do indivíduo. Uma vez que, as mudanças no cenário do mercado de trabalho e nos
anseios pessoais e culturais dos profissionais, faz com que, o planejamento individual ganhe maior
destaque nesse processo. Planejamento que deverá ter o desenvolvimento pessoal e profissional
fortemente alinhados. Conforme podemos ver na afirmação de Dutra (2010, p. 64): A carreira não deve ser entendida como um caminho rígido a ser seguido ela pessoa e sim
como uma sequencia de posições e de trabalhos por ela realizada. Essa sequencia, articulada
de forma a conciliar o desenvolvimento das pessoas como o desenvolvimento da empresa… Assim, vemos que os profissionais irão passar por diversas empresas, funções e até mesmo
por projetos ao longo de sua vida profissional. E com a constante e crescente quantidade de informa- ReCaPe | Vol. 8 | Nº. 3 | Ano 2018 | set./dez. | p. 432 GAMIFICAÇÃO E GESTÃO DE PESSOAS: UM ESTUDO DE CASO SOBRE TREINAMENTO E AMBIENTE DE DIVERSIDADE CULTURAL ções e conhecimento que a sociedade contemporânea produz, entendemos que o aprendizado toma
cada vez mais importância no processo de desenvolvimento da trajetória das carreiras. Trajetórias
que, segundo Dutra (2010), se caracterizam por atribuições e responsabilidades de uma mesma na-
tureza, de um conjunto de conhecimentos e habilidades inter-relacionados. De forma complementar, segundo Veloso, Dutra e Lima (2016), a definição de carreira con-
templa uma sequencia de experiências mais do que uma linearidade hierárquica dentro das orga-
nizações. A carreira passou a ser uma questão mais integrada com a vida pessoal dos indivíduos e
menos dependente de visão apenas da firma, visto que “… às pessoas, cabe a gestão de seu de-
senvolvimento, de sua competitividade profissional e de sua carreira.” (VELOSO, DUTRA, NAKATA. 2016, p. 90) E para o desenvolvimento de sua carreira, é exigido do trabalhador, segundo Dutra (2010),
uma série de competências individuais e de acordo com as necessidades da organização. Essas
competências, muitas vezes, são exigidas como requisito para a contratação de um funcionário. 1 TREINAMENTO, CARREIRA E APRENDIZAGEM Mas,
podemos ver que, em muitos casos parte destas competências são desenvolvidas também pelo pro-
cesso de treinamento oferecido pelas empresas. E dependendo da posição do indivíduo dentro da
organização, necessidades diferentes de conhecimento e competências são necessárias. Entendendo as características das novas formas de carreiras na sociedade contemporânea e
a necessidade de constante desenvolvimento pessoal e profissional por parte dos indivíduos, será
necessário olhar as teorias de carreira proteana e carreira sem fronteiras na ótica da aprendizagem. O conceito de carreira proteana trazida por Bendassolli (apud SALA e TREVISAN, 2014, p.159),
que nos indica apresentar como principais características: Mudança como dado de realidade; varie-
dade de experiências; adaptabilidade e resiliência; identidade como âncora. Vemos desta forma, que
um fato importante e comum é a necessidade de desenvolvimento constante. Na visão da carreira proteana, segundo Veloso e Dutra (2010), os indivíduos são impelidos a
um processo de aprendizagem contínua, uma vez que o trabalhador precisa desenvolver a habilida-
de de aprender, adquirir novas competências e de lidar com as novas demandas do ambiente. Mes-
mo que esse aprendizado não seja oferecido pelas organizações. As pessoas precisam aprender
“…o que e como aprender sobre elas mesmas, sobre as outras pessoas, sobre o trabalho e sobre o
ambiente…” (HALL, 2002 apud DUTRA 2010). Assim, no contexto da sociedade contemporânea com
maiores incertezas na estabilidade de emprego e rapidez com que os trabalhos mudam, a habilidade
de aprender constantemente trás uma maior segurança para o indivíduo. Já a teoria de carreiras sem fronteiras (ARTHUR; INKSON; PRINGLE, 1999 APUD DUTRA
2010) oferece perspectiva de carreira onde não há clara divisão entre o ambiente corporativo e a
vida pessoal de seus funcionários. A carreira sem fronteiras é uma resposta orientada para a intera- ReCaPe | Vol. 8 | Nº. 3 | Ano 2018 | set./dez. | p. 433 Matheus Matsuda Marangoni, Mauro Miguel Rodrigues Berimbau ção entre a firma e as mudanças sociais da sociedade industrial para a nova economia. Dentre as
diversas características desta nova visão de carreira, nos parece importante destacar neste artigo a
perspectiva de que as pessoas são responsáveis por suas carreiras futuras através de, entre outros
fatores, a constante busca por conhecimento. 1 TREINAMENTO, CARREIRA E APRENDIZAGEM Contudo, a partir de Peiperl e Arthur (apud Dutra 2010), entendemos que o processo de apren-
dizagem acontece mais internamente às organizações do que fora delas, uma vez que, nas orga-
nizações temos oportunidades para a transferência interfuncional de conhecimento. É importante,
também, destacar que Arthur, Inkson e Pringle (apud. Dutra 2010, p.29), nos oferecem a visão de
que nas carreiras sem fronteiras as competências são “…obtidas através de educação, trabalho e
experiência de vida…”. Segundo tais conceitos, podemos entender que, o processo de aprendizagem hoje é de res-
ponsabilidade tanto das organizações quando dos próprios indivíduos. Há uma insegurança maior
no desenvolvimento e manutenção da carreira por parte dos indivíduos, que necessitam constante-
mente busca desenvolvimento pessoal e profissional. Contudo, o processo de aprendizagem ofere-
ce desafios tanto para a firma quanto para seus funcionários, uma vez que, quais conhecimentos
e competências são necessárias muitas vezes não estão claras para ambas as partes. O acesso
a informação e as técnicas de treinamento são muitas, porém, vemos que o engajamento com tais
processos é um dos maiores desafios para o processo de aprendizagem. E a partir desta discussão,
propomos neste artigo uma visão mais aprofundada da aplicação da gamificação como uma dinâ-
mica de aprendizagem que vem oferecendo novas formas de treinamento e desenvolvimento dos
indivíduos dentro das corporações. 2 GAMIFICAÇÃO E APRENDIZAGEM O emprego de mecânicas de games nas empresas, seja para inventivo de vendas ou para
treinamento, não é necessariamente uma novidade. Há muito tempo as empresas buscam o en-
gajamento de seus funcionários na educação e treinamento corporativo. O que podemos indicar,
neste momento, é o uso mais completo das dinâmicas gamificadas. Muito desta evolução se dá pelo
desenvolvimento tecnológico dos últimos anos. A partir desde cenário, buscamos neste artigo, enten-
der mais sobre como o uso estruturado de ferramentas e modelos gamificados são uma estratégia
coerente no treinamento em um ambiente com diversos públicos em níveis diferentes dentro de uma
firma. A gamificação apresenta como conceito básico, a aplicação de mecânicas de games em si-
tuações de não game, ou seja, neste caso a construção de um game voltado para o processo de ReCaPe | Vol. 8 | Nº. 3 | Ano 2018 | set./dez. | p. 434 GAMIFICAÇÃO E GESTÃO DE PESSOAS: UM ESTUDO DE CASO SOBRE TREINAMENTO E AMBIENTE DE DIVERSIDADE CULTURAL aprendizado. Nos parece importante trazer alguns autores para discussão mais aprofundada sobre a
conceituação de gamificação relacionada à aprendizagem. É importante destacar que, a gamificação
como teoria ainda está em seu estágio inicial. Muitas das discussões a respeito do tema oferecem
abordagens diferentes. Não buscamos aqui, esgotar o assunto, uma vez que, não se trata de nosso
objetivo neste artigo. Contudo, ao buscarmos referências no principais autores do momento, enten-
demos que é possível fechar a questão necessária para analisar o estudo de caso proposto. Começamos com a definição trazida por Paharia (2013), segundo a qual a gamificação é en-
tendida enquanto ferramenta para os negócios, como uma forma de gerar lealdade, seja em públicos
internos ou externos. Paharia (2013, p. 65) temos que: …a gamificação emprega técnicas motivacionais que os designers de videogames têm usado
por anos, para motivar os jogadores, e aplica-as em contextos que não são jogos. Essas téc-
nicas incluem (mas não se limitam a): concepção de objetivos a serem alcançados, reconhe-
cimento por badges, engajamento via competição, encorajamento à colaboração em equipe,
caracterização de status por ranking e estímulo ao acúmulo de pontos. …a gamificação emprega técnicas motivacionais que os designers de videogames têm usado
por anos, para motivar os jogadores, e aplica-as em contextos que não são jogos. 2 GAMIFICAÇÃO E APRENDIZAGEM Essas téc-
nicas incluem (mas não se limitam a): concepção de objetivos a serem alcançados, reconhe-
cimento por badges, engajamento via competição, encorajamento à colaboração em equipe,
caracterização de status por ranking e estímulo ao acúmulo de pontos. Podemos entender que a motivação, ao utilizar técnicas de gamificação, busca a associação
com o engajamento através do apelo lúdico e descontraído, como se fosse mais uma forma de
entretenimento e do que um treinamento entediante. Segundo Frasca (2003), o entretenimento, a
partir de mecânicas gamificadas, se coloca como um facilitador das relações entre a máquina de
aprendizagem digital e os indivíduos que com ela interagem, fazendo com que, esses mantenham-se
engajados, ou seja, mais presentes e efetivamente participantes do processo. Aprendemos muito quando nos divertimos. Quem não gosta de assistir a um filme, ler um qua-
drinho e jogar um bom game? São experiências memoráveis, que algumas vezes nos lembramos
por décadas a seguir. Por que essas mídias, de onde originam tais conteúdos, não podem então
nos ensinar outras coisas mais interessantes? Quem trabalha com formação de pessoas costuma
se indagar desta forma, e procura as mais diferentes técnicas para transformar um conteúdo denso,
trabalhoso e complicado em algo mais palatável, acessível e agradável. É através dessas preocupa-
ções que surgem estudos e aplicações de elementos de jogos em situações diversas, incluindo na
sala de aula. Para entender a gamificação além do contexto do entretenimento, precisamos nos libertar da
observação meramente ferramental e estética do jogo (pontos, vidas, personagens animados, músi-
ca feliz, etc.) e entender a sua estrutura fundamental (estabelecimento de regras, liberdade de ação,
construção de experiência para o jogador, etc.). Além disso, é preciso estudar formas de planejamen-
to, mensuração e aplicação, sem se esquecer sobre a proposta educativa. Assim, podemos entender
que, o processo de design de um jogo voltado para a educação está segmentado em quatro cama-
das (WINN, 2009): aprendizagem, storytelling, gameplay e experiência do usuário. ReCaPe | Vol. 8 | Nº. 3 | Ano 2018 | set./dez. | p. 435 Matheus Matsuda Marangoni, Mauro Miguel Rodrigues Berimbau Como aprendizagem no contexto da gamificação, Winn (2009) nos trás a necessidade de um
alinhamento de toda a estrutura do game para com os objetivos do aprendizado em uma estrutra de
eixos. Primeiro é necessário ensinar o jogador como participar do processo, ou seja, como jogar o
game. 2 GAMIFICAÇÃO E APRENDIZAGEM Segundo Frasca (2007), todo jogo nos ensina alguma coisa, mesmo que seja apenas sobre
eles mesmos: a necessidade e significado de apertar botões, o que pode ser feito dentro dos limites
do jogo e quais seus objetivos. Os games voltados para educação buscam também transmitir uma
mensagem, reforçar um comportamento e nos ensinar algo. Como camada de storytelling podemos entender a forma como o game produz sentido. É um
elemento fundamental, mesmo que não obrigatório, para facilitar o processo educativo, uma vez que,
gera contexto e ajuda no engajamento. Temos que entender a necessidade que o game tem de gerar
uma experiência o mais agradável possível. Um jogo de mecânica simples, como perguntas de múl-
tipla escolha após um conteúdo técnico demonstrado pode engajar seus participantes o suficiente
para fixar o aprendizado. Ao tornar a narrativa do game mais lúdica, é possível gerar maior interação
do jogador e, assim, maiores chances de fixação do conteúdo oferecido. O gameplay é a camada que define o jogo em si, uma vez que, é a responsável pela mecânica
e regulação do game. Para termos um jogo é fundamental que tenhamos regras. O que o jogador
pode ou não fazer em um jogo e quais as dinâmicas que conduzem o jogador pelo tabuleiro ou tela. É
de responsabilidade do gameplay a construção da experiência e de tornar a atividade desafiadora e
equilibrada. Um jogo precisa apresentar desafios, caso contrário não prenderá a atenção do jogador,
mas tais desafios precisam ser equilibrados e alinhados com os objetivos de aprendizagem para que
gerem o engajamento necessário. Segundo Sicart (2008), as mecânicas de jogos são um conjunto de métodos invocados por
agentes, projetados para interagir com o estado do jogo. Ou seja, é o conjunto de regras colocadas
para os jogadores. Essas regras determinam todas as possíveis ações dos jogadores, quais são as
condições de vitórias, os objetivos do jogo, as recompensas, etc. Tudo o que é necessário para o
funcionamento correto do game. Por exemplo, a obrigatoriedade de assistir a um vídeo ou a neces-
sidade de cumprir uma tarefa. O processo de uma sala de aula utiliza, de certa forma, dinâmicas de gamificação. Os pro-
fessores ofereceriam as regras, os desafios e a forma como o jogador irá atingir os objetivos esta-
belecidos. 2 GAMIFICAÇÃO E APRENDIZAGEM Entendemos, contudo, que a maioria dos professores se limitem ao modelo clássico de
aprendizagem baseado em pontos (notas), missões (lições de casa) e conquistas (estrelas e outros
métodos). Sendo assim, o papel da gamificação aplicada à aprendizagem deverá trabalhar com
mecânicas para o repensar o balanceamento de ações, pontuações, permissões, objetivos e outros ReCaPe | Vol. 8 | Nº. 3 | Ano 2018 | set./dez. | p. 436 AMIFICAÇÃO E GESTÃO DE PESSOAS: UM ESTUDO DE CASO SOBRE TREINAMENTO E AMBIENTE DE DIVERSIDADE CULTURAL elementos mecânicos para equilibrar a experiência de jogo com o aprendizado. É neste eixo da me-
todologia que as teorias do game design tem mais a contribuir. A experiência do usuário (User Experience, em inglês ou simplesmente UX) é fundamental
nos jogos digitais. É através da interface que o jogo se materializa para o jogador, e tornar o sistema
didático e acessível é uma estrutura que merece uma atenção própria. Nos jogos analógicos ou de
interpretação, onde se enquadrariam hoje os modelos tradicionais de aula, essa estrutura apresenta
preocupações com tutoriais (ensinar sobre o próprio jogo) e sistemas de feedback (resultados rápi-
dos para as ações realizadas), para que o jogador tenha menos atenção em como jogar e mais no
gameplay, storytelling e na aprendizagem. Em outras palavras, não podemos deixar o jogador se
perguntar “mas como eu jogo isto?”. A tecnologia, finalmente, é um elemento viabilizador, o apoio do processo. Simulações comple-
xas podem exigir um apoio tecnológico maior, mas tudo depende dos objetivos de aprendizagem e
dos recursos disponíveis ao designer. Não deveríamos considerar apenas o suporte dos equipamen-
tos e programas digitais aqui, mas toda a estrutura que permite e facilita a aplicação dessas técnicas
como os softwares de desenvolvimento e o hardware utilizado para consumir o jogo. Essa estrutura aplicada de forma estruturada e alinhada com os objetivos de aprendizagem,
convergindo com técnicas de ensino e em um ambiente lúdico, faz com que a utilização de gamifica-
ção em processos de treinamento corporativo possam atingir melhores resultados, como pretende-
mos explorar mais a seguir na análise do estudo de caso. ReCaPe | Vol. 8 | Nº. 3 | Ano 2018 | set./dez. | p. 437 3 ESTUDO DE CASO A entrevista seguiu um roteiro semi-estruturado com o seguinte formato:
•
Conceito: Foram entrevistadas em uma única reunião, com duração de pouco mais de uma hora, as duas
colaboradoras da empresa. A entrevista seguiu um roteiro semi-estruturado com o seguinte formato: •
Conceito: •
O que é gamificação? •
Que necessidades corporativas a gamificação está atendendo hoje? Processo de contratação e desenvolvimento: •
Processo de contratação e desenvolvimento: •
Qual grau de envolvimento que a área de treinamento terá com os públicos de
interesse envolvidos na gamificação? •
Qual é a expectativa que a empresa tem do resultado da gamificação? •
Continuidade de projetos em gamificação: 3 ESTUDO DE CASO O caso escolhido para o entendimento de como a gamificação nos processos de treinamento
corporativo pode gerar melhores resultados não levará em conta resultados numéricos, uma vez que,
se trata de um projeto piloto e, portanto, não temos autorização para divulgação do nome da empresa
e de seus contatos. Contudo, não vemos essa limitação como problema para esse artigo, visto que,
nosso objetivo é demonstrar quais as motivações e o entendimento que levam as empresas na busca
por processos gamificados para o desenvolvimento de seu quadro de colaboradores. Os profissionais alvo do projeto piloto estão ligados à área de obras da empresa em questão. Contemplando desde engenheiros até cargos mais baixos da hierarquia, como ajudantes encarrega-
dos de “cavar buracos”. Entendendo que, os colaboradores de grau técnico mais elevado na hierar-
quia eram os responsáveis pelo conteúdo do treinamento. ReCaPe | Vol. 8 | Nº. 3 | Ano 2018 | set./dez. | p. 437 Matheus Matsuda Marangoni, Mauro Miguel Rodrigues Berimbau Podemos, no entanto, citar a empresa responsável pelo desenvolvimento do projeto piloto. Tra-
ta-se da Sioux Game Brasil , uma empresa que se intitula como agência multidisciplinar que combina
tecnologia, comunicação e gamificação para seus clientes. O levantamento de dados utilizou como método um levantamento qualitativo através de entre-
vistas em profundidade com profissionais da Sioux, em especial os game designers responsáveis
pelo piloto, e duas profissionais da área de treinamento da empresa que forma as sponsors do pro-
jeto perante a diretoria da empresa em questão. As entrevistas aconteceram após o desenvolvimento do piloto e, desta forma, nos ajuda a
trabalhar e analisar esse processo como fechado. Mesmos sem a necessidade de resultados es-
pecíficos ligados ao piloto, é importante avaliar a percepção de todas as partes envolvidas em um
processo encerrado. Primeiramente iremos trabalhar com a entrevista realizada com a empresa, para assim, de-
monstrar o entendimento destes profissionais para com o processo de gamificação, quais suas ex-
pectativas iniciais e como veem o produto apresentado como solução. A seguir, iremos aprofundar
nossa análise junto a um dos profissionais envolvidos no desenvolvimento da gamificação, enten-
dendo como o processo foi conduzido e quais as dificuldades encontradas. Para, por fim, concluir-
mos o estudo com a análise dos dados coletados para com a teoria apresentada anteriormente
nesse artigo. Foram entrevistadas em uma única reunião, com duração de pouco mais de uma hora, as duas
colaboradoras da empresa. GAMIFICAÇÃO E GESTÃO DE PESSOAS: UM ESTUDO DE CASO SOBRE TREINAMENTO E AMBIENTE DE DIVERSIDADE CULTURAL Inicialmente questionamos qual o entendimento das profissionais com relação ao que seria
gamificação, para que pudéssemos avaliar o conceito entendido e assim, validar a percepção quanto
ao seu valor como treinamento. De forma geral, as duas entrevistadas nos colocaram que, entediam
a gamificação apenas como um jogo, ou seja, como um programa de computador ou celular, que po-
deria ser construído para fins de treinamento. Não havia, antes de iniciarem o projeto, clareza e maior
entendimento do que a gamificação é e do que ela é capaz. Conceitos como competição e recom-
pensa foram os mais destacados pelas entrevistadas como entendimento de um jogo no começo. Contudo, uma das entrevistadas relatou ter conhecimento do termo gamificação há alguns anos. Seu
contato inicial foi por contato com uma empresa de recrutamento. Ela relatou que, seu entendimento
de gamificação nesse primeiro contato, levavam ainda em conta a questão da competitividade como
destaque principal. Em suas palavras: “A competitividade está na essência do ser humano. É ele buscar estar no topo diante de vá-
rios elementos. É gerar uma série de elementos para trabalhar uma competitividade, no caso
sadia para a organização, no processo de aprendizagem. Porque é lúdico. E uma vez que é
lúdico, você aprende bem mais fácil.” Outro fator de destaque indicado para a escolha pelo uso da gamificação em processos de trei-
namento, foi a questão tecnológica. Segundo as entrevistadas, a tecnologia favorece a otimização do
tempo. Fazendo com que o treinamento não precise ser presencial e, portanto, evitando longos pe-
ríodos de pausa no trabalho dos colaboradores. Assim como, a facilidade de oferecer o treinamento
para diversas unidades da empresa, que não necessariamente estão localizadas próximas da central
ou até mesmo reduzindo os gastos com deslocamento e estadia de professores/ instrutores para fora
de São Paulo. Contudo, é importante destacar que, segundo uma das entrevistadas, a importância
sobre o uso da tecnologia, também, está associada a um certo grau de empirismo, uma vez que, foi
citada a melhor adesão com o treinamento gamificado com sua percepção de como as pessoas tem
passado cada vez mais de seu tempo no celular. A questão de que a gamificação pode obter melhores resultados de engajamento/ motivação
para com o treinamento, é destacada por ambas as entrevistadas. •
Continuidade de projetos em gamificação: •
Quando tiveram a ideia de gamificar, tinham noção do valor necessário para
investimento? •
Como veem a receptividade da empresa para continuidade do projeto? ReCaPe | Vol. 8 | Nº. 3 | Ano 2018 | set./dez. | p. 438 GAMIFICAÇÃO E GESTÃO DE PESSOAS: UM ESTUDO DE CASO SOBRE TREINAMENTO E AMBIENTE DE DIVERSIDADE CULTURAL GAMIFICAÇÃO E GESTÃO DE PESSOAS: UM ESTUDO DE CASO SOBRE TREINAMENTO E AMBIENTE DE DIVERSIDADE CULTURAL As duas profissionais destacam
a dificuldade em motivar os colaboradores a participarem ativamente de treinamentos, que em parte
dos casos, consiste em reciclagem e/ou revisão de rotinas de segurança. Uma das entrevistadas
indica que em sua visão a gamificação derruba as barreiras que causam a falta de empatia com o
processo de treinamento. Mais uma vez, a característica indicada foi a ludicidade do processo, que
oferece o conteúdo de uma forma mais agradável para os funcionários, além de, possibilitarem uma
melhor retenção do conteúdo alvo do treinamento em contraponto com práticas presenciais, em ma-
teriais impressos ou em vídeo. ReCaPe | Vol. 8 | Nº. 3 | Ano 2018 | set./dez. | p. 439 Matheus Matsuda Marangoni, Mauro Miguel Rodrigues Berimbau A interatividade de um game também foi relevante para o processo de decisão pela escolha
da gamificação como mecânica de treinamento. A passividade encontrada nos processos ditos mais
tradicionais, como vídeo aula e aula presencial são, segundo as propositoras do projeto, outra carac-
terística que as motivou na busca por entender melhor o que seria a gamificação e por solicitar um
projeto piloto. A composição de teoria e prática e a necessidade de tomada de decisão por parte do
público-alvo, foram destacadas na entrevista. O auto-conhecimento e o reconhecimento são fatores de destaque em um processo gamifi-
cado na visão das entrevistadas. O uso de mecânicas que oferecem feedbacks mais constantes e
demonstram para os pares em que nível o colaborador se encontra, seriam questões melhores resol-
vidas a partir de um processo gamificado e, assim, possibilitam maior engajamento dos funcionários
alvos do treinamento. Ao questionarmos os objetivos da área de treinamento para a escolha da gamificação, enten-
demos que um dos principais motivos é a busca por novas formas de trabalho. Sem maior aprofun-
damento, até por não ser o objetivo deste estudo de caso, as profissionais indicaram que a há neces-
sidade de um melhor alinhamento de conteúdo por toda a empresa. Indicaram que, essa integração
deve ser total por todas as áreas da empresa, ou seja, estão desenvolvendo conteúdo de cada uma
das etapas da prestação de serviços da empresa de forma a oferecer um visão completa para todos
os departamentos de forma organizada e coesa. GAMIFICAÇÃO E GESTÃO DE PESSOAS: UM ESTUDO DE CASO SOBRE TREINAMENTO E AMBIENTE DE DIVERSIDADE CULTURAL Entendem que a gamificação é uma forma interes-
sante de gerar conteúdo que seja de fácil assimilação e que pode gerar uma visão integrada em uma
única plataforma para todas as etapas de prestação de serviços da empresa. Uma preocupação da empresa é o alto custo da plataforma, uma vez que, o processo de ga-
mificação proposto para a empresa consiste em um conjunto de conteúdos gamificados a partir do
uso de um aplicativo de celular. Não tivemos acesso aos valores de tal plataforma, contudo, como
ordem de grandeza, é possível usarmos como parâmetro um custo de aproximadamente 50 a 100
mil reais por conteúdo/ etapa do game. Segundo as entrevistadas, a empresa tem os recursos ne-
cessários para tal investimento, mas há a necessidade uma melhor parametrização das metas que
serão alcançadas com a gamificação. Contudo, o maior desafio indicado pelas profissionais, é a
organização e preparação de todo o conteúdo necessário para o projeto. Elas entendem que, a sem
um maior cuidado com essa etapa interna da empresa, qualquer game não atingiria resultados que
justificassem tal investimento. Assim sendo, a partir da entrevista com a empresa, pudemos entender que o projeto ainda está
em uma fase piloto devido a falta de cultura da empresa de forma geral em adotar novos processos
de treinamento. As profissionais encarregadas pelo projeto, relatam ainda que, o alto investimento,
a necessidade de estruturar melhor o conteúdo e um certo grau de ceticismo por parte da diretoria ReCaPe | Vol. 8 | Nº. 3 | Ano 2018 | set./dez. | p. 440 GAMIFICAÇÃO E GESTÃO DE PESSOAS: UM ESTUDO DE CASO SOBRE TREINAMENTO E AMBIENTE DE DIVERSIDADE CULTURAL são fatores que podem dificultar a adoção mais rápida da gamificação de forma mais ampla pelas
empresas. Nossa segunda ida a campo para aprofundar o estudo de caso contou com uma entrevista
junto a um dos game designers envolvidos no projeto. É importante destacar que o papel do game
designer é o entendimento das necessidades ligadas à gamificação e o desenvolvimento da mecâ-
nica e, neste caso, do storytelling utilizado para gerar o engajamento. Foi entrevistado o profissional
ligado à Sioux. A entrevista ocorreu no escritório da empresa, localizado no bairro do Brooklin em São
Paulo e contou com o questionário semi estruturado que contemplava principalmente os seguintes
tópicos: Desafios. Processo. Ganhos de um treinamento gamificado. GAMIFICAÇÃO E GESTÃO DE PESSOAS: UM ESTUDO DE CASO SOBRE TREINAMENTO E AMBIENTE DE DIVERSIDADE CULTURAL Entendemos que é importante, indicarmos o processo de construção do game, que foi dividi-
do em três etapas. Para melhor localizar a relevância na escolha de um game designer para nossa
coleta de informações. Primeiro a equipe da Sioux recebeu o briefing da empresa e montou equipe
para o desenvolvimento do projeto piloto. Tal equipe foi dividida em um time de game designers, um
de designers digitais e, por fim a equipe de programação. Nosso entrevistado participou do primeiro
grupo, que foi responsável por traduzir o conteúdo e formular o processo de aprendizagem ligado às
mecânicas gamificadas de um jogo digital para plataforma móvel. Ao questionarmos os principais desafios com esse caso, o game designer, nos indicou não ter
encontrado dificuldades diferentes de outros projetos, sejam eles maiores ou menores que nosso
caso analisado. Contudo, nos foi colocado que todo processo de gamificação tem como grande de-
safio o entendimento profundo do conteúdo que será gamificado. Esse conhecimento dependendo
do trabalho varia de complexidade e nas formas de obtenção. Neste caso, por se tratar de uma em-
presa que realiza, dentre seus serviços, obras de engenharia, foi necessário ir a campo para acom-
panhar a execução do serviço escolhido para o projeto piloto. Outro fator de atenção indicado, é a necessidade de entender a linguagem da empresa e de
seu público. Ou seja, analisar como os profissionais se comunicam e quais os termos técnicos funda-
mentais para as atividades executadas. O game designer nos indicou que uma das principais carac-
terísticas deste caso era a necessidade de trabalhar com públicos de diferentes níveis culturais. Des-
de um profissional especializado em cavar buracos até o engenheiro responsável pela obra como um
todo. O game deveria trabalhar uma linguagem que fosse adequada para essa diversidade cultural,
mas não poderia de utilizar termos técnicos bastante específicos. Entendemos essa preocupação
quando lembramos a necessidade de gerar engajamento para com o game, ou seja, o treinamento
gamificado não poderia trazer o conteúdo de forma a não conseguir se comunicar adequadamente
com um funcionário menos instruído, como um ajudante de pedreiro, ao mesmo tempo que precisa
engajar também um engenheiro com grau superior completo. ReCaPe | Vol. 8 | Nº. 3 | Ano 2018 | set./dez. | p. GAMIFICAÇÃO E GESTÃO DE PESSOAS: UM ESTUDO DE CASO SOBRE TREINAMENTO E AMBIENTE DE DIVERSIDADE CULTURAL 441 Matheus Matsuda Marangoni, Mauro Miguel Rodrigues Berimbau O processo de desenvolvimento da gamificação foi de três meses a partir do início do projeto
piloto com o briefing da empresa. A equipe de game designers, envolvida durante todo o processo,
participou de três reuniões com a empresa e foi a campo em uma ocasião para acompanhar a exe-
cução de um serviço de obra. Durante os três meses de produção do piloto, os game designers tra-
balharam pelo menos dois terços do tempo na preparação das mecânicas e adaptação do conteúdo
e um terço no acompanhamento das demais etapas de produção, o design gráfico e a programação. Desta forma, podemos entender que a mecânica de aprendizagem gamificada é a etapa do game
mais trabalhosa e que mais consome tempo. Não estamos desconsiderando a identidade visual ou
a programação, as quais são também indispensáveis para o game. Contudo, entendemos a partir da
entrevista que é a partir do entendimento do conteúdo e da elaboração da estratégia de gamificação
para o mesmo que origina o trabalho das etapas seguintes. Na visão do game designer entrevistado, um processo de treinamento gamificado de forma
correta, gera maior engajamento para com o conteúdo e melhor retenção dos conceitos transmitidos
no game. Desta ainda, a importância do storytelling para tornar o jogo lúdico e atrativo para os jo-
gadores. Sem esquecer a possibilidade de um maior controle do processo de treinamento, uma vez
que, a plataforma tecnológica possibilita a coleta de dados relevantes para metrificar os resultados,
tais como: quantidade de acesso, tempo gasto com o game, número de tentativas e número de erros
e acertos. Dados que ajudarão a entender se o game é relevante, para controlar a adesão ao treina-
mento e para ajudar a empresa a metrificar seus resultados. Por fim, entendemos a partir da visão do game designer que a gamificação nos treinamentos
corporativos tendem a crescer nos próximos anos e que com o aumento de projetos e evolução da
tecnologia utilizada, o custo deve cair substancialmente, ajudando assim na melhoria do processo. 4 CONSIDERAÇÕES FINAIS Conforme indicamos anteriormente, a carreira é um conjunto de competências adquiridas ao
logo da vida pessoal e profissional das pessoas. Juntando a esse fato, a definição de gamificação
como aplicação de técnicas de game buscando o melhor engajamento, entendemos o emprego da
gamificação como uma forma de favorecer o contínuo aprendizado. Tanto em função da ludicidade,
que torna o processo mais agradável, quando do engajamento em si, buscando maior interação e
retenção do que se deseja educar. O caso apresentado, nos ajuda a destacar tais questões, visto que indica a busca da firma
por formas diferentes do treinamento presencial e/ ou video-aula. Formas, segundo as profissionais
entrevistadas, que tomam tempo e não favorecem a retenção do conteúdo apresentado. Quando ReCaPe | Vol. 8 | Nº. 3 | Ano 2018 | set./dez. | p. 442 GAMIFICAÇÃO E GESTÃO DE PESSOAS: UM ESTUDO DE CASO SOBRE TREINAMENTO E AMBIENTE DE DIVERSIDADE CULTURAL vemos a questão do tempo, podemos destacar a dificuldade do processo dito tradicional que, requer
deslocamento físico e horas de trabalho. Já na utilização de uma plataforma gamificada, há a busca
da otimização do tempo. Tanto os profissionais não precisam realizar deslocamento, quanto o tempo
não é o mesmo para todos e, tão pouco, necessita que todos os colaboradores estejam ligados na
plataforma no mesmo momento. O game, ao ser lúdico, tende a transpassar a divisão entre a vida profissional e a vida pessoal,
uma vez que, o storytelling, tão importante no processo de gamificação, buscará, de certa forma,
suavizar o conteúdo do treinamento corporativo. Conceito de destaque nas teorias de carreiras sem
fronteiras. Os indivíduos serão conduzidos de forma lúdica e interativa pelo game/ treinamento. Duas
características colocadas como altamente relevantes pela empresa do estudo de caso, para definir
pela escolha desta dinâmica. Contudo, o uso de uma plataforma tecnológica para o emprego da gamificação não apresenta
apenas questões positivas. O principal ponto de atenção, segundo as entrevistas tanto com a em-
presa contratante, quanto indicado pelo profissional da Sioux, é a preparação do conteúdo. As en-
trevistadas da firma, indicaram a importância deste alinhamento interno e para com a Sioux. O game
designer, demonstrou como maior desafio a necessidade de aprender conteúdos desconhecidos e
técnicos para desenvolver o game. 4 CONSIDERAÇÕES FINAIS Por fim, devemos indicar a continuidade de novas pesquisas ligadas ao treinamento corpo-
rativo gamificado, uma vez que, esse artigo inicial, objetiva abrir a discussão de como o uso de
mecânicas gamificadas estão alinhadas com as perspectivas de desenvolvimento de pessoas nas
firmas. Assim como, de que forma, as empresas brasileiras estão preparadas e dispostas a investir,
em muitos casos altos recursos financeiros e tempo, na promessa de maior engajamento e eficácia
na aprendizagem corporativa. REFERÊNCIAS BIBLIOGRÁFICAS AARSETH, E. (1997). Cybertext: Perspectives on Ergodic Literature. Maryland: The Johns Hopkins
University Press. DUTRA, Joel Souza (2010). Gestão de Carreiras na Empresa Contemporânea. São Paulo. Editora
Atlas. DUTRA, Joel Souza (2010). Gestão de Carreiras na Empresa Contemporânea. São Paulo. Editora
Atlas. FRASCA, G. (2003). Simulation vs Narrative: Introduction to Ludology. Nova Iorque: Routledge. Disponivel em: <http://ludology.org/articles/VGT_final.pdf>. Acesso em: ago. 2018. FRASCA, G. (2003). Simulation vs Narrative: Introduction to Ludology. Nova Iorque: Routledge. Disponivel em: <http://ludology.org/articles/VGT_final.pdf>. Acesso em: ago. 2018. FRASCA, G. (2007). Play the message: play, game and videogame rethoric. IT University of
Copenhagen. FULLERTON, T. (2008) Game Design Workshop: a playcentric approach to creating innovative
games. Burlington: Elsevier. ReCaPe | Vol. 8 | Nº. 3 | Ano 2018 | set./dez. | p. 443 Matheus Matsuda Marangoni, Mauro Miguel Rodrigues Berimbau KOSTER, R. (2005). A theory of fun for Game Design. Arizona: Paraglyph Press. KOSTER, R. (2005). A theory of fun for Game Design. Arizona: Paraglyph Press. PAHARIA, Rajat (2013). Loyalty 3.0 - How to revolutionize customer and employee engagement with
big data and gamification. Canadá: McGraw-Hill Education. SALA, Otávia Travençolo Muniz e TREVISAN, Leonardo Nelmi (2014). A construção de carreira em
ambientes inovativos - um estudo nas empresas de base tecnológica. Revista de Administração e
Inovação, São Paulo, v. 11, n.2, p. 154-178. SALEN, K.; ZIMMERMAN, E. (2004). Rules of Play: Game design fundamentals. Cambridge: MIT
Press. SCHELL, J. (2008). The Art of Game Design: A Book of Lenses. Nova Iorque: CRC Press. VELOSO, Elza Fátima Rosa, DUTRA, Joel Souza e NAKATA, Lina Eiko (2016). Percepção sobre
carreiras inteligentes: diferenças entre as gerações y, x e baby boomers. REGE - Revista de Gestão,
23, p. 88–98. YIN, Robert K. (2018). Case Study Research and Applications: Design and Methods. SAGE
Publications. Kindle Edition. WERBACH, K.; HUNTER, D. (2012). For the Win: How Game Thinking Can Revolutionize Your
Business. Filadélfia: Wharton Digital Press. WINN, B. (2009). The Design, Play, and Experience Framework. In: FERDIG, R. Handbook of
Research on Effective Electronic Gaming in Education. 1a. ed. Nova Iorque: Information Science
Reference, v. III, Cap. LVIII, p. 1010-1024. ReCaPe | Vol. 8 | Nº. 3 | Ano 2018 | set./dez. | p. 444
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Theorizations about the child's Play and Movent: implications for the pedagogical practice of Physical Education in Early Childhood Education and other problematizations
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Pro-Posições
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cc-by
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2 References correction and bibliographic normalization services: Vera Lúcia Fator Gouvêa Bonilha -
verah.bonilha@gmail.com
3 English version: Viviane Ramos - vivianeramos@gmail.com 1 Responsible editor: Carmen Lúcia Soares - https://orcid.org/0000-0002-4347-1924 (ii) Universidade Federal do Espírito Santo – UFES, Centro de Educação Física e Desportos – CEFD,
Departamento de Ginástica, Vitória, ES, Brasil. http://orcid.org/0000-0002-4056-5159,
fqalmeida@hotmail.com http://dx.doi.org/10.1590/1980-6248-2020-0139EN http://dx.doi.org/10.1590/1980-6248-2020-0139EN Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022 3 English version: Viviane Ramos - vivianeramos@gmail.com 3 English version: Viviane Ramos - vivianeramos@gmail.com 2 References correction and bibliographic normalization services: Vera Lúcia Fator Gouvêa Boni
verah.bonilha@gmail.com (iii) Universidad de Valparaíso, Facultad de Medicina, Escuela de Educación Parvularia, Viña del Mar,
Chile. https://orcid.org/0000-0002-4277-0535, alberto.moreno@uv.cl e-ISSN 1980-6248 http://dx.doi.org/10.1590/1980-6248-2020-0139EN Abstract This article analyzes the theorization that has been undertaken about "playing and
movement" and that can be considered as a possibility to support the intervention
of Physical Education with Early Childhood Education. In methodological terms,
it operates with some keys of reading and interpretation, attempting to understand
childhood concepts; of body and movement which dialogue with "playing and
moving", as well as identifying the definition of what would be the object of teaching
Physical Education in Early Childhood Education and the definition of the teacher's
role in this context. Then, it makes some considerations regarding the potentials,
limits, and challenges of this theorization for Physical Education in Early Childhood
Education. Keywords: Physical Education, Childhood Education, Play and Move e-ISSN 1980-6248 e-ISSN 1980-6248 Teorizações sobre o Brincar e o Se-movimentar da criança:
implicações para a prática pedagógica da Educação Física na
Educação Infantil e outras problematizações Teorizaciones sobre Jugar y Moverse de los niños y niñas:
implicaciones para la práctica pedagógica de la Educación Física
en la Educación Infantil y otras problematizaciones Rocha, Maria Celeste(i)
Almeida, Felipe Quintão(ii)
Moreno, Alberto(iii) (i) Universidade Federal do Espírito Santo – UFES, Centro de Educação Física e Desportos – CEFD,
Programa de Pós-Graduação em Educação Física – PPGEF; Prefeitura Municipal de Vitória, Secretaria
Municipal de Educação, Gerência de Formação e Desenvolvimento em Educação. Vitória, ES, Brasil. https://orcid.org/0000-0002-2572-9098, mcrocha@prof.edu.vitoria.es.gov.br (ii) Universidade Federal do Espírito Santo – UFES, Centro de Educação Física e Desportos – CEFD,
Departamento de Ginástica, Vitória, ES, Brasil. http://orcid.org/0000-0002-4056-5159,
fqalmeida@hotmail.com (iii) Universidad de Valparaíso, Facultad de Medicina, Escuela de Educación Parvularia, Viña del Mar,
Chile. https://orcid.org/0000-0002-4277-0535, alberto.moreno@uv.cl (iii) Universidad de Valparaíso, Facultad de Medicina, Escuela de Educación Parvularia, Viña del Mar,
Chile. https://orcid.org/0000-0002-4277-0535, alberto.moreno@uv.cl Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022
1/26 Resumo Resumo
Este artigo analisa a teorização que vem sendo empreendida sobre “brincar e se-movimentar” e que
pode ser considerada como uma das possibilidades de fundamentar a intervenção da Educação
Física com a Educação Infantil. Em termos metodológicos, opera com algumas chaves de leitura e
interpretação, na tentativa de compreender as concepções de infância; de corpo e movimento com as
quais o “brincar e se-movimentar” dialogam, assim como, identificar a definição do que seria o
objeto de ensino da Educação Física na Educação Infantil e qual o papel do professor nesse
contexto. Em seguida, tece algumas considerações a respeito das potencialidades, dos limites e dos
desafios dessa teorização para a Educação Física na Educação Infantil. Palavras-chaves: Educação Física, Educação Infantil, Brincar e Se-movimentar e-ISSN 1980-6248 e-ISSN 1980-6248 Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022 Resumen Este artículo analiza la teorización que se ha emprendido sobre “jugar y moverse” y que se puede
considerar como una de las posibilidades para sustentar la intervención de la Educación Física con
la Educación Infantil. En términos metodológicos, opera con algunas claves de lectura e
interpretación, en un intento por comprender los conceptos de la infancia; de cuerpo y movimiento
con los que dialogar “jugar y moverse”, así como identificar la definición de cuál sería el objeto de
la enseñanza de la Educación Física en Educación Infantil y la definición del rol del docente en
este contexto. Luego, hace algunas consideraciones sobre las potencialidades, límites y desafíos de
esta teorización de Educación Física en Educación Infantil. Palabras Clave: Educación física, Educación infantil, Jugar y moverse Palabras Clave: Educación física, Educación infantil, Jugar y moverse Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022 Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022 http://dx.doi.org/10.1590/1980-6248-2020-0139EN 1. Introduction In the Brazilian context, the reflection on Physical Education (P.E.) in Childhood
Education is permeated by extremely challenging questions, mainly when we consider the
specificity of this educational segment. Rocha (2011) argues that we cannot ignore the existing
tensions when discussing the legitimacy of a "school subject", conducted by a specialist teacher
in Childhood Education. In other words, we would like to call attention to the existing
implications when a curricular component such as Physical education, as a subject responsible
for a piece of specific knowledge, becomes part of the curriculum of a segment of K-12
education, which is not organized by specific subjects or knowledge but by fields of experience. If, on one hand, these issues were strongly present in the academic debates in the area,
on the other, the fact that P.E. is already established as a practice in many contexts of Brazilian
Childhood Education, including the work of a specialist teacher, has potentialized the progress
of discussions. In this direction, it is possible to follow a considerable increase of studies and
publications in the area to reflect on their articulation with this educational segment. Thus, they
show the need and the possibilities to build an integrated work, not fragmented, which considers
children's specificities and new concepts of Childhood Education. The exercise of analyzing the academic production about P.E. in Childhood Education
has indicated that some theoretic-methodological perspectives have been built to think of
pedagogical interventions for children from 0 to 5 years old. Thus, in this article, we have chosen
to present, discuss, and problematize the efforts of the theorization about early childhood
education that has been conducted in the field of Physical education from the dialogue with the
Human Self-Movement Theory (HSMT) and phenomenology itself. The option to analyze such theorization, in particular, comes from the fact that this
exercise of thinking about the contributions of P.E. in Childhood education has taken place
from reasoning and dialogue as knowledge accumulated and produced by the field o Physical
Education itself. Its main author is Elenor Kunz, author of Abordagem Crítico-emancipatória and
disseminator of HSMT in Brazil. In methodological terms, this study is a theoretical study focused on the analysis of
Kunz's texts and those co-written by him with his master's and doctorate students. About this
production, we highlight that there is no specific text about HSMT with a theoretical- Pro-Posições | Campinas, SP | V. e-ISSN 1980-6248 e-ISSN 1980-6248 e-ISSN 1980-6248 methodological perspective for P.E. in Childhood Education. However, there is a group of texts
on issues inherent to P.E. in Childhood education, children’s play, and movement. Nevertheless, though it is not a clearly systematized proposal, it is possible to perceive
the indication that HSMT works, which establishes a theorization about "Playing and Self-
movement" that could eventually have a status of a propositional framework for action
perspectives on P.E. teachers' work in Childhood Education. In other words, from the counter
position of a certain type of intervention, the studies analyzed indicate another modus operandi
for teachers, providing didactic-pedagogical elements that help teachers assume play as an
element of P.E. and how to work with them. Thus, in a sense, we understand that this
production indicates which formative dimension play should have in children’s lives. With that said, our first criterion to select texts was based on an analysis of the titles of
bibliographic works in Kunz' Lattes curriculum. We have then selected those that mentioned in
the title the terms: “play and move”, “children/childhood” and/or “childhood education”. When we had doubts, the reading of the abstract provided a second criterion. In this way, we
have identified books, book chapters, articles, and complete articles published in congresses. However, we have excluded the texts from congresses because we understand that they were
similar to book chapters and articles already published and which were part of the sample. In total, we selected 25 productions divided among the organization of a book, the
writing of 13 book chapters, and the publication of 12 articles (see the table in the appendix). These productions were concentrated between the years 2005 and 2019, showing, therefore,
that they were current publications and, in a way, recurrent in the production of the author in
this time framework, corresponding to 12% of all his article production and 33% of his
publication of book chapters. Besides this, these publications are mostly from collaborations
with masters and Ph.D. students. Only 2 book chapters were individually published by Kunz. We have also seen that the main arguments of the co-authors’ texts derive from the authors’
individual productions, published in 2005 and 2007. 1. Introduction 33 | e20200139EN| 2022
3/26 Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022 3/26 http://dx.doi.org/10.1590/1980-6248-2020-0139EN Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022
4/26 e-ISSN 1980-6248 Regarding the method of analysis, we were based on Bardin (2011) to analyze content
and propose the creation of categories that allow us to identify and group common elements in
the analyzed texts and that, to a certain measure, can be seen in the ensemble of productions
that intend to guide pedagogical practices. Therefore, we read the texts based on the
identification of some important categories to understand this proposal, such as conceptions of Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022 4/26 http://dx.doi.org/10.1590/1980-6248-2020-0139EN Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022
5/26 Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022 e-ISSN 1980-6248 e-ISSN 1980-6248 childhood, body, and movement with which they dialogue; the definition of the teaching
object/specificity of P.E. in Childhood education; and the definition of teachers’ roles in this
context. These analytical categories emerge from a double movement: first, our gaze does not
start from a specific perspective or model of proposition for P.E. in Childhood Education but,
as said, part of more general aspects that we believe are pertinent. Second, we consider the
elements and the arguments present in the analyzed sources. Therefore, we organize our dialogue with the selected production intending to
understand the effort made by the authors and create a synthesis of the practices of P.E. in
Childhood Education to, later, present our analyses on these studies. The text is structured into
two topics, followed by the final remarks. The first topic refers to the proposal of "playing and
moving" from the previously described reading keys. After, we write some considerations on
the potentials, limits, and challenges of this proposal for P.E. in Childhood Education. 2. From the Human Self-Movement Theory to “Play and
Move”: fundaments for Physical Education in Childhood
Education In the 1990s, based on the works of Trebels, Elenor Kunz presented HSMT as a
possibility to ground pedagogical theory for Physical Education. Though the author had already
been discussing the concepts of children's "play and move" since 1991, it was only in 2005 that
we find publications in which he thinks specifically about the issue of P.E. in Childhood
Education. Besides his publications, Kunz has been conducting studies on this topic with his
master's and doctorate students. Before presenting the theorization exercise, we should
conceptualize one of the key concepts of his work, also used to think about education in early
childhood. Kunz defends an understanding of human movement from its dialogic potential
because, for him, it manifests itself through sensibility, perception, and human intuition. Summing up, this conception considers not only the biomechanical movement of the subject
but the human being that moves (Kunz, 2005). The expression “self-movement” characterizes Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022
5/26 Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022 5/26 5/26 http://dx.doi.org/10.1590/1980-6248-2020-0139EN e-ISSN 1980-6248 e-ISSN 1980-6248 the relation of the sense and meaning that human beings establish with the world to move
(Kunz, 2005). This “self-movement” refers to an expression translated from German focusing
on the "self", i.e., the subject of the movement. The pedagogical proposal developed by Kunz for P.E. is called Critical-Emancipatory. According to the author, "[...] Physical Education should contribute to developing certain
competencies that cannot be summed up in the objective competence of know-how but include
the social, linguistic, and creative competence" (Kunz, 2005, p.16). Therefore, Kunz considers
students’ formation to transcend technical and instrumental formation. In the proposal of Self-
movement Theory, grounding the pedagogical practice of Physical Education, the author
operates with some concepts of phenomenology, especially the “Phenomenology of
Perception” developed by Merleau-Ponty (Almeida et al., 2013). When analyzing these concepts and phenomenological ideas that compose the HSMT
framework and discussing their use in Brazilian Physical education, Almeida et al. (2013)
recognize its importance but problematize the fact that in his works Kunz did not give the due
attention to the reviews produced by Merleau-Ponty. This means that “Kunz continues to
operate, in the scope of HSMT, with theses revised by Merleau-Ponty himself” (Ghidetti et al.,
2014, p.329). Some problematization on the appropriation of Merleau-Ponty’s works by Kunz in the
field of P.E. can be found in the works of Ghidetti (2012), Almeida et al. (2013), and Guidetti
et al. (2014). For them, by basing himself mainly on the “Phenomenology of perception”, Kunz
disregards that Merleau-Ponty himself reviewed his thoughts affecting the theses presented in
the 1945 book. From the perspective of these authors, it means saying that HSMT is "stuck" to
some ambiguities of Merleau-Ponty’s thought. This can be seen by the fact that HSMT affirms that “the production of senses and
meanings without explaining the connection between perception and language” (Ghidetti et al.,
2014, p.329). In other words, contrary to what was problematized by Merleau-Ponty himself,
HSMT still operates with the idea of a pre-reflexive contact of the perceptive awareness with
oneself as previous to language. As an example, we can cite Kunz's focus on sensitiveness,
spontaneity, and childhood as possibilities to contrapose the instrumental logic of play and
human movement. Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022 http://dx.doi.org/10.1590/1980-6248-2020-0139EN e-ISSN 1980-6248 The child before thinking and perceiving the world around perceives exactly this world before
being reflected by our thought (…) this process that starts in the world previous to reflection
(pre-reflexive world) is what provides the arguments for us to understand the process of
awareness. (Surdi & Kunz, 2010, pp. 264-265) The authors also highlight that the body, understood as a body-subject, moves itself intending to perceive things by living them. We could say that this living and free
movement is an expressive act, meaningful, and unique. The expression can be enacted through
our bodies. It can reveal the meaning of our pure experiences (Surdi & Kunz, 2010, p. 274) As argued by Ghidetti et al. (2014), it is not clear how the meaning of our pure
experiences articulates with language that, in this case, would continue to be conceived as a
result of an original awareness. In other words, the passage from the perceptive scope to the
meaning referring to language, from behavior to thematization, is not clear in HSMT
framework. We highlight these questions because we identify that, in the scope of Physical
Education in Childhood Education, HSMT has also been used as an important foundation using
these same phenomenological concepts and ideas that were problematized in the field of
Physical Education. However, besides HSMT and Merleau-Ponty’s Phenomenology, we could
perceive that the theorization about early childhood education proposed by Kunz and
collaborators has interfaces, in practically all their texts, with the references of Humberto
Maturana and Gerda Verden-Zöller (2004), mainly the work Amar e brincar: fundamentos esquecidos
do humano, as well as Violet Oaklander (1980) and Honoré (2009). In the more recent texts,
mainly after 2016, we have also seen a dialogue with childhood phenomenology, disseminated
and discussed by Machado (2010, 2013). Thus, regarding HSMT and Physical Education in Childhood Education, we have
perceived that Kunz has incorporated in the concept of self-movement other discussions and
presented the “Playing and Moving” as a theoretical perspective that can ground the work with
small children. Reading the texts written by Kunz and his collaborators allows us to say that, in
general, these theories have been thematized as “playing and moving”, articulating it to the
discussions about being-child, the importance of playing and moving freely and spontaneously,
and the questions of time, creativity, art, and imagination. We have also noticed that the
proposition of “playing and moving” continues to be grounded in some criticisms of science
and rationalization, the logic of schooling and formal education, the didactic play, the adult-
centric perspective, and the impositions and the controls to which children are submitted. Kunz 7/26 http://dx.doi.org/10.1590/1980-6248-2020-0139EN 4 For more details, see Kunz (2007). e-ISSN 1980-6248 e-ISSN 1980-6248 (2007), before presenting the concepts of childhood with which he dialogues, criticizes the
studies that treat childhood as a scientific category in social, anthropological, and educational
sciences, arguing that these studies say little about children as a subject. Therefore, grounded on
the work Child Psychology and Pedagogy: The Sorbonne Lectures 1949-1952 by Merleau-Ponty (2006),
the author writes about the complexes children go through, emphasizing those he considers
important to be considered in the context of Childhood Education, such as the Weaning
Complex or Disruption, as well as talking about Perception in Children4. Considering this, we can affirm that the concept of childhood present in the theorization
of "play and self-movement" takes place through a phenomenological perspective. The child is
understood as a being-in-the-world and recognized by what she is, by how she expresses and
presents herself in the world. In other words, children are considered by their childhood nature
and as a subject that have world perceptions different than the ones of adults, thus, one should
value imagination, fantasy, and emotions (Stavisk et al., 2013). Though we have not identified a punctual definition of the concepts of body and
movement with which they operate, it is possible to infer from his writings about "Playing and
Moving" a dialogue with the ideas of the body itself and the relational body of Merleau-Ponty. In this case, there is an understanding of the body as a child that experiences it or the
understanding of the body through its expressivity and totality. Such a way to understand the
body is directly associated with the dialogic concept of movement (Costa et al., 2018; Gomes-
Da-Silva, 2007). In this perspective, playing and moving freely and spontaneously are emphasized as
what characterizes the specificity of Physical Education in Childhood Education. In our
opinion, considering propositional terms, they can be characterized as a teaching object of
Physical Education for early childhood. Besides this, according to Costa et al. (2016): Physical Education should open and broaden a valuable and ample field of teaching and research
and could, maybe in the future, become one of the most important and valued areas in the field
of Education because it teaches children, i.e., the human being in its early age, 'the art of knowing
how to live better' [original highlight] (p. 51) 8/26 Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022 8/26 e-ISSN 1980-6248 e-ISSN 1980-6248 Amidst the defense of the importance of children’s free, autonomous, and creative play
and movement, Kunz (2007) distinguishes spontaneous play and didactic play. With assertive
arguments, he affirms that play is spontaneous by nature, however, adults, aiming to prepare
children for the future, have given it a didactic dimension. For the author, the latter would
represent a play with specific educational ends, which is interested in the future of the child
from the perspective and gaze of the adult and has been greatly used in Childhood Education. Kunz (2007) argues that didactic playing, much covered in the literature even with
elements of interpretation and practical application, has been transforming the "play and
moving" into activities empty of meaning for the children and full of adult impositions with
activities recognized and accepted by the latter. As an example, the author states that, in
literature, this type of play is represented under different names, such as motor-skill learning,
psychomotricity, motricity, and game, among others, concluding that the didactic play is more concerned with content and the use of play than with the child who
plays. As in the theories of Human Movement in Physical education which focus more on the
possibilities of copying and imitating already created movements than on the child, the being
that moves. (p.20) Though established the relationship between the world of life and the experience of
free, autonomous, and spontaneous movement, when taken as a teaching object of Physical
Education in Childhood Education, and the ways they are approached in the texts, the concept
of free and spontaneous play and movement deserves more attention and explanation from the
authors, on how they are daily supported in educational institutions, at the risk of being confused
with a certain spontaneity. We have observed that, by emphasizing this free and spontaneous
play and movement of children with no clear articulation of its meaning with the pedagogical
proposals and practices that compose Childhood Education, the texts leave doubts about the
role of teachers in this process. This can be seen in a citation present in the texts of Costa et al. (2016) and Surdi et al. (2016) affirming that "children, in general, develop normally, we do not
have to do anything special for this. We simply have to like them, what happens effortlessly
most of the time" (retrieved from Verden-Zöller, 2004, p. 237). Pro-Posições | Campinas, SP | V. e-ISSN 1980-6248 http://dx.doi.org/10.1590/1980-6248-2020-0139EN e-ISSN 1980-6248 e-ISSN 1980-6248 Though we do not find a clear definition of what would be this “art of living better”,
when analyzing the texts written by Kunz and his students, we perceive a certain connection
between this expression with the possibility of playing freely and spontaneously. By
understanding children in their "being-in-the-world" and their need to fully live the present, the
authors, dialoguing with Oaklander (1980) and Maturana and Verden-Zoller (2004), consider
playing a valid activity in itself, manifested in children’s spontaneous action, in what they are,
feel, and experience during its enactment, that is, in the present and with no foreign purposes
and interference (Costa et al., 2016). With that said, we have identified that free and spontaneous playing and movement,
understood as an object of teaching or specificity of Physical Education in Childhood
Education, comes from the understanding that “playing is established as a type of
communication and dialogue of children with the world and with themselves” (Kunz, 2015,
p.10) and that it only happens through movement. However, given the centrality that the
concepts "free and spontaneous" have in the texts analyzed, we still have doubts about their
meaning in the context of pedagogical practice in Childhood Education, because the lack of a
greater explanation gives space for some problematizations, mainly, about teacher’s role in this
play that, as stated, should not suffer any external influence. We could see that Kunz (2005) uses the concept of 'life world' to criticize the use of
rationality/science in the references to the movement presented to the children, as well as to
reaffirm the importance of building references to movement and experiences that are inherent
to the subject, in these cases, free and spontaneous. In this direction, the author continues "the
sensibility, the perceptions, and human intuition develop more openly and intensely, the greater
are the degrees and life opportunities and experiences with activities established by a
spontaneous, autonomous and free self-movement" (p.20). Thus, based on these concepts, Kunz (2007) understands that the specialization of
knowledge can fragment children’s experiences in Childhood Education. In Kunz's (2005)
perspective, the experiences provided to children should not always start from an external
reference to themselves or be used without their respective authorization because, to him, all
that is external hinders children to discover the world, others, and themselves through their
resources and conditions. Pro-Posições | Campinas, SP | V. e-ISSN 1980-6248 33 | e20200139EN| 2022 http://dx.doi.org/10.1590/1980-6248-2020-0139EN 5 In her dissertation, Gomes-da-Silva (2010) deepens the theme and reflects on teachers' role from the semiotics
of Charles Pierce; however, in the article co-written with Kunz, these elements are just mentioned. e-ISSN 1980-6248 33 | e20200139EN| 2022 10/26 10/26 http://dx.doi.org/10.1
wishes and recognizing thei
teachers not to deny them
on of time and attention t
but chosen by them to dialog
Simon & Kunz, 2014, p.38
cern for children, adding to
). These obligations include seve
. (Castro & Kunz, 2015, p. ms understanding what w
Childhood Education co
onomy of children, seem
ny pedagogical work. B http://dx.doi.org/10.1590/1980-6248-2020-0139EN e-ISSN 1980-6248 http://dx.doi.org/10.1590/1980-6248-2020-0139EN e-ISSN 1980-6248 e-ISSN 1980-6248 explain the meaning of this active help, the authors only affirm that “an active help does not
mean to be a conductor or guide of the activities that child does” (p.32). Faced with this, the category that aims to understand teachers' roles, following the
theories of play and self-movement, showed itself as the weakest category and even a little
confusing, hard to define. From our perspective, it is not clear to say that teachers of Physical
Education in Childhood Education should potentialize and help free and spontaneous play and
self-movement, nor that they should help children "in the struggle to survive as children in a
world that wants to quickly transform them in a miniature adult" (Kunz, 2007, p.14). In this context, though we agree that a great part of the criticisms towards adult-centric
perspectives, it seems that, in the texts analyzed, they are, in some moments, exaggerated. When
operating with the criticism of the process of formal schooling and extending it to Childhood
Education, Kunz and Costa (2015) propose an “active and intelligent follow-up of the general
and full development of the child” (p. 32). Despite not giving further explanations about what
this would be, the authors argue that this follow-up is more adequate than the schooling process. Therefore, we identified that a great part of the criticism of this process is because the authors
consider that in Childhood Education "children are educated with purposes and objectives
formulated by adults" (p.33). It seems that adults' presence and role in the educational process
of children are always considered from a problematic viewpoint. In our perspective, this can
create a polarization between adults and children, as they attribute a negative characteristic to
adults’ actions and, it seems to us, an excessive autonomy to children. Considering this, we wish to highlight two points. First, amidst the many criticisms made
by Kunz and his collaborators towards Childhood education, few consider the specificity of this
educational segment as, some of them, seem to ignore the debate that already takes place in the
sphere of the education of young children and the proposals of Childhood Education
curriculum documents. Besides this, we found in other texts: Besides this, we found in other texts: This way, becoming sensitive to children’s wishes and recognizing their needs, without depriving
them of their right to live what they want, is a way for teachers not to deny them [our highlight]. (Staviski
et al., 2013, p.123) The educational process demands dedication of time and attention to plays and variations
developed by the children, non-guided activities, but chosen by them to dialogue with the world, with
others, and with themselves [our highlight]. (Simon & Kunz, 2014, p.384) What we perceive nowadays is a strong concern for children, adding to that an imposition of
precocious obligations (Kunz & Costa, 2015). These obligations include several activities that, often, do
not result from the children's wishes [our highlight]. (Castro & Kunz, 2015, p. 48) Based on these excerpts, we have problems understanding what would be the teachers'
role in the educational process of children in Childhood Education considering that these
arguments, besides pointing out an excessive autonomy of children, seem to place their desires
and wishes as fundamental determinants for any pedagogical work. Before continuing, we
should say that we are not questioning the importance of hearing children and considering their
needs and specificities in the context of pedagogical practices of Childhood Education. However, we think that this is different from “the right to live what they want”, or contemplate
only activities chosen by them, that is, that answer to their wills. Or even, the fact that believing
that there is no need to do anything, as children will develop normally, can lead to the idea or
interpretation that teachers' role is dispensable. Nonetheless, in other texts, such as those of Gomes-da-Silva et al. (2010), Simon and
Kunz (2014), and Kunz and Costa (2015), it is possible to identify arguments different from
those previously described. In these texts, the authors affirm that it is up to the teacher to
integrate the planning and to create adequate conditions for children's discovery and learning. The texts also preconize that the adult/teacher should be sometimes an interlocutor and not a
mediator, according to Gomes-da-Silva et al. (2010)5, while in other times an active helper in
the experiences of children’s lives, as said by Kunz and Costa (2015). However, even with such
appointments, it is also not clear what would be the teachers’ role because, in the attempt to 11/26 11/26 11/26 Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022 6 To better understand the discussion on these concepts, see Arendt (1990). e-ISSN 1980-6248 We understand that this might show some fragilities of these criticisms,
mainly, because of the current debates on the education of young children in Brazil, as well as
some assumptions of curriculum documents that have been based on studies of Childhood
Sociology and Pedagogy. That is, the current perspectives of Childhood Education have
indicated a refusal of the adult-centric perspective. They consider play as a central and
fundamental axis in the educational process of children, defending a pedagogical practice that
considers children and their interests, and aiming to overcome the understanding of children as Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022 Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022
12 12/26 http://dx.doi.org/10.1590/1980-6248-2020-0139EN Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022 To better understand the discussion on these concepts, see Arendt (1990). Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022
13/26 e-ISSN 1980-6248 e-ISSN 1980-6248 e-ISSN 1980-6248 e-ISSN 1980-6248 a "beings-to-be", to see them as a "beings-that-are" (Sarmento, 2013). Therefore, though starting
from different references, when we think about the context of pedagogical practice in
Childhood Education, we have identified some similarities in the discourses present in the
theorization of "Play and Self-movement", which do not sustain part of the criticisms presented. The second point to be highlighted refers to the polarization between adults/teachers
and children that emerge from the criticism of the adult-centric perspective, as well as the strong
valuing and idealization granted to children's autonomy, even proposing that they decide about
what they want to experience in the context of Childhood Education. In this case, the way they
criticize adults does not seem to distinguish well authority and authoritarianism6. Therefore, we
have doubts if the search for childhood autonomy can, in fact, be established from a project of
emancipation and liberation of children from what is "imposed" by adults, mainly if we believe
that they are beings under formation and, thus, need adults' care and protection (Arendt, 1990). Arendt (1990) consider absurd the ideas that treat children as an oppressed minority by
the dictates of rationality and the “world” of adults, from which they need to free themselves. In her line of thought, abolishing adults’ authority would be leaving children to their own luck,
having to deal with a greater tyranny, the one of the majority. Besides this, supposing children’s
autonomy would be transferring our responsibilities to this generation (Arendt, 1990). Furthermore, based on this author, it is wrong to think children's education by emancipating
them from adult authority or even to think of the existence of a specific world for children and
another for adults. According to Arendt (1990), there is only a world shared by all, that is,
children and adults. In this sense, we could say that it is up to the teachers to present the world to children. Therefore, in the context of P.E. in Childhood education, we see no problem in the fact that
teachers organize the activities, guide the process to elaborate knowledge that circulates in the
institutions, and propose experiences and plays to present children with different body practices
that are part of the body movement culture. This does not mean silencing children or thinking
education according to an adult-centric model! http://dx.doi.org/10.1590/1980-6248-2020-0139EN http://dx.doi.org/10.1590/1980-6248-2020-0139EN e-ISSN 1980-6248 Part of human problems, we deduce, emerges from the lack of "outlet" for creative impulses,
because the rational, functional logic of our way of thinking, has a "dehumanizing effect" and
trivializes the human, as it disrespects intuitive intelligence, present in childhood when playing
or drawing. Intuitive intelligence, which leads to creation, originates from imagination and
fantasy. Therefore, it emerges in the moments the person is completely passive or when the
conditions of the environment allow a free and spontaneous expression, such as in plays and
games, in general [our highlights]. If we take as a reference the reflection of Vaz (1995), we could question if this
theorization about "Play and Self-movement", when considering children's education, would
not be operating with a perspective that outlines a type of reduction to sensations and
perceptions. As they defend sensitivity as a condition for children's freedom and, as something
that precedes rationality, would they not be forgetting language mediation? Would devaluing
rationality be a more promising pathway in the educational process with children? Though we understand that the dialogue with Maturana allows the Theory of Play and
Self-movement to understand emotion as something that precedes reason in the sense that it
gives direction or freedom and spontaneity as something that can potentialize rational
development, we identify that the emphatic way they operate the criticisms to rationality seems
to disregard this relation between emotion, sensibility, and reason. Highlighting only the
negative aspects of rationality, or not clearly explaining how they think this relation and the issue
of language in this context, could be a big gap in this theorization. Although we understand that the theme of language is not deeply developed in the
analyzed texts, we could thematize here a ponderation already created by Almeida et al. (2013,
p.12), about how this expressive sense, which precedes meaning, is related to language. In this
direction, citing Sérgio (1987), the authors argue that the “transit from perception to concept,
the zone of pre-constitution to constitution” (p.94) can be seen as something that Merleau-
Ponty could not explain. Vaz (1995) believes that the passage from the sensitive to the intelligible implies the
practical mediation of language and emphasizes that overcoming the dichotomy of body-
thought has been one of the concerns in the field of Physical Education. 3. Playing and moving: other problematizations When analyzing the writings that outline the concept of playing and self-movement as
a theoretical-methodological perspective for Physical Education in Childhood education, we
have observed that some ideas and arguments are often repeated. Be it in the sense of presenting
the conception of children and the concepts with which they operate, be it in the perspective of
making some criticisms towards formal school, rationality, and science to reaffirm such
concepts. Therefore, we can say, more precisely, that the proposal for Physical Education in
Childhood Education undertaken by Kunz and his collaborators suppose rescuing play and self-
movement, understood by the authors as natural elements inherent to children, counterposing
what is called the "adult world". In this direction, Kunz (2007) argues that his understanding of
play as a natural element comes from the understanding of human beings as beings born to be
free and creative. In other words, the author says that freedom, recognized as the power and
the desire to decide its accomplishments, and creativity, understood as the possibility of building
senses and meanings from what happens, can only be manifested in the exercise of play (Kunz,
2007). Though we consider the contributions of the theorization “Play and Self-movement”
for the work context in Physical Education with Childhood Education and that we also agree
with part of the criticism of “didactic” play, we believe that some arguments are too emphatic
and leave room to some questions, such as the understanding of play and self-movement as
natural and inherent to children, or, play as the only form, original, and authentic of children
learning. Therefore, some statements require prudence and care in their use because the way the
arguments appear in the texts may show some dichotomies that can be problematic. For instance, when defending children's freedom to play and move freely,
autonomously, and spontaneously, Kunz and co-authors frequently consider playing in
opposition to rationality. Or, in other moments, besides establishing a hierarchical relationship,
consider that the dimensions of human sensitivities, freedom and human creativity precede
rationality. Thus, Costa et al. affirm in 2015 (p.27) and in 2018 (pp.204-205) that: Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022
14/26 14/26 e-ISSN 1980-6248 e-ISSN 1980-6248 However, Vaz (1995) highlights exactly the opposite. It is through the work of the
concept, that is, "through the possibility that human being to place himself as the one who
understands the regularity of nature (his own and the surrounding one), and over which he can
place his humanity, is how freedom can be built" (p. 21). Furthermore, “[...] the criticism to
instrumental reason is necessary and pertinent, but we cannot forget that one can only reach it
through the work of reason” (p.21). Despite recognizing the importance of the sensitive, ludic, and play dimension in the
context of children's education, we should question the way they have been approached. We
have identified that opposition between the terms 'ludic and play' against 'rationality/logic' has
been discussed and problematized in the field of Physical Education on the studies about leisure
(Bracht, 2019). According to Bracht (2019), it is common to use the term ludic in opposition to
logic/rationality, giving ludic a subversive and/or utopic character, constantly associated with
corporality. Thus, similarly to what Bracht (2019) identifies in the studies of leisure and Santin’s
(1994) work, we perceive that free and spontaneous play is always understood through its
eminently positive, pleasant, interesting, and autonomous characteristics. More specifically, the
theorization of “Play and Self-movement”, by attributing those positive characteristics to free
and spontaneous play, thematizes the ‘didactic’ play, or rationality elements present in the
education of children from very negative aspects. This theorization makes explicit a notion of
play in opposition to scientific rationality and rational logic. Quoting Bracht (2019), we could
also say that, in Kunz’s writings, there is a connotation that assuming ludic is, in a way, denying the mechanism and the objectification
of humans typical of scientific rationality, for example, in the mechanical conception of the
human body that strongly grounded the intervention of Physical Education. Affirming the ludic
would be affirming the humanity of men, highlighting extremely desirable characteristics, such
as freedom, autonomy, creativity, and pleasure. (p. 72) However, we have doubts about if the suspension/denial of didactic play and rationality
could not lead to an idea that the human dimension only takes place in play because, for Kunz
and collaborators, reason “dehumanizes”. In this sense, we agree with Bracht (2019), when
affirming that the victimization of ludic play, having reason as an executioner, is extremely
problematic. When we think about
the propositions for Physical Education in Childhood Education proposed by Kuns and
collaborators, we identify that the criticism of reason and scientific rationality arises from the
understanding that human reality would be better understood through sensitivity, a similar idea
to Santin (1994). Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022
15/26 Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022
15/2 Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022 http://dx.doi.org/10.1590/1980-6248-2020-0139EN e-ISSN 1980-6248 e-ISSN 1980-6248 rational dimension subtracted, as well as cannot dispense nature. Or better, in a certain sense, it
is related to widening our understanding of rationality, understanding it in the direction of the
body, the ludic, the sensitive, the intensities, and affections. rational dimension subtracted, as well as cannot dispense nature. Or better, in a certain sense, it
is related to widening our understanding of rationality, understanding it in the direction of the
body, the ludic, the sensitive, the intensities, and affections. It is interesting to think of this question, based on the analogy Bracht (2019) makes
about aporia present in Adorno’s Aesthetic Theory (AT). When criticizing this Theory, Bracht
(2019) highlights the ambiguity in the relation between arts and rationality and argues that they
are not opposed but complementary. When operating a criticism towards rationality, this is not
done to subtract it. Or even, based on Adorno, Bracht (2019) states that art is defined as a form
of knowledge, it is also rational. In the relation between reason and body, the author argues that aporia would be connected to the fact that reason that believes to have a body, that transfers
the “mimetic impulse” for the rationality that objectifies the body (or propose its total control
through instrumental reason) indicates at the same time a return to nature (to game, to magic),
and this is what prescribes the law of movement (of body practices)– an aporia or ambiguity
that cannot be overcome. We could even question if this attachment to the nature of the body,
to the mimetic moment of the game (of movement), for example, could be considered a reaction
to the bad irrationality of the ration world as managed, Would that be the reason for the appeal
to a (romantic) rescue of ludic in Physical Education? [original highlight]. (p.159) Therefore, Bracht (2019) speculates about the experiences of movement and play and
their relation with nature. In another moment, Bracht (2000) argued that the counter position
to the hegemony of instrumental reason should not take place only through the affirmation of
its opposite. For the author, we should prioritize sensitivity, or play, as an attempt to return to
a "primordial unit (nature/man; world/man)” (p. XVII). e-ISSN 1980-6248 According to the author, the aesthetic and sensitive dimension cannot have its Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022
16/26 Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022 http://dx.doi.org/10.1590/1980-6248-2020-0139EN e-ISSN 1980-6248 http://dx.doi.org/10.1590/1980-6248-2020-0139EN e-ISSN 1980-6248 e-ISSN 1980-6248 One of the justifications presented by texts about "Play and Self-Movement" in the
defense of free and spontaneous play refers to the recognition of play as a natural need, as a
possibility of direct dialogue with the world, as well as of human nature that is implicit in
children. We believe that these texts miss some considerations on the relation between culture
and nature, and the mediation of language. The emphasis given to the rescue of human nature
in children's education, associated with criticisms of rationality, seems to disregard the role
culture plays in the mediation between human beings and nature. Or even, they leave doubts
about what that direct dialogue with the world would be if language is also established based on
culture. Paraphrasing Bracht (2019), we consider the need to think about play not in the sense
of original purity, as its potential should not be only in the freely and spontaneously play and
the denial of what composes rationality and the didactic play proposed by the adult. Children
are not only nature. They are born and inserted into a universe of culture. This is even shown
by the appeal of the cultural industry for a “natural” interest of children in games and play
(Bracht, 2019). The risks of denying teachers’ intervention, or some didactic plays in Childhood
education, can create the false illusion of freedom and spontaneity, as in a context under the
influence of cultural interest, the appeal to children’s natural interest to play might take place,
as warned by Bracth (2019), from a logic that, when resignifying this understanding, consider
children much more as an object than the protagonist of a greater game. Thus, the author
continues, children's cultural contexts are often inserted in the universe of plays through the
offering of certain fantasies, objects, and delimitations of time and space. e-ISSN 1980-6248 An overcoming of instrumental reason
should take place through mediation and “through the recognition of the ambiguity of our being
in the world and being the world”, therefore, not only through a “return to the original
sensitivity” (p. XVII). With this, we want to emphasize that thinking about children's educational process in
Childhood Education institutions is also to have in mind some ambiguities and contradictions
present in the relations between free and spontaneous play and didactic play, between
sensitiveness and rationality, and between the relationships of adults/teachers with children. Besides spontaneity and freedom, play can also be experienced from intentionality, that is, we
cannot eliminate all the contradictions and ambiguities of the educational process but think of
them based on a dialogic process. Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022 Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022 e-ISSN 1980-6248 e-ISSN 1980-6248 e-ISSN 1980-6248 adult-centric education and the valuing of children's creative and participative potentialities,
these perspectives differ in their understanding of the body. If on one hand, as affirmed by Sarmento in an interview with Richter et al. (2015),
Childhood sociology, by emphasizing the social and historic dimensions of childhood,
understood children's bodies as guided only by cultural practices, ignoring that the child is also
body and nature. On the other hand, it seems that Kunz and collaborators, when criticizing
rationality, propose a return to this lost nature of children, to perception, and sensitiveness. Faced with this, we have identified that the dilemma nature and culture which has been broadly
discussed in the field of Physical education can also encompass the discussions on childhood
and its education regarding the body, the movement, and children's expressions. In the specific case of the field of Physical Education, Bracht and Almeida (2019) argue
that the dilemma between nature and culture is an “articulation problem”. Thus, the process of
articulation and mediation between the ‘sayable’ and the ‘unsayable’, between reason and
emotion, between ‘thought’ and ‘movement’”[original highlights] (Bracht & Almeida, 2019,
p.12) has been one of the main challenges in the production of knowledge in Physical
Education, mainly in the sphere of critical perspectives in the area. We could also extend this issue of articulation, as a challenge to be faced, through the
theorization of play and self-movement. In the direction of what was argued by Bracht and
Almeida (2019), thinking this process of articulation in the scope of children's education in
Childhood Education would mean softening or even filling some gaps between nature and
culture, sensitiveness and rationality, play and reason, body and language, adult and child. In
their discussions, Bracht and Almeida (2019) point out that we should “talk about an interaction
between the lived situation, our pre-reflexive experience, our individual articulation, and the
cultural repertoire of interpretative standards” (p. 10). Thus, we understand that it is exactly in
this “sphere that new meanings (new understandings of movement) can be produced because
they are articulated” (p. 10). Before finishing, we should highlight that, despite choosing an analytical cut that
privileged elements we consider problematic in the theorization in question, the writings about
"Play and Self-movement" also offered important contributions to the debate about Physical
Education in Childhood Education. 4. Final remarks The theorization about "Play and Self-movement" establishes another way of thinking
about the pedagogical work with the body and children's movement in Childhood Education. Thus, instead of 'didactic' plays, or activities indicated by adults, the proposal is for children's
free and spontaneous play and self-movement. Besides that, even if it might have some
assumptions closer to those of Childhood Sociology and Pedagogy, such as the rupture with an Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022
18/26 Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022 http://dx.doi.org/10.1590/1980-6248-2020-0139EN e-ISSN 1980-6248 e-ISSN 1980-6248 e-ISSN 1980-6248 In this sense, we highlight the understanding, the value, and recognition of children for
what they are in the present; the idea of an education that intends to be integral and not
fragmented; the thematization of the game as a central axis in the process of children’s learning
and full development; the recognition of children’s protagonism; the value of knowledge
produced by children; and, a conception of body and movement that, understood as language,
also contemplates the possibilities of children’s communication and expression. To conclude, we end this text by reiterating that, despite the contributions identified,
we cannot deny that the theorization about playing and moving can risk certain inflections
regarding the relationship between body, rationality, and language in children's educational
process. Thus, we should ask if there is not something positive in reason, in the process of
humanization, or the proposal of educational activities proposed by adults in Childhood
Education. We also stress the importance and the need for the theory of "Play and Self-
movement" to dialogue more with the specificity and assumptions of Childhood education,
showing how the concepts presented are supported in the scope of the professional intervention
of Physical Education in Childhood Education. e-ISSN 1980-6248 The keys of reading/interpretation with which we operated
show some fundamental aspects of the context of Childhood Education, contemplated by such
theorization. Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022
19/26 Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022 19/26 http://dx.doi.org/10.1590/1980-6248-2020-0139EN References Almeida, F. Q., Ghidetti, F. F., & Bracht, V. (2013). A presença da fenomenologia na/da Teoria
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manifestação
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20160824.pdf Vaz, A. F. (1995). A filosofia na educação física: soltando as amarras e a capacidade de ser
negatividade. In A. Ferreira Neto, S. V. Goelnner, & V. Bracht. (Orgs.), As Ciências do
Esporte no Brasil (pp. 165-191). Autores Associados. Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022 23/26 23/26 http://dx.doi.org/10.1590/1980-6248-2020-0139EN Appendix Table 1: Studies that are part of the theorization exercise on “Play and move”
Titles
Authors
Type of production
Year
Práticas
didáticas
para
um
“conhecimento de si” de crianças e
jovens na Educação Física
Kunz, E. Book chapter:
Didática da Educação Física 2
2005
Educação Física a questão da
Educação Infantil
Kunz, E. Book chapter:
Educação
Física,
Esporte
e
Sociedade: temas emergentes – Vol. 1. 2007
Brincar e se-movimentar. Kunz, E.;
Santos, L.M.E
Book chapter:
Física:
Conhecimento
e
Saber
Escolar
2009
A liberdade no brincar e se-
movimentar da criança com uma
perspectiva teórica para a educação
(física) infantil
Kunz, E.;
Kuhn, R.;
Santos, V. B. Book chapter:
Educação
Física,
Esporte
e
Sociedade: Temas Emergentes – Vol. 3
2009,
Educação
(Física)
Infantil:
Território
de
Relações
Comunicativas. Gomes-da-
Silva, E.; Kunz,
E.;
Sant'agostinho,
L. H. Article:
RBCE, v. 32, pp. 29-41. 2010
Fenomenologia,
movimento
humano e a educação física. 2010. KUNZ,
E;
SURDI, A. G. Article:
Movimento, v. 16, n. 4, p. 263-
290. 2010
Crianças não são adultos em
Miniatura
Kunz,
E.;
Müller,
U.;
Costa, A.R. Book chapter:
Didática da Educação Física 2
2012
Sem tempo de ser criança: a pressa
no contexto da educação de
crianças e implicações nas aulas de
Educação Física
Stavisk, G.;
Surdi, A. C.;
Kunz, E. Article:
RBCE, v. 35, pp. 113-128. 2013
Relações
Comunicativas
como
Processo Pedagógico na Educação
(Física) Infantil
Gomes-da-
Silva, E.;
Kunz,
E.;
Santagostinho,
L. H. F. Book chapter:
Educação Física Escolar: Pesquisas e
Reflexões
2014
O brincar como diálogo/Pergunta e
não como resposta à prática
pedagógica
Simon, H. S.;
Kunz, E. Article:
Movimento, v. 20, pp. 375-394. 2014
Apresentação e organização do
Livro
Kunz, E. Book:
Brincar & Se-Movimentar: Tempos
e Espaços de vida da criança
2015
A imprescindível e vital necessidade
da criança: brincar e se-movimentar
Kunz, E.;
Costa, A. R. Book chapter:
Brincar & Se-Movimentar: Tempos
e Espaços de vida da criança
2015 Table 1: Studies that are part of the theorization exercise on “Play and move” http://dx.doi.org/10.1590/1980-6248-2020-0139EN Corresponding author: Maria Celeste Rocha - Rua Dr. Arlíndo Sodré, 512 – Itararé, Vitória, ES, 29047-
500, Brasil. e-ISSN 1980-6248
O
brincar
como
construção
racional nas aulas de Educação
Física
Costa, A. R.;
Barros, T.E.S.;
Kunz, E. Article:
Motrivivência, v. 30, pp. 196-208. 2018
Educação e sensibilidade: o brincar
e o se-movimentar da criança
pequena na escola
Surdi, A. C.;
Rodrigues, W. C. M. F.; Freire,
E. J.S.M.;
Kunz, E. Article:
Motrivivência, v. 31, pp. 1-22. 2019 Submission data: Submitted to evaluation November 9, 2020; revised May 17, 2021; approved for publication
October 13, 2021. e-ISSN 1980-6248
Sem tempo de Ser Criança
Stavisk, G.;
Kunz, E. Book chapter:
Brincar & Se-Movimentar: Tempos
e Espaços de vida da criança
2015
Criança e o brincar como obra de
arte:
O
sentido
de
um
esclarecimento
Cunha, A. C.;
Kunz, E. Book chapter:
Brincar & Se-Movimentar: Tempos
e Espaços de vida da criança
2015
A
sensibilidade
na
Educação
Infantil. Surdi, A. C.;
Pereira, D. A.;
Kunz, E. Book chapter:
Brincar & Se-Movimentar: Tempos
e Espaços de vida da criança
2015
A Curiosidade da criança: quem
fomenta? Souza, C. A.;
Kunz, E. Book chapter:
Brincar & Se-Movimentar: Tempos
e Espaços de vida da criança
2015
O fazer da experiência do ser-
criança: entre o estímulo e a
descoberta
Castro, F. B.;
Kunz, E. Book chapter:
Brincar & Se-Movimentar: Tempos
e Espaços de vida da criança
2015
Brincar e se-movimentar da criança:
a
imprescindível
necessidade
humana em extinção? Costa,
A.R.;
Souza, M. F;
Miranda,
D.;
Kunz, E. Article:
Corpoconsciência, v. 19, pp. 45-52,
2015. 2015
O controle da subjetividade e das
experiências corporais sensíveis:
implicações para o brincar e se-
movimentar da criança. Castro, F.B.;
Kunz, E. Article:
Motrivivência, v. 27, pp. 44-57. 2015
Autonomia nas Aulas de Educação
Física: para pensar a prática
pedagógica com crianças
Souza, C. A.;
Kunz, E. Book chapter:
Iniciação à Docência, Reflexões e
Produção do Conhecimento: PIBID
Educação
Física
no
CEFD/UFSM
2016
O brincar e o se-movimentar da
criança
como
manifestação
artística
Surdi, A.C.;
Melo, J. P.;
Kunz, E. Article:
Licere, v. 19, pp. 225-252. 2016
O brincar e o se-movimentar nas
aulas de educação física infantil:
realidades e possibilidades
Surdi, A.C.;
Melo, J.P.;
Kunz, E. Article:
Movimento, v. 22, pp. 1-16. 2016
Sobre como tolhemos a curiosidade
das crianças
De
Souza,
C.A.; Donadel,
T. B.; Kunz, E. Article:
Motrivivência, v. 29, pp. 192-204. 2017
Elementos da fenomenologia como
uma
das
possibilidades
de
compreender o jogo como um
movimento humano significativo
Cunha,
A.C.T.N; Surdi,
A. C.; Marques,
D.A.P; Kunz,
E.; Moreira, E
Article:
Revista Portuguesa de Educação, v. 31, pp. 54-67. 2018
As
relações
do
brinquedo
industrializado com o brincar e se-
movimentar:
uma
reflexão
na
Educação Física
Burckardt,
E.V.;
Costa,
L.C.;
Kunz, E. Article:
Motrivivência, v. 30, pp. 278-294. 2018 Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022
25/26 Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022 25/26 25/26 http://dx.doi.org/10.1590/1980-6248-2020-0139EN Authors’ contributions: Rocha, Maria Celeste: Conceptualization (Equal), Data curation (Leader), Formal
Analysis (Equal), Grant demand (Equal), Investigation (Equal), Methodology (Equal,
Project Management (Equal), Resources (Equal), Software (Equal), Supervision (Equal),
Validation (Equal), Visualization (Equal), Writing-original draft (Leader), Writing-
review and edition (Leader). Almeida, Felipe Quintão: Conceptualization (Equal), Data curation (Equal), Formal
Analysis (Equal), Grant demand (Equal), Investigation (Equal), Methodology (Equal),
Project Management (Equal), Resources (Equal), Software (Equal), Supervision (Equal),
Validation (Equal), Visualization (Equal), Writing-original draft (Equal), Writing- review
and edition (Equal). Moreno, Alberto: Conceptualization (Equal), Data curation (Support), Formal
Analysis (Equal), Grant demand (Equal), Investigation (Equal), Methodology (Equal),
Project Management (Equal), Resources (Equal), Software (Equal), Supervision (Equal),
Validation (Equal), Visualization (Equal), Writing-original draft (Support), Writing-
review and edition (Equal). Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022
26/26 Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022
26/26 Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022
|
https://openalex.org/W2486036269
|
https://europepmc.org/articles/pmc4974648?pdf=render
|
English
| null |
Small angle x-ray scattering with edge-illumination
|
Scientific reports
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cc-by
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Small angle x-ray scattering with
edge-illumination Peter Modregger1, Tiziana P. Cremona2, Charaf Benarafa3, Johannes C. Schittny2,
Alessandro Olivo1 & Marco Endrizzi1 received: 14 April 2016
accepted: 11 July 2016
Published: 05 August 2016 Sensitivity to sub-pixel sample features has been demonstrated as a valuable capability of phase
contrast x-ray imaging. Here, we report on a method to obtain angular-resolved small angle x-ray
scattering distributions with edge-illumination- based imaging utilizing incoherent illumination
from an x-ray tube. Our approach provides both the three established image modalities (absorption,
differential phase and scatter strength), plus a number of additional contrasts related to unresolved
sample features. The complementarity of these contrasts is experimentally validated by using
different materials in powder form. As a significant application example we show that the extended
complementary contrasts could allow the diagnosis of pulmonary emphysema in a murine model. In support of this, we demonstrate that the properties of the retrieved scattering distributions are
consistent with the expectation of increased feature sizes related to pulmonary emphysema. Combined
with the simplicity of implementation of edge-illumination, these findings suggest a high potential for
exploiting extended sub-pixel contrasts in the diagnosis of lung diseases and beyond. In small angle x-ray scattering sensitivity to sub-pixel sample features provides access to information on micro-
scopic scales with macroscopic pixel sizes. In addition to complementary information about the sample this offers
the opportunity for faster scans and/or dose reduction by exploiting larger pixel sizes. The ability to obtain con-
trasts related to sub-pixel sample structures was demonstrated with analyzer-based imaging1,2, grating interfer-
ometry (GI)3,4 and edge-illumination (EI)5,6. Currently, the potential of phase sensitive x-ray imaging for clinical
application is investigated in the fields of mammography7–9, cartilage imaging10, bone structure determination11,
imaging the airway surface liquid12 and the diagnosis of pulmonary emphysema13,14. Sub-pixel information is
typically accessed through the broadening of the scattering distribution underlying the signal within a pixel, and
is usually referred to as scatter strength. Scatter strength can be regarded as a measure for sample inhomogeneity
within a detector pixel and its relation to sub-pixel sample morphology is the subject of ongoing research15–19. In this study, we applied a deconvolution-based data analysis approach previously used in GI20,21 to EI, which
increases the number of complementary contrasts related to sub-pixel sample features. EI is a non-interferometric
and incoherent phase sensitive x-ray imaging technique22 utilizing a set of two apertured masks (Fig. 1). www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Small angle x-ray scattering with
edge-illumination The
pre-sample mask shapes the incident radiation into an array of well defined beamlets, which are distorted by the
sample. These distortions are transformed into detectable intensity variations by the detector mask, which covers
approximately half of each pixel. Since the optical elements are achromatic, EI is readily compatible with inco-
herent x-ray tubes, where the full spectrum of the source contributes to the signal at the detector23,24. The com-
paratively large structure sizes of the apertured masks (typically tens of microns) render EI easily scalable, robust
against environmental vibrations and thermal stress. Established scan and data procedures simultaneously yield
absorption contrast, differential phase contrast25,26 with nano-radiant sensitivity27 and scatter contrast6. Recently,
EI was also successfully combined with tomography28,29. Extended Number of Contrasts with EI-SAXS In the experiment, illumination curves (IC) were obtained by recording the detected intensity for each pixel while
the sample mask was laterally scanned in M steps separated by a fraction of the mask pitch. This was performed
with and without a sample present yielding the sample IC (s) and the flat IC (f), respectively. We express the ICs
in terms of the scatter angle α (i.e., s(α) and f(α)), which is related to the lateral offset Δx of the sample mask and 1Department of Medical Physics and Bioengineering, University College London, Gower Street, WC1E 6BT London,
United Kingdom. 2Institute of Anatomy, University of Berne, Baltzerstrasse 2, 3012 Bern, Switzerland. 3Theodor
Kocher Institute, University of Berne, Freiestrasse 1, 3012 Bern, Switzerland. Correspondence and requests for
materials should be addressed to P.M. (p.modregger@ucl.ac.uk) Scientific Reports | 6:30940 | DOI: 10.1038/srep30940 www.nature.com/scientificreports/ Figure 1. Sketch of the experimental set-up for EI. Figure 1. Sketch of the experimental set-up for EI. the sample to detector distance z by α = Δx/z. In analogy with the procedure described in20, we assumed that the
distortion of the sample IC can be modelled by the sample to detector distance z by α = Δx/z. In analogy with the procedure described in20, we assumed that the
distortion of the sample IC can be modelled by α
α
α
=
⊗
s
f
g
( )
( )
( )
(1) (1) with the small-angle x-ray scattering (SAXS) distribution g(α) being determined by the sub-pixel structure of the
sample. Therefore, g(α) can be retrieved by deconvolving the sample IC s with the flat IC f. In order to distinguish
this analysis method from others we will refer to it as EI-SAXS.l with the small-angle x-ray scattering (SAXS) distribution g(α) being determined by the sub-pixel structure of the
sample. Therefore, g(α) can be retrieved by deconvolving the sample IC s with the flat IC f. In order to distinguish
this analysis method from others we will refer to it as EI-SAXS.l For this proof of concept experiment, we used a dragon fly as a biological sample expected to provide a variety
of differently shaped scattering distributions. During the experimental scan M = 10 images were acquired on
equally spaced positions of the IC. Each image consisted of 5 frames with 5 s exposure time to ensure sufficient
signal to noise ratio. Extended Number of Contrasts with EI-SAXS In order to reduce possible aliasing effects due to partial illumination of the sample within
one period of the sample mask, the sample was dithered 8 times (i.e., additional translations of the sample by a
fraction of the sample mask pitch between the dithering steps were performed), and the corresponding images
averaged prior to data analysis. g
p
y
Data analysis was performed by deconvolving s(α) with f(α) using iterative Lucy-Richardson deconvolution30,31,
where the k-th iteration is performed by computing =
⋅
⊗
⊗
+
g
g
s
g
f
f
(2)
k
k
k
1 (2) and f denotes f mirrored at the origin. In practice the occurring convolutions are computed utilizing fast Fourier
transform. Lucy-Richardson deconvolution has an implicit positive constraint, converges to the maximum likeli-
hood solution in the case of Poisson statistics, and is known to deliver stable results in the presence of photon shot
noise. The starting value was chosen to be
α
α
α
=
∑
∆
α
g
s
f
( )/(
( )
)
0
, which provides the correct normalisation of
the retrieved scattering distribution g. The number of iteration steps was 1000 in order to ensure convergence. The
deconvolution provided the scattering distribution g(α) on the half-open interval α
α
α
∈[
,
[
min
max . For the sub-
sequent moment analysis (see below) it was convenient to symmetrize the scattering distribution
(i.e.,
α
α
=
g
g
(
)
(
)
max
min ), which is justified by the periodicity of the discrete Fourier transform employed by
Lucy-Richardson deconvolution. and f denotes f mirrored at the origin. In practice the occurring convolutions are computed utilizing fast Fourier
transform. Lucy-Richardson deconvolution has an implicit positive constraint, converges to the maximum likeli-
hood solution in the case of Poisson statistics, and is known to deliver stable results in the presence of photon shot
noise. The starting value was chosen to be
α
α
α
=
∑
∆
α
g
s
f
( )/(
( )
)
0
, which provides the correct normalisation of
the retrieved scattering distribution g. The number of iteration steps was 1000 in order to ensure convergence. The
deconvolution provided the scattering distribution g(α) on the half-open interval α
α
α
∈[
,
[
min
max . Scientific Reports | 6:30940 | DOI: 10.1038/srep30940 Extended Number of Contrasts with EI-SAXS For the sub-
sequent moment analysis (see below) it was convenient to symmetrize the scattering distribution
(i.e.,
α
α
=
g
g
(
)
(
)
max
min ), which is justified by the periodicity of the discrete Fourier transform employed by
Lucy-Richardson deconvolution. y
Figure 2 provides examples of the utilized ICs and of the retrieved scattering distributions inside and outside
the sample. As expected, the scattering distribution outside the sample shows an approximate δ-function shape,
where the finite width is due to finite sampling with respect to the scatter angle. The distribution inside of the
sample is both shifted and broadened, which are both in accordance with expectations. Since the deconvolution
procedure yields a scattering distribution g(α) for each pixel, scatter images (see Supplementary Figure 1) are
available for each scattering angle α. This demonstrates an extension of the number of contrast from the previ-
ous 3 to 10 for EI. Ultimately, the number of contrast modalities is limited by the stability of the imaging system
and by the resolution of the motor used for scanning the sample mask. Later in this study, we will utilize M = 32
sample points. In GI, the retrieval of angular-resolved scattering distributions involves the deconvolution of noisy, periodic
signals with suppressed even harmonics20, which is intrinsically a challenging task32. Moreover, owing to the
coherence intrinsic to the contrast formation process in GI, parts of the broad spectrum provided by an x-ray tube
contribute substantially less to the signal collected by a detector that integrates over photon energies33. In contrast,
the ICs in EI are approximately Gaussian-shaped, and the achromaticity of the optical elements significantly
improves the signal to noise ratio24. Thus, the deconvolution procedure is simpler for EI. However, due to the
geometry of experimental set-ups angular sampling of the scattering distribution is about an order of magnitude
larger for EI (Δα = 25 μrad here) than for GI (Δα = 1.7 μrad in20). Therefore, the two methods retrieve the scat-
tering distribution on essentially complementary, and possibly expanding scales. For EI, finer angular sampling
can be achieved by either increasing the distance between sample and detector or by increasing the number of
acquired sample points M on the IC. The latter will be ultimately limited by the stability of the set-up and the
resolution of utilized scanning motors. Scientific Reports | 6:30940 | DOI: 10.1038/srep30940 2 www.nature.com/scientificreports/ Figure 2. Extended Number of Contrasts with EI-SAXS Recovery of scattering distributions. (a) Flat IC and sample ICs for a pixel outside and inside of the
sample, respectively. (b) SAXS distribution as retrieved by deconvolution of the ICs shown in (a). As expected
the retrieved distribution outside of the sample is approximately δ-shaped, while the distribution inside of the
sample is shifted and broadened. Figure 2. Recovery of scattering distributions. (a) Flat IC and sample ICs for a pixel outside and inside of the
sample, respectively. (b) SAXS distribution as retrieved by deconvolution of the ICs shown in (a). As expected
the retrieved distribution outside of the sample is approximately δ-shaped, while the distribution inside of the
sample is shifted and broadened. Complementarity of contrasts provided by EI-SAXS. Based on the assumption that the flat and the
sample IC can be expressed as Gaussian functions, it was demonstrated that three contrast modalities (i.e., trans-
mission t, refraction ΔxR and scatter strength σ) can be retrieved from three experimental images6. In the fol-
lowing, we will use this established data analysis procedure for comparison. Please refer to the reference for more
details.h The three contrast modalities can be regarded as parameters of the shape of the scattering distribution and can
be retrieved from the deconvolved scattering distributions by calculating the moments Mn (with n ∈ N being the
order of the moment) according to34 ∫α
α
=
M
d g ( )
(3)
0
∫αα
α
=
M
d
g
M
( )/
(4)
1
0 (3) ∫αα
α
=
M
d
g
M
( )/
1
0 ∫αα
α
=
M
d
g
M
( )/
1
0 (4) ∫α α
α
=
−
≥
. Extended Number of Contrasts with EI-SAXS M
d
M
g
M
n
(
)
( )/
for
2
(5)
n
n
1
0 (5) While M0, M1 and M2 correspond to absorption, differential phase and scatter strength contrasts, respectively,
higher order moments (n > 2) constitute additional descriptive parameters for the scattering distribution and,
thus, provide increased structural information about the sample on a sub-pixel scale.f While M0, M1 and M2 correspond to absorption, differential phase and scatter strength contrasts, respectively,
higher order moments (n > 2) constitute additional descriptive parameters for the scattering distribution and,
thus, provide increased structural information about the sample on a sub-pixel scale.f p
p
p
Figure 3 shows a comparison of different contrast modalities as retrieved by the established data analysis to
the corresponding moments of deconvolved scattering distributions at the example of a dragon fly. We found an
excellent agreement for M0 and the transmission t (correlation coefficient: 0.999) as well as for M1 and the refrac-
tion contrast ΔxR (correlation coefficient: 0.954). M2 and the scatter strength σ show the same sample morphol-
ogy (correlation coefficient: 0.926), while they differ only in the magnitude of the retrieved scatter strength. This
deviation can attributed to the fact that the shape of the sample and flat IC differ from a Gaussian, which leads the
established data analysis to wrongly estimate the scatter strength.h y
g y
g
This establishes EI-SAXS as a viable alternative to the data analysis methods previously used in imaging with
edge-illumination. It should be noted that, while previously reported data methods made assumptions about the
underlying scattering distribution (e.g., negligible scattering in25,26), the proposed deconvolution method does
not assume a specific shape of the involved IC and, thus, produces reliable results for arbitrarily shaped scatter
distributions. In order to investigate the information content provided by higher order moments we investigated the scatter-
ing properties of three household powders: salt, coffee and sugar. The same parameters for the scan and analysis
procedures as above were used, except for the lack of dithering (i.e, the sample was not moved). Figure 4(a,b)
show the 2nd and 4th moment of the scattering distributions, respectively retrieved by deconvolution. The comple-
mentarity of M2 and M4 is clearly demonstrated by the corresponding pixel-wise scatter plot, shown in Fig. 4(c). Extended Number of Contrasts with EI-SAXS Therefore, EI-SAXS provides the opportunity to exploit extended sub-pixel contrasts, which could be useful in
areas as diverse as biomedical research, materials science and security screening. As an example of a significant
application, we will take a first step into exploring the potential of EI-SAXS for the diagnosis of lung diseases. EI-SAXS for the diagnosis of pulmonary emphysema. Chronic obstructive pulmonary disease
(COPD) is characterized by severe changes in lung morphology and is a major and increasing cause of death
associated with cigarette smoking35. The micro-structure of lungs is defined by alveolar walls and distal airspaces,
which have typical sizes on the micrometer scale36. Pulmonary emphysema is a frequent constituent of COPD and
a condition of the lung “characterized by abnormal, permanent enlargement of air spaces distal to the terminal
bronchiole, accompanied by destruction of their walls, and without obvious fibrosis”37. Scientific Reports | 6:30940 | DOI: 10.1038/srep30940 3 www.nature.com/scientificreports/ Figure 3. Comparison of an established data analysis procedure with the proposed deconvolution-based
method for a dragon fly. The top row shows the transmission t (a), refraction μ (c) and scatter strength σ (e)
retrieved according to6. The bottom row shows the first three moments of deconvolved scattering distribution:
(b) M0 corresponds to transmission, (d) M1 to refraction and (f) M2 to scatter strength, respectively. Scale bars
are 1 cm. An excellent agreement between M0 and M1 and their corresponding contrasts retrieved from the
established data analysis was found. M2 and σ show the same sample morphology while slightly differing in
the magnitude of retrieved values (factor of ≈1.5). The deviation is attributed to a violation of the strict
assumptions of the previously established data analysis. Figure 3. Comparison of an established data analysis procedure with the proposed deconvolution-baselh Figure 3. Comparison of an established data analysis procedure with the proposed deconvolution-based
method for a dragon fly. The top row shows the transmission t (a), refraction μ (c) and scatter strength σ (e)
retrieved according to6. The bottom row shows the first three moments of deconvolved scattering distribution:
(b) M0 corresponds to transmission, (d) M1 to refraction and (f) M2 to scatter strength, respectively. Scale bars
are 1 cm. An excellent agreement between M0 and M1 and their corresponding contrasts retrieved from the
established data analysis was found. M2 and σ show the same sample morphology while slightly differing in
the magnitude of retrieved values (factor of ≈1.5). Extended Number of Contrasts with EI-SAXS Based upon this observation, the ratio of the 4th and the 2nd moment (i.e., M4/M2), which appears as the slope
of lines connecting the plotted points to the origin, was used as an illustration model for the potential diagnos-
tic power of EI-SAXS. The physical interpretation of this ratio is as follows. For a constant M2, larger values of
M4 indicate stronger tails of the scattering distribution (see equation 5). In turn, stronger tails indicate a larger
contribution of large scattering angles to the scattering distribution compared to small scattering angles. Since
large scattering angles are associated with smaller sample features, high values of the M4/M2-ratio are related to
larger relative contributions of smaller sample features. Potentially, the other moments (i.e., M3,M5,…) could carry
additional information about the sub-pixel sample morphology. However a detailed analysis of their possible
contributions lies outside the scope of this preliminary study and will be the subject of future investigations. In the following pilot experiment with a mouse model of cigarette smoke exposure-induced emphysema,
we demonstrate that EI-SAXS can detect this increase in feature sizes by exploiting the complementarity of the
second and fourth moment of deconvolved scattering distributions. Two murine lungs, one control (N = 1) and
one emphysematous (N = 1) were imaged. The same scan parameters and data analysis procedures as described
above were used except for the number of sample points on the IC (M = 32) and the fact that dithering was not
performed. Images for the M0, M2 and M4 can be found in the Supplementary material (supplementary Figure 2).h p
g
0,
2
4
pp
y
(
pp
y
g
)
The scatter plot of M2 and M4 (Fig. 5a) reveals a clear distinction between control and emphysematous lung. Due to the small number of samples, we can only consider this as preliminary evidence for the utility of higher
order moments provided by EI-SAXS in the context of pulmonary emphysema diagnosis. Further, for each lung
sample an approximate linear relationship between M2 and M4 was found (also visible for the powders in Fig. 4c). Based upon this observation, the ratio of the 4th and the 2nd moment (i.e., M4/M2), which appears as the slope
of lines connecting the plotted points to the origin, was used as an illustration model for the potential diagnos-
tic power of EI-SAXS. The physical interpretation of this ratio is as follows. Extended Number of Contrasts with EI-SAXS The deviation is attributed to a violation of the strict
assumptions of the previously established data analysis. Figure 4. Complementary of moments extracted from different powders with EI-SAXS. M2 (a) and M4
(b) for salt (blue rectangle), coffee (yellow rectangle) and sugar (red rectangle). Scale bars are 5 mm. The
pixel-wise scatter plot of M2 and M4 (c) demonstrates the complementarity of those moments. Figure 4. Complementary of moments extracted from different powders with EI-SAXS. M2 (a) and M4
(b) for salt (blue rectangle), coffee (yellow rectangle) and sugar (red rectangle). Scale bars are 5 mm. The
pixel-wise scatter plot of M2 and M4 (c) demonstrates the complementarity of those moments. Scientific Reports | 6:30940 | DOI: 10.1038/srep30940 4 www.nature.com/scientificreports/ Figure 5. Diagnosis of lung emphysema with EI-SAXS. A clear separation of the control and the
emphysematous lung in the M2–M4 scatter plot (a) is indicated by arrows. The ratio M4/M2 of the control (b) is
larger than that of the emphysematous lung (c), which is most noticeable in the emphysematous left lung. Scale
bars are 1 cm and the background was masked by thresholding. Comparison between scattering distributions
(d) confirm that the emphysematous lung provides features smaller tails than that of the control one, which is
consistent with increased average feature sizes in the former. Figure 5. Diagnosis of lung emphysema with EI-SAXS. A clear separation of the control and the
emphysematous lung in the M2–M4 scatter plot (a) is indicated by arrows. The ratio M4/M2 of the control (b) is
larger than that of the emphysematous lung (c), which is most noticeable in the emphysematous left lung. Scale
bars are 1 cm and the background was masked by thresholding. Comparison between scattering distributions
(d) confirm that the emphysematous lung provides features smaller tails than that of the control one, which is
consistent with increased average feature sizes in the former. In the following pilot experiment with a mouse model of cigarette smoke exposure-induced emphysema,
we demonstrate that EI-SAXS can detect this increase in feature sizes by exploiting the complementarity of the
second and fourth moment of deconvolved scattering distributions. Two murine lungs, one control (N = 1) and
one emphysematous (N = 1) were imaged. Scientific Reports | 6:30940 | DOI: 10.1038/srep30940 Extended Number of Contrasts with EI-SAXS The same scan parameters and data analysis procedures as described
above were used except for the number of sample points on the IC (M = 32) and the fact that dithering was not
performed. Images for the M0, M2 and M4 can be found in the Supplementary material (supplementary Figure 2). The scatter plot of M2 and M4 (Fig. 5a) reveals a clear distinction between control and emphysematous lung. Due to the small number of samples, we can only consider this as preliminary evidence for the utility of higher
order moments provided by EI-SAXS in the context of pulmonary emphysema diagnosis. Further, for each lung
sample an approximate linear relationship between M2 and M4 was found (also visible for the powders in Fig. 4c). Based upon this observation, the ratio of the 4th and the 2nd moment (i.e., M4/M2), which appears as the slope
of lines connecting the plotted points to the origin, was used as an illustration model for the potential diagnos-
ti
f EI SAXS Th
h
i
l i t
t ti
f thi
ti i
f ll
F
t
t M l
l
f In the following pilot experiment with a mouse model of cigarette smoke exposure-induced emphysema,
we demonstrate that EI-SAXS can detect this increase in feature sizes by exploiting the complementarity of the
second and fourth moment of deconvolved scattering distributions. Two murine lungs, one control (N = 1) and
one emphysematous (N = 1) were imaged. The same scan parameters and data analysis procedures as described
above were used except for the number of sample points on the IC (M = 32) and the fact that dithering was not
performed. Images for the M0, M2 and M4 can be found in the Supplementary material (supplementary Figure 2). The scatter plot of M2 and M4 (Fig. 5a) reveals a clear distinction between control and emphysematous lung. Due to the small number of samples, we can only consider this as preliminary evidence for the utility of higher
order moments provided by EI-SAXS in the context of pulmonary emphysema diagnosis. Further, for each lung
sample an approximate linear relationship between M2 and M4 was found (also visible for the powders in Fig. 4c). Extended Number of Contrasts with EI-SAXS For a constant M2, larger values of
M4 indicate stronger tails of the scattering distribution (see equation 5). In turn, stronger tails indicate a larger
contribution of large scattering angles to the scattering distribution compared to small scattering angles. Since
large scattering angles are associated with smaller sample features, high values of the M4/M2-ratio are related to
larger relative contributions of smaller sample features. Potentially, the other moments (i.e., M3,M5,…) could carry
additional information about the sub-pixel sample morphology. However a detailed analysis of their possible
contributions lies outside the scope of this preliminary study and will be the subject of future investigations. Scientific Reports | 6:30940 | DOI: 10.1038/srep30940 5 www.nature.com/scientificreports/ Figure 6. CI separation (ΔCI) of M4/M2 values for the control and the emphysematous lung as a function
of delivered dose. The dashed line represents a linear extrapolation. Figure 6. CI separation (ΔCI) of M4/M2 values for the control and the emphysematous lung as a function
of delivered dose. The dashed line represents a linear extrapolation. The images of M4/M2-ratios for the control and the emphysematous lung (Fig. 5b,c) appear generally flat and
the emphysematous lung has noticeably darker appearance–especially in the left lung. Faint horizontal stripes
are residuals from bridges in the mask design that stabilize the structure38. These minor artefacts can be easily
removed by e.g. improved mask design or data analysis. The flatness of the M4/M2-contrasts is remarkable for two
reasons. First, due to the opening in the sample mask only 10 μm/79 μm ≈ 12.7% of the sample was illuminated
within a pixel (no dithering was used in this case) indicating the robustness of M4/M2-ratio. Second, sample
movement does affect but not destroy a quasi-homogeneous contrast modality as it was shown with breathing
during dark-field imaging of mouse lungs13. Since M4/M2 is homogeneous we speculate that breathing during
exposure should only have a minor impact on EI-SAXS, as has been observed in the case of GI. p
y
p
The mean M4/M2-ratios for the two lungs, determined over the entire sample, were
=
M
M
(
/
)control
4
2
. ±
. ⋅
−
(5 535
0 006
10
) rad
9
2 and
=
. ±
. ⋅
−
M
M
(
/
)
(5 128
0 004
10
) rad
emphysematous
4
2
9
2, where the uncertainties
are given at 0.68 confidence interval (CI). Extended Number of Contrasts with EI-SAXS Thus, the two ratios were separated by more than 40 CIs. While the
specific values of M4/M2 could potentially be affected by variations in experimental parameters, this very signifi-
cant separation suggests a high potential of EI-SAXS for reliable diagnosis of emphysema at different stages of
development.hi The scattering distributions in Fig. 5(d) confirmed that the tails of the control were stronger than the tails of
the emphysematous lung. Since smaller scattering angles are associated with larger sample features, the smaller
tails of the diseased lung are consistent with increased average feature sizes due to emphysema. Since the higher
order moments correspond to scattering of sub-pixel sample features, the pixel size could be increased without
losing access to this information. In this proof of concept study no special attention was paid to dose optimization. Using an ion chamber the
dose rate was estimated as 0.2 mGy/s entrance air kerma. This implies a delivered dose of 400 mGy for the dragon
fly (Fig. 3), 50 mGy for the powders (Fig. 4) and 160 mGy for the lungs (Fig. 5). However, the dose can be signifi-
cantly reduced by optimizing aperture sizes8, adequate filtering of low energy photons29 and increasing pixel sizes. In order to demonstrate this potential, we simulated experiments with decreased dose by using 1, 2, 3, 4 or 5 out
of the 5 accumulations that were acquired for the murine lungs as the input for data analysis. Figure 6 shows the
resulting separation of the M4/M2-ratio for the control and the emphysematous lung in terms of CIs as a function
of simulated dose. It is obvious that the separability of healthy and emphysematous lungs is preserved if the dose
is reduced significantly. If a separation of 5 CIs is taken as sufficient, then a rough linear extrapolation (dashed
line in Fig. 6) implies a dose of only 6.3 mGy with this un-optimized set-up. Thus, EI-SAXS offers the potential to
diagnose pulmonary emphysema at low dose. Methods Sample preparation. The dragon fly was air-dried over several days prior to scanning in order to ensure
stable sample features and fixed at its tail for imaging. The three household powders were put on top of each
other in a rectangular plastic holder to ensure consistent sample thicknesses. Wild-type 129S6/SvEv/Tac mice
were exposed to air or to cigarette smoke 5 hours/day, 5 days/week for 4 months. Smoke of 3R4F research ciga-
rettes (University of Kentucky, Lexington, KY) was generated by a TE10z smoking machine (Teague Enterprises,
Woodland, CA) connected to whole-body exposure chambers as described previously39. All animal studies were
approved by the Cantonal Veterinary Office of Bern, Switzerland and carried out in accordance with relevant
guidelines. Lungs were instilled with 1.5% paraformaldehyde-1.5% glutaraldehyde in 0.15 M HEPES pH 7.35
through a tracheal cannula at a constant pressure of 20 cm H2O. The trachea was ligated and lungs were placed in
fresh fixative for at least 24 hours to complete fixation. Following super-critical drying, samples were placed in the
x-ray system using sample holder with membranes for imaging. Instrumentationh The experiment was carried out with a laboratory-based set-up at University College London (London, UK). The
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This project has received funding from the European Union’s Horizon 2020 research and innovation programme
under the Marie Skłodowska-Curie Grant Agreement No. 657621 and from the EPSRC (grant EP/I021884/1. This
project was also supported by the Royal Academy of Engineering under the RAEng Research Fellowships scheme. Part of this work has been supported by the Flight Attendant Medical Research Institute (FAMRI) and the Swiss
National Science Foundation (310030-127464 and 310030-149790). Author Contributionsh The experiment was devised by P.M., A.O. and M.E. and the measurements as well as the data analysis was carried
out by P.M. Murine lung samples were prepared by T.P.C., C.B. and J.S. The manuscript was written by P.M. with
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39. Cremona, T. P., Tschanz, S. A., von Garnier, C. & Benarafa, C. Additional Information Supplementary information accompanies this paper at http://www.nature.com/srepi Competing financial interests: The authors declare no competing financial interests. How to cite this article: Modregger, P. et al. Small angle x-ray scattering with edge-illumination. Sci. Rep. 6,
30940; doi: 10.1038/srep30940 (2016). 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) 2016 Scientific Reports | 6:30940 | DOI: 10.1038/srep30940 8
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TGF-β is elevated in hyperuricemic individuals and mediates urate-induced hyperinflammatory phenotype in human mononuclear cells
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Arthritis research & therapy
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Arthritis Research & Therapy Arthritis Research & Therapy Klück et al. Arthritis Research & Therapy (2023) 25:30
https://doi.org/10.1186/s13075-023-03001-1 Open Access Abstract Background Soluble urate leads to a pro-inflammatory phenotype in human monocytes characterized by increased
production of IL-1β and downregulation of IL-1 receptor antagonist, the mechanism of which remains to be fully
elucidated. Previous transcriptomic data identified differential expression of genes in the transforming growth factor
(TGF)-β pathway in monocytes exposed to urate in vitro. In this study, we explore the role of TGF-β in urate-induced
hyperinflammation in peripheral blood mononuclear cells (PBMCs). Methods TGF-β mRNA in unstimulated PBMCs and protein levels in plasma were measured in individuals with nor-
mouricemia, hyperuricemia and gout. For in vitro validation, PBMCs of healthy volunteers were isolated and treated
with a dose ranging concentration of urate for assessment of mRNA and pSMAD2. Urate and TGF-β priming experi-
ments were performed with three inhibitors of TGF-β signalling: SB-505124, 5Z-7-oxozeaenol and a blocking antibody
against TGF-β receptor II. Results TGF-β mRNA levels were elevated in gout patients compared to healthy controls. TGF-β-LAP levels in serum
were significantly higher in individuals with hyperuricemia compared to controls. In both cases, TGF-β correlated posi-
tively to serum urate levels. In vitro, urate exposure of PBMCs did not directly induce TGF-β but did enhance SMAD2
phosphorylation. The urate-induced pro-inflammatory phenotype of monocytes was partly reversed by blocking
TGF-β. Conclusions TGF-β is elevated in individuals with hyperuricemia and correlated to serum urate concentrations. In
addition, the urate-induced pro-inflammatory phenotype in human monocytes is mediated by TGF-β signalling. Future studies are warranted to explore the intracellular pathways involved and to assess the clinical significance of
urate-TGF-β relation. Keywords TGF-β, Hyperuricemia, Inflammation, Mononuclear leukocytes 2 Radboud Institute for Molecular Life Sciences (RIMLS), Nijmegen, The
Netherlands
3 Department of Medical Genetics, “Iuliu Haţieganu” University
of Medicine and Pharmacy, Cluj Napoca, Romania
4 Departement of Rheumatology, Radboud UMC, Nijmegen, The
Netherlands TGF‑β is elevated in hyperuricemic
individuals and mediates urate‑induced
hyperinflammatory phenotype in human
mononuclear cells Viola Klück1,2†, Georgiana Cabău3†, Linda Mies1, Femke Bukkems4, Liesbeth van Emst1, René Bakker4,
Arjan van Caam4, HINT consortium, Tania O. Crişan3 and Leo A. B. Joosten1,2,3* †Viola Klück and Georgiana Cabău share first authorship. *Correspondence:
Leo A. B. Joosten
Leo.Joosten@radboudumc.nl
1 Department of Internal Medicine, Radboud UMC, Nijmegen, The
Netherlands *Correspondence:
Leo A. B. Joosten
Leo.Joosten@radboudumc.nl
1 Department of Internal Medicine, Radboud UMC, Nijmegen, The
Netherlands *Correspondence:
Leo A. B. Joosten
Leo.Joosten@radboudumc.nl
1 Department of Internal Medicine, Radboud UMC, Nijmegen, The
Netherlands @
1 Department of Internal Medicine, Radboud UMC, Nijmegen, The
Netherlands Background When TGF-β is secreted, it is in inactive form bound to
its latency-associated peptide (LAP) of which it can get
separated via ROS, metalloproteinases and integrins
[20]. The active form of TGF-β can subsequently bind
to TGF-β receptor II (TGF-βRII). Upon binding of TGF-
β, two TGFβRII receptors form a heterotetramer with
TGFβRI, forming a signalling-competent complex able to
induce the C-terminal phosphorylation of receptor-acti-
vated SMADs [19]. The activated SMADs form a com-
plex with SMAD4 and translocate to the nucleus, where
they regulate the expression of a large number of genes
involved in e.g. fibrosis and immune signalling. Indepen-
dently of SMADs, TGF-β can also signal via, e.g. TAK1,
ERK and the PI3K-Akt pathway, of which the latter was
also shown to be involved in urate priming [15]. g
Hyperuricemia, defined as elevated serum urate levels
above the saturation threshold, is the major risk factor
for gout [1]. Supersaturated serum urate precipitates into
monosodium urate (MSU) crystals which deposit within
joints leading to recurrent inflammatory arthritis. These
gout flares are initiated by interleukin (IL)-1β produc-
tion by macrophages within the synovium. In these cells,
stimulation of a Toll-like receptor (TLR), by free fatty
acids for instance, results in the synthesis of pro-IL-1β,
while MSU crystals activate the NLRP3 inflammasome
leading to active caspase-1, which processes this pro-
IL-1β to mature IL-1β [2]. Based on this finding, several
therapies targeting IL-1β demonstrated efficacy in treat-
ing gout flares [3].l In myeloid cells, the function of TGF-β depends on
the specific nature of the activating conditions. Gener-
ally, TGF-β stimulates cells at the resting state, whereas
activated cells are inhibited [21]: in activated monocytes,
TGF-β inhibits MyD88-dependent TLR- and IL-1R sig-
nalling pathways by promoting MyD88 degradation [22]. However, TGF-β alone can induce gene expression of
IL-1 in peripheral blood monocytes [23–25].l l
Although presenting as an intermittent flaring condi-
tion, gout is a chronic disease [4]. It affects about 2.5–
3.9% of the Western population and has become more
prevalent in the last decades [5]. In addition, hyperurice-
mia is associated with higher incidence of comorbidities
such as cardiovascular disease, type 2 diabetes, metabolic
syndrome, chronic kidney disease, cancer and premature
ageing [6–9]. Moreover, gout patients have an increased
mortality rate of 2.21 compared with the total popula-
tion, and this increase is associated with high urate levels
[10]. Background Therefore, elucidating the mechanisms responsible
for this enhanced risk to develop comorbidities caused by
hyperuricemia is crucial. Taken together, hyperuricemia has pro-inflammatory
effects in human monocytes and is a risk factor for gout
and its associated comorbidities. Previous findings sug-
gest TGF-β pathway might be a relevant target to assess
in relationship to the inflammation elicited by urate. Therefore, in this study, we explore the role of TGF-β in
the context of hyperuricemia and urate induced repro-
gramming of myeloid cells. Crişan et al. previously demonstrated that soluble
urate leads to a pro-inflammatory phenotype in primary
human monocytes characterized by increased produc-
tion of IL-1β, a classical pro-inflammatory cytokine, and
downregulation of IL-1 receptor antagonist (IL-1Ra), the
natural inhibitor of IL-1 [11–13]. Features of proinflam-
matory reprogramming after urate exposure persisted
for up to 6 days in PBMCs in vitro and were associated
with epigenetic changes [14]. Previously published tran-
scriptomic assessment revealed several differentially
enriched pathways in primary monocytes treated with
urate for 20 hours, including the TGF-β signalling path-
way [15]. In line with this, several genetic variants in
genes encoding activin receptors and inhibins belonging
to the TGF-β superfamily were found to be associated
with serum urate concentrations [16]. In addition, IL-37,
an anti-inflammatory cytokine with an important role in
gout, also functions via an interaction with SMAD3, a
major intracellular signalling effector of TGF-β, further
enforcing the importance of this signalling pathway in
gout [17, 18].l © 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. Klück et al. Arthritis Research & Therapy (2023) 25:30 Klück et al. Arthritis Research & Therapy (2023) 25:30 Page 2 of 12 Page 2 of 12 Materials and methods
Volunteers For the discovery cohort, blood from 9 gout patients (8
male, 1 female, mean age 66.2 years old) was used for
identification experiments. Blood from 7 healthy volun-
teers was used as a control (6 male, 1 female, mean age
60.4 years old). All volunteers gave informed consent
to use leftover blood for research purposes. Blood draw
from healthy volunteers were approved by the Ethical
Committee of the Radboud University Medical Center
(no. NL32357.091.10 and no. NL42561.091.12) Our validation cohort consisted of 197 individuals with
normouricemia, 179 individuals with hyperuricemia
without gout, and 195 patients with gout. All study par-
ticipants in the gout group were included if they corre-
sponded to the ACR/EULAR 2015 classification criteria
with a score of 8 or higher. Thirty-six patients presented
to the rheumatologist with acute flares. For gout man-
agement, allopurinol was used either alone or in com-
bination with NSAIDs and/or colchicine. Volunteers in
the hyperuricemia group were included based on serum TGF-β is generally considered an anti-inflammatory
cytokine with pro-fibrotic properties which can be
secreted by most immune cells [19]. It consists of three
paralogs of which paralog 1 is expressed in monocytes. Klück et al. Arthritis Research & Therapy (2023) 25:30 Page 3 of 12 for Western blotting. Protein concentrations were deter-
mined using Pierce BCA Protein Assay Kit (ThermoFis-
cher; Cat#23227) following manufacturer’s instructions,
and equal amounts of protein were loaded in Laemmli
sample buffer and separated on a 10% SDS/PAGE gel
for 2 h at 120V. After running the gel, the proteins
transferred to a 0.45-μM nitrocellulose membrane (GE
Healthcare; Cat#10600002) using wet transfer in Tow-
bin buffer on ice. The membrane was blocked for unspe-
cific binding with 5% BSA-TBST followed by incubation
with the primary antibody (Table S2). After overnight at
4 °C, incubation blots were washed and incubated with
the secondary antibody for 30 min at RT (Table S3). After
another washing step, the blots were developed using the
Odyssey CLX Infrared imaging system (Licor). Quan-
titative assessment of band intensity was performed by
Image Lab software (Bio-Rad). urate levels of 7 mg/dl or higher and negative history of
gout flares. None of the individuals with hyperuricemia
were treated with urate-lowering therapies. Ex vivo mRNA expression experimentsh The PBMCs from patients and matched healthy controls
were seeded on flat-bottom 96-well plates at a density of
0.5 × 106 cells per well and incubated at 37 °C with 5%
CO2 for an hour. Subsequently, non-adhering cells were
washed away using pre-warmed PBS and the adherent
monocytes were incubated with RPMI for 4 h before cells
were stored in TRIzol reagent. RNA purification was per-
formed according to manufacturer’s instructions. Sub-
sequently, RNA concentrations were determined using
NanoDrop software and cDNA was synthesized using
iScript cDNA Synthesis Kit. The SYBR Green method
was used to determine the mRNA expression of TGFB1,
TGFBR1, TGFBR2, MMP9, ITGAV and SMAD7 relative
to reference gene B2M (primer sequences Table S1) To inhibit TGF-β receptor signalling, three inhibitors
were used. The ALK4/5/7-kinase inhibitor SB-505124
(Sigma) in a concentration of 5 μM with DMSO as sol-
vent control, 5Z-7-oxozeaenol (100 nM) dissolved in
DMSO (Tocris Bioscience) and a blocking antibody
against TGF-β receptor II (AF-241-NA, R&D systems)
with mouse IgG1 as the isotype control (10 μg/mL). Cells
were pre-incubated with the inhibitor for 0.5 h before
adding urate. Materials and methods
Volunteers All partici-
pants were included in Cluj-Napoca, Romania, as part
of the HINT project (supported by the Romanian Minis-
try of European Funds; P_37_762, MySMIS 103587), and
both clinical characteristics and blood were collected for
analysis. For in vitro experiments, buffy coats from healthy
donors were obtained after written informed consent
(Sanquin blood bank, Nijmegen, the Netherlands). Urate priming experiments For urate priming experiments, adherent monocytes
were primed for 24 h in RPMI supplemented with 10%
human pool serum with or without urate (Sigma, 69-93-
2) and recombinant TGF-β1 (R&D Systems, Catalogue
number 7754-BH-005). After 24 h, cells were restimu-
lated with 10 ng/mL ultra-pure E. coli LPS (InVivogen,
Catalogue number tlrl-pelps). Subsequently, cell-free
supernatants were collected. Secretion of cytokines was
measured in supernatants using ELISA kits for IL-1β,
IL-6, IL-1Ra and TGF-β (R&D Systems, Catalogue num-
ber DY201, DY206, DY280 and DY240 respectively). In vitro TGF‑β1 signalling experiments For mRNA expression, adherent monocytes from healthy
volunteers were treated with dose-ranging concentra-
tions of urate for 24 h. Subsequently, cells were stored in
TRIzol and RNA isolation and qPCR were performed as
described above. Cell isolation Peripheral blood mononuclear cells (PBMCs) were iso-
lated using Ficoll-gradient from whole blood of volun-
teers and were resuspended in RPMI 1640 supplemented
with 50 μg/mL gentamycin, 2 mM L-glutamine and 1
mM pyruvate medium. Monocytes were further enriched
by either adherence for 1 hour followed by washout of
non-adherent lymphocytes or using Percoll gradient. Proteomics Serum samples from controls (N = 196), hyperurice-
mic (N = 173) and gout patients (N = 213) collected
and stored at – 80 °C were used for commercial targeted
serum proteomics analysis (Olink, Uppsala, Sweden). Olink Target 96 Inflammation panel measures 92 protein
biomarkers and four internal control samples, using 1 μl
serum sample, by multiplex proximity extension assays
[26]. The method uses two specific DNA-labelled anti-
bodies for each protein that upon target binding come in
close proximity to each other and allow the formation of
a PCR reporter sequence that is quantified by real-time
PCR (qPCR). Results are generated from cycle threshold For the pSMAD2 assessment, Percoll monocytes iso-
lated from healthy volunteers were seeded into a 12-wells
plate (1 × 106 cells/well) and treated overnight with urate
and, subsequently, TGF-β1 was added for the last hour. For collection of cell lysates, cells were kept on ice and
lysed with lysis buffer (Cell signalling; Cat#9803) contain-
ing 1x Complete Protease Inhibitor Cocktail (PIC; Roche
Diagnostics, #11697498001). Lysates were centrifuged at
25.000×g for 15 min at 4 °C, and supernatants were taken Klück et al. Arthritis Research & Therapy (2023) 25:30 Klück et al. Arthritis Research & Therapy (2023) 25:30 Page 4 of 12 Page 4 of 12 RNA. Fragmented target RNA was reverse transcribed
to cDNA using random N6 primers followed by end-
repair and A tailing for adaptor ligation. Purified liga-
tion products were enriched using PCR amplification
followed by denaturation and cyclization of ssDNA by
splint oligos and DNA ligase generating DNA nanoballs
(DNBs). Sequencing of DNBs was performed on DNBseq
platform. (Ct) values. The normalized protein expression (NPX)
values are arbitrary (log2 scale) units in which 1 NPX dif-
ference equals a two-fold change in protein abundance. Data pre-processing to minimize any technical intra- and
inter-assay variation is performed using internal plate
controls. Quality control was performed at both sample
and protein levels and samples that did not pass QC were
excluded. All proteomic data was corrected for age and
gender before targeted analysis. In case of LIF where the
majority of data were below the lower limit of detection,
we chose to use the actual data as was recommended by
Olink. Raw data was generated by removing reads mapped
to rRNAs. Transcriptomics Peripheral blood mononuclear cells were isolated using
whole blood from normouricemic or hyperuricemic
controls and from patients with gout by density gra-
dient centrifugation using Ficoll-Paque PLUS (Sigma
Aldrich). Freshly isolated cells were kept in TRIzol rea-
gent (Invitrogen), stored at – 80 °C and were later used
for commercial RNA-Seq analysis (Beijing Genomics
Institute, BGI, Beijing, China). The integrity of extracted
RNA was assessed using Agilent 2100 Bio. Oligo dT
magnetic beads were used to capture mRNA from total Read counts were normalized using DESeq2 (Version:
DESeq2_1.34.0) median of ratios method using R pack-
age (Version: R4.0.4.) and were used for downstream tar-
geted gene expression statistical analysis. Proteomics Clean reads were generated using SOAPnuke
software (version:v1.5.2) by removing reads with adap-
tors, reads with unknown bases > 10% and low-quality
reads, defined as reads with a quality score less than 15
in over 50% bases. Clean reads were mapped to human
transcriptome assembly GRCh37 (hg19) using bowtie2. Statistical analysis In ex vivo experiments, Mann-Whitney U test and
Welch ANOVA were performed to compare means
between groups. For correction of multiple comparisons, Fig. 1 TGF-β mRNA is upregulated in gout patients and correlates to serum urate levels in the discovery cohort. PBMCs from patients with gout
and matched healthy controls (HC) were isolated and adhered to a flat-bottom plate, mRNA was isolated and compared to the mean dCT of
healthy controls by Mann-Whitney U tests *p < 0.05 (A, B). Serum urate levels were correlated to relative mRNA expression levels and analysed by
Spearman’s correlation (C, D) Fig. 1 TGF-β mRNA is upregulated in gout patients and correlates to serum urate levels in the discovery cohort. PBMCs from patients with gout
and matched healthy controls (HC) were isolated and adhered to a flat-bottom plate, mRNA was isolated and compared to the mean dCT of
healthy controls by Mann-Whitney U tests *p < 0.05 (A, B). Serum urate levels were correlated to relative mRNA expression levels and analysed by
Spearman’s correlation (C, D) Klück et al. Arthritis Research & Therapy (2023) 25:30 Page 5 of 12 Klück et al. Arthritis Research & Therapy (2023) 25:30 Klück et al. Arthritis Research & Therapy Games-Howell correction was employed for proteomic
data (n > 50 within each group) and Tamhane T2 for tran-
scriptomics. Spearman analyses were used for correla-
tions. For in in vitro experiments, Wilcoxon signed rank
tests were employed to compare means. Differences with
adjusted p-value < 0.05 were considered statistically sig-
nificant. All analyses were done in GraphPad Prism 5. unstimulated PBMCs from individuals with normour-
icemia, hyperuricemia and patients with gout (HINT
study). Within the group of gout patients we observed no
differences between intercritical and flaring patients. We
observed no significant difference in receptor expression
or TGFB1, ITGAV and SMAD7, but the downstream tar-
get MMP9 was increased in patients with gout compared
to controls (Fig. 2A-D). For further assessment on a protein level, serum TGF-
β-LAP and two downstream targets LIF [27] and VEGFA
[28] levels were determined in the same individuals with
normouricemia, hyperuricemia and gout. All were signifi-
cantly higher in hyperuricemic individuals compared to
controls (Fig. 3A–C). Serum LIF was significantly higher
in gout patients during gout flare compared to intercriti-
cal gout. Statistical analysis However, for TGF-β-LAP no differences were
observed between controls and patients with gout, suggest-
ing they are more related to high urate levels than to gout. To test for this hypothesis, TGF-β-LAP was correlated with
serum urate levels. In line with the results observed in the
discovery experiments, serum urate levels were positively
correlated with serum TGF-β-LAP in all cohorts combined
(Pearson’s correlation = 0,19; p < 0,0001; Fig. 3D). TGF‑β is elevated in hyperuricemic individuals
and correlates positively to serum urate levels TGF‑β is elevated in hyperuricemic individuals
and correlates positively to serum urate levels As a first exploration, mRNA levels of TGFB1, TGFBRI,
TGFBRII and three TGF-β target genes ITGAV, MMP9
and SMAD7 were compared between untreated adher-
ent monocytes of patients with gout and age and sex
matched healthy controls. Expression levels of TGFB1
were increased in individuals with gout compared to
controls, and ITGAV mirrored this expression of TGFB1
(Fig. 1A, B). We identified no change for TGFBRI, TGF-
BRII, MMP9 and SMAD7. Moreover, serum urate lev-
els correlated positively to TGFB1 mRNA expression in
patients with gout (Fig. 1C).i To validate these findings in a larger cohort, mRNA
expression of the same genes (TGFB1, TGFBRI, TGF-
BRII, ITGAV, MMP9 and SMAD7) was assessed in Fig. 2 mRNA expression of TGF-β and downstream targets in PBMCs from individuals with hyperuricemia or gout. PBMCs from individuals with
normoruricemia (n = 110), hyperuricemia (n = 22) and gout (n = 72) of which 15 flaring (marked in red) were isolated and transcriptomics were
analysed. Relative mRNA expression of TGFB1 (A), MMP9 (B), ITGAV (C) and SMAD7 (D) are shown. Lines represent means with SD. Means were
compared by Welch ANOVA with Tamhane’s T2 multiple comparisons test. **p < 0.01 Fig. 2 mRNA expression of TGF-β and downstream targets in PBMCs from individuals with hyperuricemia or gout. PBMCs from individuals with
normoruricemia (n = 110), hyperuricemia (n = 22) and gout (n = 72) of which 15 flaring (marked in red) were isolated and transcriptomics were
analysed. Relative mRNA expression of TGFB1 (A), MMP9 (B), ITGAV (C) and SMAD7 (D) are shown. Lines represent means with SD. Means were
compared by Welch ANOVA with Tamhane’s T2 multiple comparisons test. **p < 0.01 Page 6 of 12 (2023) 25:30 Klück et al. Arthritis Research & Therapy (2023) 25:30 Klück et al. Arthritis Research & Therapy Fig. 3 Serum TGF-β-LAP, LIF and VEGF-A levels are elevated in individuals with hyperuricemia and TGF-β-LAP correlates positively to serum urate. Serum proteins were analysed by Olink proteomics panel. Flaring gout patients (n = 36) are marked in red dots. NPX was shown for TGF-β-LAP (A),
LIF (B) and VEGF-A (C). Means were compared by Welch ANOVA with Games-Howell’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001. Spearman correlation was used to analyse the correlation between serum TGF-β-LAP to urate levels (D). TGF‑β is elevated in hyperuricemic individuals
and correlates positively to serum urate levels Serum LIF levels were significantly higher in
flaring gout patients compared to intercritical gout patients (Welch’s t-test p < 0.0001) Fig. 3 Serum TGF-β-LAP, LIF and VEGF-A levels are elevated in individuals with hyperuricemia and TGF-β-LAP correlates positively to serum urate. Serum proteins were analysed by Olink proteomics panel. Flaring gout patients (n = 36) are marked in red dots. NPX was shown for TGF-β-LAP (A),
LIF (B) and VEGF-A (C). Means were compared by Welch ANOVA with Games-Howell’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001. Spearman correlation was used to analyse the correlation between serum TGF-β-LAP to urate levels (D). Serum LIF levels were significantly higher in
flaring gout patients compared to intercritical gout patients (Welch’s t-test p < 0.0001) Urate induces TGF‑β signalling in vitro To assess the functional consequences of enhanced
TGF-β signalling, in vitro priming experiments inves-
tigating the combined effects of urate and TGF-β on
cytokine production were performed. Human mono-
cytes from healthy volunteers were treated with urate,
TGF-β or a combination of the two for 24 h, washed
and subsequently stimulated with LPS. Cytokine
release was measured in supernatant. Both TGF-β and
urate priming increased the release of IL-1β and IL-6. Whereas urate lowered IL-1Ra release, TGF-β had no
effect on the production of IL-1Ra. Interestingly, we
observed a small additive effect, but no synergistic
effect between TGF-β and urate (Fig. 5).h To explore whether urate may drive TGF-β expression
or production, further in vitro studies were performed. Human primary monocytes isolated from healthy vol-
unteers treated with urate showed no elevated TGFB1
mRNA or TGF-β1 protein production after 24 h as
assessed by qPCR, ELISA and luciferase bioassay (Sup-
plemental Figures 1, 2, 3). Interestingly, in monocytes
treated with urate, mRNA expression of MMP9, which
can activate latent TGF-β to its active form, was upreg-
ulated. Moreover, the expression of SMAD7, a nega-
tive regulator of TGF-β signalling, was significantly
downregulated compared to RPMI control condition
(Supplemental Figure 1). To further assess intracel-
lular TGF-β signalling, C-terminally phosphorylated
SMAD2 was measured in urate and/or TGF-β treated
monocytes. Strikingly, both urate and TGF-β induced
C-terminal phosphorylation of SMAD2 showing
that intracellularly TGF-β signalling was more active
(Fig. 4). The lack of synergy between TGF-β and urate prim-
ing led us to hypothesize that the urate-induced inflam-
matory phenotype of the monocytes is mediated via
TGF-β. Therefore, we primed human monocytes with
urate in the presence of an antibody against the TGF-β
receptor II. Blocking the TGF-β receptor II partly Page 7 of 12 Klück et al. Arthritis Research & Therapy (2023) 25:30 Page 7 of 12
Klück et al. Arthritis Research & Therapy (2023) 25:30
Fig. 4 Protein expression of pSMAD2 (Ser465/467) in urate primed monocytes. The monocytes were primed overnight with no, 6.25 or 12.5 mg/dL
urate followed with a stimulation of 1 ng/mL TGFβ for 1 h. Cell lysates were used for western blotting. Relative pSMAD2 expression (n = 7) (A) and a
representative blot (B) are shown. Wilcoxon signed-rank test was used to compare means. *p < 0.05 Fig. 4 Protein expression of pSMAD2 (Ser465/467) in urate primed monocytes. Urate induces TGF‑β signalling in vitro The monocytes were
urate followed with a stimulation of 1 ng/mL TGFβ for 1 h. Cell lysates were used for western blotting
representative blot (B) are shown. Wilcoxon signed-rank test was used to compare means. *p < 0.05 Fig. 4 Protein expression of pSMAD2 (Ser465/467) in urate primed monocytes. The monocytes were primed overnight with no, 6.25 or 12.5 mg/dL
urate followed with a stimulation of 1 ng/mL TGFβ for 1 h. Cell lysates were used for western blotting. Relative pSMAD2 expression (n = 7) (A) and a
representative blot (B) are shown. Wilcoxon signed-rank test was used to compare means. *p < 0.05 induced elevated IL-1β production can be partly reversed
by blocking TGF-β and several TGF-β receptor blockers
ameliorate the urate-induced reduction of the monocyte
IL-1Ra production. reversed the urate induced phenotype. This was shown
by the fact that IL-1β production was greatly reduced
and that IL-1Ra levels were partly restored (Fig. 6A). Also, SB-505124, a kinase inhibitor of TGF-β I recep-
tors ALK 4, 5, and 7, inhibited urate induced IL-1β and
restored IL-1Ra (Fig. 6B). In addition, 5Z-7-oxozeaenol,
a TGF-β-activated kinase 1 inhibitor (TAK1), reduced
urate-induced IL-1β, but did not affect IL-1Ra (Fig. 6C). Together, these findings pinpoint TGF-β as a potential
mediator in urate-induced pro-inflammatory pheno-
type of human primary monocytes. In this study, we explored the expression levels of
TGFB1, its two receptors TGFBRI and TGFBRII and
three TGF-β target genes ITGAV, MMP9 and SMAD7,
the latter being a negative regulator of TGF-β signalling. In two different groups, we observed either an increased
expression of TGF-β itself in gout patients or an increase
in TGF-β-LAP protein in hyperuricemic individuals. Also, the expression of the downstream targets ITGAV
and MMP9 were increased in gout patients. Discussion In our in vitro setting, we observed no change to
TGFB1 expression, a decrease in TGFBRI and SMAD7
expression, while MMP9 expression was again increased
after treatment of PBMC with soluble urate for 24 h. Although both findings point towards enhanced TGF-β
signalling pathway, prolonged exposure of PBMCs to ele-
vated urate levels in vivo results to different TGF-β sig-
nalling kinetics compared to 24 h urate priming in vitro. By showing SMAD2 phosphorylation and blocking In this study, we assessed TGF-β in the context of hyper-
uricemia and gout and found that there is a role for
TGF-β in urate-induced pro-inflammatory status of
monocytes. In two different populations, TGF-β was
elevated in subjects with hyperuricemia or gout and cor-
related to serum urate concentrations. In vitro, urate
exposure did not directly induce TGF-β transcription
or protein release in human monocytes but did induce
C-terminally phosphorylated SMAD2. Moreover, urate Page 8 of 12 Page 8 of 12 Klück et al. Arthritis Research & Therapy Klück et al. Arthritis Research & Therapy (2023) 25:30 (2023) 25:30 Fig. 5 Both TGF-β and urate demonstrate pro-inflammatory
effects in a priming model without a synergistic effect. Adherent
monocytes isolated from healthy volunteers (n = 6) were treated with
dose-ranging concentrations of recombinant TGF-β and/or urate (50
mg/dL) for 24 h after which cells were washed and stimulated with
LPS (10 ng/mL) for 24 h. IL-1β (A), IL-6 (B) and IL-Ra (C) were measured
in the supernatant after 48 h culture flares. In contrast, we observed no differences in TGFB1
expression or serum TGF-β-LAP within our gout cohort
between patients during gout flare and intercritical gout
patients. Possibly, TGF-β has a local effect at the site of
arthritis but does not result in changes in serum protein
or transcripts in circulating PBMCs. However, serum LIF
protein was significantly elevated in gout patients dur-
ing flare. Apart from TGF-β, LIF can also be induced by
IL-1β during arthritis [33, 34], which could account for
these differences. In vitro, we found that co-incubation
with LPS, MSU crystals and TGF-β reduces pro-inflam-
matory cytokines (data not shown). However, priming
monocytes with TGF-β before stimulation with LPS has
pro-inflammatory effects similar to urate priming. A
pro-inflammatory role for TGF-β has previously been
described in adaptive immunity where TGF-β is a key
regulator of T helper 17 differentiation. Discussion f
Confirming our in vivo findings, a positive correla-
tion between serum TGF-β and urate was also observed
in patients with coronary artery disease [35]. This raises
the question what the functional consequences are of
elevated urate and TGF-β in humans. In mice, hyper-
uricemia induces TGF-β in renal tubular tissue [36,
37]. In parallel, allopurinol withdrawal in patients with
chronic kidney disease leads to worsening of hyperten-
sion, acceleration of the rate of loss of kidney function
and an increase in the urinary excretion of TGF-β [38]. The observed increase in fibrosis by urate is not limited
to renal disorders. Serum urate has been described as
being predictive of pulmonary arterial hypertension, a
severe complication in patients with systemic sclerosis
[39]. Moreover, Febuxostat, a urate-lowering drug, was
shown to suppress angiotensin II-induced aortic fibrosis
in mice [40]. We observed that the pro-inflammatory effects of urate
were partly mediated by TGF-β. Treating monocytes
with both urate and TGF-β had no obvious synergistic
effect on cytokine production. Potentially, this could be
explained by the presence of human serum in the culture
medium which accounts for 3–4 ng/mL TGF-β already. Blocking the TGF-β pathway with several inhibitors
reduced the production of IL-1β in human monocytes
after stimulation with LPS. Combined with the observed
SMAD phosphorylation, this suggests urate activates
TGF-β signalling. One of the possible underlying mecha-
nisms is that urate activates the TGF-β activated kinase
1 (TAK1), an important kinase in the TGF-β pathway. Uric acid molecules are capable of arresting TAK1 in an
active-state conformation, resulting in sustained TAK1
kinase activation [41]. Both TGF-β induced SMAD2/3
and SMAD1/5 phosphorylation are mediated by TAK1
kinase activity. By using different inhibitors with each
slightly different targets in the TGF-β signalling cascade, Fig. 5 Both TGF-β and urate demonstrate pro-inflammatory Fig. 5 Both TGF-β and urate demonstrate pro-inflammatory
effects in a priming model without a synergistic effect. Adherent
monocytes isolated from healthy volunteers (n = 6) were treated with
dose-ranging concentrations of recombinant TGF-β and/or urate (50
mg/dL) for 24 h after which cells were washed and stimulated with
LPS (10 ng/mL) for 24 h. IL-1β (A), IL-6 (B) and IL-Ra (C) were measured
in the supernatant after 48 h culture TGF-β in vitro, we were still able to demonstrate its rel-
evance in urate-induced inflammatory phenotype. TGF-β in vitro, we were still able to demonstrate its rel-
evance in urate-induced inflammatory phenotype. Discussion Previously, TGF-β was studied in the context of gout. MSU crystals induce TGF-β in macrophages [29] and
exogenous TGF-β inhibits MSU-crystal induced inflam-
mation in vivo [30]. In synovial fluid, TGF-β1 is sig-
nificantly elevated in acute gouty arthritis compared to
osteoarthritis [31] and increases during duration of gout
flare [32]. These data combined suggests an anti-inflam-
matory role for TGF-β in the resolution phase of gout l
Previously, TGF-β was studied in the context of gout. MSU crystals induce TGF-β in macrophages [29] and
exogenous TGF-β inhibits MSU-crystal induced inflam-
mation in vivo [30]. In synovial fluid, TGF-β1 is sig-
nificantly elevated in acute gouty arthritis compared to
osteoarthritis [31] and increases during duration of gout
flare [32]. These data combined suggests an anti-inflam-
matory role for TGF-β in the resolution phase of gout Page 9 of 12 Klück et al. Arthritis Research & Therapy Klück et al. Arthritis Research & Therapy (2023) 25:30 Page 9 of 12
Klück et al. Arthritis Research & Therapy (2023) 25:30
we could potentially pinpoint the pathways effected by
urate. SB-505124, targeting ALK5, inhibits gene expres-
sion downstream of both pSMAD2/3 and pSMAD1/5,
whereas (5Z)-7-Oxozeaenol, a TAK1 inhibitor, does so
[42]. In our in vitro urate priming models, SB-505124
both reduced IL-1β and restored IL-1Ra, whereas (5Z)-
7-Oxozeaenol only inhibited IL-1β without affecting
IL-1Ra. Possibly the difference in target gene expression
Fig. 6 Blocking TGF-β signalling pathway partly reverses urate priming effects. Adherent monocytes isolated from healthy volunteers (An = 10; B-C
n = 6) were treated with dose-ranging concentrations of urate (50 mg/dL) in the presence or absence of a TGF-β inhibitor for 24 h after which cells
were washed and stimulated with LPS (10 ng/mL) for 24 h. IL-1β and IL-Ra were measured in the supernatant after 48 h culture. TGF-β inhibitors: a
blocking antibody against TGF-β receptor II with mouse IgG1 as the isotype control (10 μg/mL), SB-505124 (5 μM) and 5Z-7-oxozeaenol (100nM)
both with DMSO as solvent control. Wilcoxon signed rank test was applied to compare means. *p < 0.05 Fig. 6 Blocking TGF-β signalling pathway partly reverses urate priming effects. Adherent monocytes isolated from healthy volunteers (An = 10; B-C
n = 6) were treated with dose-ranging concentrations of urate (50 mg/dL) in the presence or absence of a TGF-β inhibitor for 24 h after which cells
were washed and stimulated with LPS (10 ng/mL) for 24 h. Conclusions In conclusion, TGF-β is elevated in individuals with
hyperuricemia correlating to serum urate levels and the
urate induced pro-inflammatory phenotype in human
monocytes is mediated by TGF-β signalling. Future stud-
ies are warranted to explore the intracellular pathways
involved and to assess the clinical significance of the rela-
tion between serum urate and TGF-β. Abbreviations
TGF
Transforming growth factor
TAK1
TGF-β-activated kinase 1 inhibitor
IL
Interleukin
IL-1Ra
Interleukin 1 receptor antagonist
PBMC
Peripheral blood mononuclear cells
mRNA
Messenger ribonucleic acid
SMAD
An acronym from the fusion of Caenorhabditis elegans Sma genes
and the Drosophila Mad, mothers against decapentaplegic
MSU
Monosodium urate
TLR
Toll-like receptor
LAP
Latency-associated peptide
MMP
Matrix metallopeptidase
ITGAV
Integrin subunit alpha V
LPS
Lipopolysaccharide
PCR
Polymerase chain reaction
LIF
Leukemia inhibitory factor
VEGF
Vascular endothelial growth factor Another unexplored mechanism of enhanced TGF-β
signalling in urate treated monocytes is the regulation of
integrin αvβ8. Human CD14+ monocytes activate TGF-β
via the expression of the integrin αvβ8 and matrix metal-
loproteinase 14 [44]. Since TGF-β is always secreted as a
latent complex, the function of TGF-β in the regulation
of immune responses is controlled by mechanisms that
regulate latent TGF-β activation. Finally, the cellular responses to TGF-β have been
shown to be altered by pro-inflammatory cytokines
such as IL-1β. In chondrocytes, TGF-β induced SMAD7
could be reversed by IL-1β treatment [45]. Likewise, we
observed a significant reduction in SMAD7 after urate
treatment which is known to lower IL-1Ra expression. Potentially, not urate itself, but a reduction in IL-1Ra
could modulate the cellular response to TGF-β similar
to IL-1β. In our priming model, blocking TGF-β signal-
ling with SB-505124 enhanced IL-1Ra release independ-
ent of urate, further suggesting a link between IL-1Ra and
TGF-β signalling. Supplementary Information The online version contains supplementary material available at https://doi.
org/10.1186/s13075-023-03001-1. Additional file 1: Table S1. Primer sequences ex vivo experiments. Table S2. Primary antibodies for western blot. Table S3. Secondary
antibodies for western blot. Figure S1. mRNA expression of genes in
the TGF-β signalling pathway in adherent monocytes treated with urate
in vitro. PBMCs of healthy volunteers were isolated, adhered to a flat-
bottom plated and cultured in medium supplemented with 10% HPS with
dose-ranging concentrations of urate. mRNA was isolated after 24h and
compared to control condition by Wilcoxon matched-pairs signed rank
test. *p < 0.05, **p < 0.01. Figure S2. Urate does not increase TGF-β release
of human monocytes. Acknowledgements We would like to thank Dr. Sander van Leuven (Department of Rheumatology,
Radboudumc) for his input in this project. Also, a special thanks to all patients
and volunteers who donated blood for this study. Consortia HINT consortium: Leo A. B. Joosten, Ioan V. Pop, Radu A. Popp, Simona Rednic,
Cristina Pamfil, Tania O. Crişan, Marius Farcaş†, Dragoş H. Marginean, Orsolya I. Gaal, Medeea O. Badii, Ioana Hotea, Loredana Peca, Andreea-Manuela Mirea,
Georgiana Cabău, Valentin Nica, Doina Colcear, Mariana S. Pop, Ancuta Rus Discussion IL-1β and IL-Ra were measured in the supernatant after 48 h culture. TGF-β inhibitors: a
blocking antibody against TGF-β receptor II with mouse IgG1 as the isotype control (10 μg/mL), SB-505124 (5 μM) and 5Z-7-oxozeaenol (100nM)
both with DMSO as solvent control. Wilcoxon signed rank test was applied to compare means. *p < 0.05 [42]. In our in vitro urate priming models, SB-505124
both reduced IL-1β and restored IL-1Ra, whereas (5Z)-
7-Oxozeaenol only inhibited IL-1β without affecting
IL-1Ra. Possibly the difference in target gene expression
accounts for this observed difference. we could potentially pinpoint the pathways effected by
urate. SB-505124, targeting ALK5, inhibits gene expres-
sion downstream of both pSMAD2/3 and pSMAD1/5,
whereas (5Z)-7-Oxozeaenol, a TAK1 inhibitor, does so
to a lesser extent and in a more limited number of genes Page 10 of 12 Page 10 of 12 Klück et al. Arthritis Research & Therapy (2023) 25:30 Klück et al. Arthritis Research & Therapy (2023) 25:30 Another possible intracellular mechanism involved in
urate and TGF-β priming is the PI3K/Akt/mTOR path-
way. Although we demonstrated pSMAD2 involvement,
TGF-β can also activate PI3K resulting in phosphoryla-
tion of Akt, independently of SMADs [43]. Similarly,
urate priming induces pAkt in monocytes which was
reversed by a PI3K inhibitor [15]. Both urate and TGF-β
activate the mammalian target of rapamycin (mTOR)
via PI3K/Akt pathway, thereby presumably inhibiting
autophagy. Since urate induces phosphorylation of Akt
within 15 min, it is uncertain whether PI3K activation
is mediated via TGF-β. Further research should explore
whether these shared pathways are regulated dependent
or independent of each other. Conclusions PBMCs were isolated from healthy volunteers and
adherent monocytes were primed for 24 hours in RPMI supplemented
with 10% human pool serum with or without urate. TGF-β was measured
in the supernatant by ELISA (R&D standard). Figure S3. Urate does not
affect TGF-β bioactivity. PBMCs were isolated from healthy volunteers and
adherent monocytes were primed for 24 hours in RPMI supplemented
with 10% human pool serum with or without urate. Supernatant was used
in a CAGA12-luciferase bioassay. β
The unexplored intracellular signalling is a clear limi-
tation of this study. Moreover, the consequence of ele-
vated TGF-β was only evaluated in vitro. Exploring the
clinical relevance of urate induced changes in TGF-β
signalling is not only important for gout but also for
other rheumatic diseases as osteoarthritis and systemic
sclerosis. In osteoarthritis, changes in in TGF-β signal-
ling are known to contribute to the pathogenesis [46],
and serum urate was also identified as a risk factor for
symptomatic knee osteoarthritis and joint space nar-
rowing [47, 48]. Elevated serum urate is also associated
with increased risk for pulmonary arterial hypertension
in patients with systemic sclerosis, a complex connec-
tive tissue disease characterized by inflammation, vas-
culopathy and excessive fibrosis, meditated by TGF-β
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tory factor (LIF) and p21 mediate the TGFβ tumor suppressive effects in
human cutaneous melanoma. BMC Cancer. 2015;15(1):200. 6. Athyros VG, Mikhailidis DP. Uric acid, chronic kidney disease and type
2 diabetes: a cluster of vascular risk factors. J Diabetes Complications. 2014;28(2):122–3. 6. Athyros VG, Mikhailidis DP. Uric acid, chronic kidney disease and type
2 diabetes: a cluster of vascular risk factors. J Diabetes Complications. 2014;28(2):122–3. 28. Availability of data and materials The datasets supporting the conclusions of this article are included within the
article and its additional file. 16. Kottgen A, Albrecht E, Teumer A, Vitart V, Krumsiek J, Hundertmark C, et al. Genome-wide association analyses identify 18 new loci associated with
serum urate concentrations. Nat Genet. 2013;45(2):145–54. Received: 12 May 2022 Accepted: 29 January 2023 22. Naiki Y, Michelsen KS, Zhang W, Chen S, Doherty TM, Arditi M. Transform-
ing growth factor-β differentially inhibits MyD88-dependent, but not
TRAM- and TRIF-dependent, lipopolysaccharide-induced TLR4 signaling*. J Biol Chem. 2005;280(7):5491–5. Funding This work was supported by a Competitiveness Operational Programme grant
of the Romanian Ministry of European Funds (P_37_762, MySMIS 103587). VK was supported by a 2016 Radboudumc Master-PhD grant. These funding
sources had no role in the design, collection, analysis, interpretation of data
nor in writing the manuscript. 14. Badii M, Gaal OI, Cleophas MC, Klück V, Davar R, Habibi E, et al. Urate-
induced epigenetic modifications in myeloid cells. Arthritis Res Ther. 2021;23(1):202. 15. Crisan TO, Cleophas MCP, Novakovic B, Erler K, van de Veerdonk FL,
Stunnenberg HG, et al. Uric acid priming in human monocytes is driven
by the AKT-PRAS40 autophagy pathway. Proc Natl Acad Sci U S A. 2017;114(21):5485–90. Consent for publication
Not applicable 19. Derynck R, Zhang YE. Smad-dependent and Smad-independent path-
ways in TGF-beta family signalling. Nature. 2003;425(6958):577–84. Declarations 17. Nold MF, Nold-Petry CA, Zepp JA, Palmer BE, Bufler P, Dinarello CA. IL-37 is a fundamental inhibitor of innate immunity. Nat Immunol. 2010;11(11):1014–22. Authors’ contributions VK, LM, FB, LE and AC performed the in vitro experiments. GC, the HINT
consortium and TOC designed, performed and collected all the data from Page 11 of 12 Klück et al. Arthritis Research & Therapy (2023) 25:30 Klück et al. Arthritis Research & Therapy (2023) 25:30 the cohort of individuals with normouricemia, hyperuricemia and gout. RB
was involved in the acquisition of patient material. VK, AC, TOC and LABJ the cohort of individuals with normouricemia, hyperuricemia and gout. RB
was involved in the acquisition of patient material. VK, AC, TOC and LABJ
contributed to the design of the work and interpretation of data. VK and GC
drafted the manuscript. All authors revised the manuscript and approved the
submitted version. the cohort of individuals with normouricemia, hyperuricemia and gout. RB
was involved in the acquisition of patient material. VK, AC, TOC and LABJ cytokine production by human primary cells via inhibition of IL-1Ra. Ann
Rheum Dis. 2016;75(4):755–62. 12. Cabau G, Crisan TO, Kluck V, Popp RA, Joosten LAB. Urate-induced
immune programming: consequences for gouty arthritis and hyperurice-
mia. Immunol Rev. 2020;294(1):92–105. contributed to the design of the work and interpretation of data. VK and GC
drafted the manuscript. All authors revised the manuscript and approved the
submitted version. 13. Joosten LAB, Crisan TO, Bjornstad P, Johnson RJ. Asymptomatic hyperuri-
caemia: a silent activator of the innate immune system. Nat Rev Rheuma-
tol. 2020;16(2):75–86. Competing interests 20. Taylor AW. Review of the activation of TGF-beta in immunity. J Leukoc
Biol. 2009;85(1):29–33. The authors declare that they have no competing interests. 21. Li MO, Wan YY, Sanjabi S, Robertson A-KL, Flavell RA. Transforming
growth factor-β regulation of immune responses. Annu Rev Immunol. 2006;24(1):99–146. Received: 12 May 2022 Accepted: 29 January 2023 Received: 12 May 2022 Accepted: 29 January 2023 Received: 12 May 2022 Accepted: 29 January 2023 Ethics approval and consent to participate All ethics approval and consent are described in the methods section of this
manuscript. 18. Kluck V, van Deuren RC, Cavalli G, Shaukat A, Arts P, Cleophas MC,
et al. Rare genetic variants in interleukin-37 link this anti-inflammatory
cytokine to the pathogenesis and treatment of gout. Ann Rheum Dis. 2020;79(4):536–44. References Arthritis Rheumatol. 2017;69(6):1213–20. 49. van Caam A, Vonk M, van den Hoogen F, van Lent P, van der Kraan P. Unraveling SSc pathophysiology; the myofibroblast. Front Immunol. 2018;9:2452. 49. van Caam A, Vonk M, van den Hoogen F, van Lent P, van der Kraan P. Unraveling SSc pathophysiology; the myofibroblast. Front Immunol. 2018;9:2452. •
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? Choose BMC and benefit from: References 35. Chen C, Lei W, Chen W, Zhong J, Gao X, Li B, et al. Serum TGF-beta1 and
SMAD3 levels are closely associated with coronary artery disease. BMC
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37. Kim SM, Choi YW, Seok HY, Jeong KH, Lee SH, Lee TW, et al. Reduc-
ing serum uric acid attenuates TGF-beta1-induced profibrogenic
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transforming growth factor beta and the progression of chronic kidney
disease. Am J Nephrol. 2007;27(5):435–40. p
39. Coghlan JG, Denton CP, Grunig E, Bonderman D, Distler O, Khanna D, et al. Evidence-based detection of pulmonary arterial hypertension in systemic
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40. Kondo M, Imanishi M, Fukushima K, Ikuto R, Murai Y, Horinouchi Y, et al. Xanthine oxidase inhibition by febuxostat in macrophages suppresses
angiotensin II-induced aortic fibrosis. Am J Hypertens. 2019;32(3):249–56. 41. Singh AK, Haque M, O’Sullivan K, Chourasia M, Ouseph MM, Ahmed S. Suppression of monosodium urate crystal-induced inflammation by
inhibiting TGF-beta-activated kinase 1-dependent signaling: role of the
ubiquitin proteasome system. Cell Mol Immunol. 2021;18(1):162–70. 42. van Caam A, Madej W, Garcia de Vinuesa A, Goumans MJ, Ten Dijke P,
Blaney Davidson E, et al. TGFβ1-induced SMAD2/3 and SMAD1/5 phos-
phorylation are both ALK5-kinase-dependent in primary chondrocytes
and mediated by TAK1 kinase activity. Arthritis Res Ther. 2017;19(1):112. 43. Zhang YE. Non-Smad pathways in TGF-beta signaling. Cell Res. 2009;19(1):128–39. 44. Kelly A, Gunaltay S, McEntee CP, Shuttleworth EE, Smedley C, Houston
SA, et al. Human monocytes and macrophages regulate immune
tolerance via integrin αvβ8-mediated TGFβ activation. J Exp Med. 2018;215(11):2725–36. 45. Roman-Blas JA, Stokes DG, Jimenez SA. Modulation of TGF-β signaling by
proinflammatory cytokines in articular chondrocytes. Osteoarthr Cartil. 2007;15(12):1367–77. 46. Thielen NGM, van der Kraan PM, van Caam APM. TGFβ/BMP signaling
pathway in cartilage homeostasis. Cells. 2019;8(9):969. 47. Wang S, Pillinger MH, Krasnokutsky S, Barbour KE. The association
between asymptomatic hyperuricemia and knee osteoarthritis: data from
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Cartil. 2019;27(9):1301–8. 48. Krasnokutsky S, Oshinsky C, Attur M, Ma S, Zhou H, Zheng F, et al. Serum
urate levels predict joint space narrowing in non-gout patients with
medial knee osteoarthritis. References Shi X, Guo LW, Seedial SM, Si Y, Wang B, Takayama T, et al. TGF-β/Smad3
inhibit vascular smooth muscle cell apoptosis through an autocrine
signaling mechanism involving VEGF-A. Cell Death Dis. 2014;5(7):e1317-e. 7. Kim SY, Guevara JP, Kim KM, Choi HK, Heitjan DF, Albert DA. Hyperurice-
mia and coronary heart disease: a systematic review and meta-analysis. Arthritis Care Res (Hoboken). 2010;62(2):170–80. 29. Yagnik DR, Evans BJ, Florey O, Mason JC, Landis RC, Haskard DO. Mac-
rophage release of transforming growth factor beta1 during resolution of
monosodium urate monohydrate crystal-induced inflammation. Arthritis
Rheum. 2004;50(7):2273–80. 8. Chaudhary K, Malhotra K, Sowers J, Aroor A. Uric acid - key ingredient
in the recipe for cardiorenal metabolic syndrome. Cardiorenal Med. 2013;3(3):208–20. 30. Liote F, Prudhommeaux F, Schiltz C, Champy R, Herbelin A, Ortiz-Bravo
E, et al. Inhibition and prevention of monosodium urate monohydrate
crystal-induced acute inflammation in vivo by transforming growth fac-
tor beta1. Arthritis Rheum. 1996;39(7):1192–8. 9. Feldman N, Rotter-Maskowitz A, Okun E. DAMPs as mediators of sterile
inflammation in aging-related pathologies. Ageing Res Rev. 2015;24(Pt
A):29–39. 31. Chen Y, Huang Y, Li X, Xu M, Bi Y, Zhang Y, et al. Association of arterial
stiffness with HbA1c in 1,000 type 2 diabetic patients with or without
hypertension. Endocrine. 2009;36(2):262–7. 10. Disveld IJM, Zoakman S, Jansen T, Rongen GA, Kienhorst LBE, Janssens
H, et al. Crystal-proven gout patients have an increased mortality due to
cardiovascular diseases, cancer, and infectious diseases especially when
having tophi and/or high serum uric acid levels: a prospective cohort
study. Clin Rheumatol. 2019;38(5):1385–91. 32. Scanu A, Oliviero F, Ramonda R, Frallonardo P, Dayer JM, Punzi L. Cytokine
levels in human synovial fluid during the different stages of acute gout:
role of transforming growth factor beta1 in the resolution phase. Ann
Rheum Dis. 2012;71(4):621–4. y
11. Crisan TO, Cleophas MC, Oosting M, Lemmers H, Toenhake-Dijkstra H,
Netea MG, et al. Soluble uric acid primes TLR-induced proinflammatory Page 12 of 12 Klück et al. Arthritis Research & Therapy (2023) 25:30 Klück et al. Arthritis Research & Therapy (2023) 25:30 33. Enomoto H, Saito S, Yabe H, Toyama Y, Tomatu T. The levels of leukemia
inhibitory factor in synovial tissues of patients with rheumatoid arthritis:
inflammation and other proinflammatory cytokines. Mod Rheumatol. 2003;13(2):121–8. 34. Lotz M, Moats T, Villiger PM. Leukemia inhibitory factor is expressed in car-
tilage and synovium and can contribute to the pathogenesis of arthritis. J
Clin Invest. 1992;90(3):888–96. •
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Extensive Mucocutaneous Verruca Vulgaris in a Nonimmunocompromised Patient
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Jaypee's international journal of clinical pediatric dentistry
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IJCPD CASE REPORT
10.5005/jp-journals-10005-1084 Extensive Mucocutaneous Verruca Vulgaris
in a Nonimmunocompromised Patient 1Vela D Desai, 2Rajeev Sharma, 3Durgesh N Bailoor 1Vela D Desai, 2Rajeev Sharma, 3Durgesh N Bailoor
1Professor and Head, Department of Oral Medicine and Radiology, Jaipur Dental College, Jaipur, Rajasthan, India
2Senior Lecturer, Department Oral Medicine and Radiology, Jaipur Dental College, Jaipur, Rajasthan, India
3Professor, Department of Oral Medicine and Radiology, Karnavati Dental College, Ahmedabad, Gujarat, India Correspondence: Vela D Desai, Professor and Head, D-302, Trimurthy Apartments, Opp. BSNL Telecom Colony, Model Town
Malviya Nagar, Jaipur, Rajasthan-302017, India, Fax: +91 1426-234176, e-mail: veladesai@hotmail.com, drsunilmv@hotmail.com ABSTRACT Papilloma virus infections of the oral cavity have been long recognized with various clinical expressions characterized as verruca vulgaris,
Heck’s disease, multiple papilloma and condyloma acuminata. In this paper, we are highlighting a case of verruca vulgaris involving the oral
cavity with extensive skin lesions in a nonimmunocompromised 9-year-old boy. Different treatment modalities are discussed in this article. Keywords : Verruca vulgaris, Warts, Human papilloma virus. INTRODUCTION Fig. 1: A 9-year-old boy presenting with papillary growths on the
forehead, inner canthus of eye, left angle of mouth and on vermilion
border of lip Verruca vulgaris is a relatively uncommon oral lesion which
is caused by an infectious agent HPV (human papilloma
virus). Verruca vulgaris (VV) of oral mucosa is rare and
is typically a childhood problem. The lesions of verruca
vulgaris are circumscribed, firm, elevated papule with
papillomatous hyperkeratotic surface. They occur most
commonly on the dorsal aspect of the fingers and hand. They are also found on sole of feet. Lesions of verucca occur
extensively in immunocompromised host. Here, we present
a case of severe form of verruca in a healthy individual. CASE REPORT A 9-year-old boy reported to the Department of Oral
Medicine, Radiology and Diagnosis with multiple, small,
whitish growth on his legs, hands, fingers and face
(Fig. 1). The first symptom of this appeared 2 year ago. Lesions were asymptomatic except for slight occasional
itching. Similar type of lesion also had been noticed in his
elder brother and peers, which regressed spontaneously
without any treatment. Systemic review was noncontributory. Patient neither suffer from any long-term illness in the past
nor was on any medications. On extraoral examination,
numerous keratotic, well-defined whitish colored, papules
varying in size from 2 to 4 mm were found on dorsum of
hand, feet, face, chin and angle of mandible (Figs 1 to 2B). On left hand they were found in groups of more than 20 in
number. Some of the lesions on left hand were in a linear
fashion and exhibited Koebner phenomenon. Mild discharge
of blood was noticed on scraping the lesion (Fig. 3). Fig. 1: A 9-year-old boy presenting with papillary growths on the
forehead, inner canthus of eye, left angle of mouth and on vermilion
border of lip Examination of the oral cavity revealed whitish solitary
lesion on right buccal mucosa with finger like projection and
multiple small (4-5 in number) lesions were also appreciable
on lower labial mucosa and on alveolar ridge in the region
of lower left lateral incisor and canine (Fig. 4). On palpation
they were firm in consistency and nontender. Patient had an
otherwise good oral hygiene. Examination of other systems
was noncontributory. A provisional diagnosis of verruca
vulgaris was made. 65 International Journal of Clinical Pediatric Dentistry, January-April 2011;4(1):65-68 8/13/2016 5:09:20 PM Art-12.indd 65 Vela D Desai et al Figs 2A and B: Multiple grayish white lesions on the hands and legs
A
B
A B Figs 2A and B: Multiple grayish white lesions on the hands and legs The patient underwent a complete blood investigation
including Elisa test, which were within normal range. Excisional biopsy was performed with the patients parents
consent (as he was a minor) from two different sites, i.e. dorsum of hand and intraorally from the right buccal
mucosa. Histopathological examination revealed acanthosis
with proliferative, hyperkeratotic, stratified squamous
epithelium showing finger like projection and elongated
rate pegs converging towards the center, producing cupping
effect suggestive of verruca vulgaris. There were foci of
vacuoled cells referred to as koilocytotic cells. CASE REPORT Vertical
tier of parakeratotic cells and foci of clumped keratohyalin
granules were also seen (Figs 5A and B). Patient underwent
a thorough oral prophylaxis only. Fig. 4: Intraorally small flat lesion on right buccal mucosa Fig. 4: Intraorally small flat lesion on right buccal mucosa DISCUSSION Fig. 3: Coalesced linear occurring lesions showing bleedi A variety of papillary lesions occur in the oral cavity
and while many can be differentiated on the basis of
histopathological features, they may closely resemble
one another clinically1. Verruca vulgaris, condyloma
acuminate, Heck’s disease, squamous papilloma are all local
papillary lesions that share similar clinical feature yet their
microscopic patterns differ.1,2 Verruca vulgaris commonly
occurs in children and usually regresses spontaneously over
a period of time.2,3 Papilloma virus type 2 and 4 are the most
prevalent isolates from cutaneous warts, and these same
viral genotypes are identifiable when verruca valgaris arise
on the lip or on mucous membrane. Whereas condylomas
are usually diagnosed in teenagers and young adults and are
more frequently encountered in the mucosa membranes of
the anogenital region, it may also be seen in the oral cavity
perhaps arising therein as a consequence of oragenital sexual
transmission.1 They are bigger in size and appear pink to red
as a results of less keratinization. 66 JAYPEE JAYPEE J 8/13/2016 5:09:20 PM 8/13/2016 5:09:20 PM Art-12.indd 66 IJCPD Extensive Mucocutaneous Verruca Vulgaris in a Nonimmunocompromised Patient A reports.4,5 Kirchner has speculated that a certain kind of
lesion is more frequently found before puberty and links its
presence to a genetic defect that retards the establishment
of an effective defense mechanism until puberty leaving
some children more susceptible than others to the infection.6
Verruca vulgaris may not require any treatment, but is done
for cosmetic purposes and to prevent their spread.7 Most people develop an immune response that causes
verruca vulgaris to vanish by themselves.8 The patient’s
sibling’s lesions vanished in a similar way and in the present
case also, no treatment was initiated as the patient denied
any treatment. Skin verruca vulgaris is treated effectively by liquid
nitrogen, cryotheraphy, conservative surgical excision or
curettage or topical application of keratinolytic agents
containing salicylic acid and lactic acid.7,8 Oral lesions are
successfully surgically excised or they may be destroyed by
laser, cryotheraphy or electrosurgery. Recurrence is seen in
a small proportion of treated case. Verruca vulgaris does not
transform into malignancy with or without treatment and two
third will disappear spontaneously within 2 years especially
in children.7 Detailed genetic study should be carried out to
rule out any genetic origin in all the suspected cases. Figs 5A and B: Histopathology showing the classical lesion of
verucca vulgaris
B CONCLUSION Due to paucity of cases of oral verruca vulgaris being
reported, information regarding malignant potential is
limited. In the current case despite an extensive skin
lesion there was no evidence of underlying disease (AIDS/
dysplasia on histopathology). On basis of a single case, it
is very difficult to infer that they may occur in nonimmuno-
compromised patient and have no malignant potential. More
studies need to be carried out to corroborate that they occur
in immunocompromised patient. Any patient with presence
of extensive verruca vulgaris lesion must be eyed with
suspicion of AIDS/compromised immune system or internal
malignancy and thorough blood investigation including
ELISA/immunohistochemistry/in situ hybridization/
southern blot hybridization should be carried out. Fortunately
in our case patient did not have any of this life threatening
condition. The association of oral lesion with HPV should
be extensively examined to clarify the pathogenesis of HPV
infection in oral cavity. B Figs 5A and B: Histopathology showing the classical lesion of
verucca vulgaris The case herein represent classical form of oral verruca
vulgaris with extensive multiple skin involvement. Such
type of exacerbated involvement is usually observed
in immunocompromised host.4 There have been very
few reported cases of verruca vulgaris in a nonimmuno
compromised patients. Thus, presented case is unique as
there was no underlying immune supression. 1. Eversole LR, Laipis PJ,. Oral squamous papillomas: Detection
of HPV DNA by in situ hybridization. Oral Surg Oral Med Oral
Pathol 1988 May;65(5):545-550. 2. Sharma G, Nagpal A, Veagal M, Pai K. Multiple Papilloma: A
case report and review of literature. J Indian Acad Oral Med
Radiol 2007;19(3):384-388. 1. Eversole LR, Laipis PJ,. Oral squamous papillomas: Detection
of HPV DNA by in situ hybridization. Oral Surg Oral Med Oral
Pathol 1988 May;65(5):545-550.
2. Sharma G, Nagpal A, Veagal M, Pai K. Multiple Papilloma: A
case report and review of literature. J Indian Acad Oral Med
Radiol 2007;19(3):384-388. International Journal of Clinical Pediatric Dentistry, January-April 2011;4(1):65-68 4. Adler-Storthz K, Newland JR, Tessin BA, Yeudall WA, Shillitoe
EJ. Identification of human papilloma virus types in oral verruca
vulgaris. J Oral Pathol 1986 Apr;15(4):230-233. 3. Sivapathasundharam B, Shifa S. Oral verruca vulgaris: Report
of a rare case. Indian J Dent Res 2004 Jan-Mar;15(1):32-34. 5. Green TL, Eversole LR, Leider AS. Oral and labial verruca vulgaris:
Clinical, histological and immunohistochemical evaluation.
Oral Surg Oral Med Oral Pathol 1986 Oct:62(4):410-416. 6. Kirchner H. Immunobiology of human papillomavirus infection.
Prog Med Virol 1986;33:1-41. 3. Sivapathasundharam B, Shifa S. Oral verruca vulgaris: Report
of a rare case. Indian J Dent Res 2004 Jan-Mar;15(1):32-34.
4. Adler-Storthz K, Newland JR, Tessin BA, Yeudall WA, Shillitoe
EJ. Identification of human papilloma virus types in oral verruca
vulgaris. J Oral Pathol 1986 Apr;15(4):230-233.
5. Green TL, Eversole LR, Leider AS. Oral and labial verruca vulgaris:
Clinical, histological and immunohistochemical evaluation.
Oral Surg Oral Med Oral Pathol 1986 Oct:62(4):410-416. REFERENCES From clinical observation, we found that the lesions were
mainly located on the buccal mucosa, lower labial mucosa,
also autoinoculation from a lesion located on hand has been
observed. A slightly higher incidence was encountered for
OVV in children and young adults and differs from other 2. Sharma G, Nagpal A, Veagal M, Pai K. Multiple Papilloma: A
case report and review of literature. J Indian Acad Oral Med
Radiol 2007;19(3):384-388. 67 International Journal of Clinical Pediatric Dentistry, January-April 2011;4(1):65-68 Art-12.indd 67 8/13/2016 5:09:21 PM Vela D Desai et al 6. Kirchner H. Immunobiology of human papillomavirus infection. Prog Med Virol 1986;33:1-41. 7. Neville.; B, Damm.; DD, Allen.; CM, et al. In: Oral and
maxillofacial pathology. 2nd edition. Saunders: Elsevier, 2002. p. 317-318. Chapter 8. Moondragon Health [Internet]. Available from: http://www. moondragon.org/health/disorders/warts.html 68 JAYPEE Art-12.indd 68 Art-12.indd 68 8/13/2016 5:09:21 PM
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English
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Behaviour of Distribution Grids with the Highest PV Share Using the Volt/Var Control Chain Strategy
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Energies
| 2,019
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cc-by
| 18,772
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Received: 30 August 2019; Accepted: 10 October 2019; Published: 12 October 2019 Received: 30 August 2019; Accepted: 10 October 2019; Published: 12 October 2019 Abstract: The large-scale integration of rooftop PVs stalls due to the voltage limit violations they
provoke, the uncontrolled reactive power flow in the superordinate grids and the information and
communications technology (ICT) related challenges that arise in solving the voltage limit violation
problem. This paper attempts to solve these issues using the LINK-based holistic architecture,
which takes into account the behaviour of the entire power system, including customer plants. It focuses on the analysis of the behaviour of distribution grids with the highest PV share, leading to
the determination of the structure of the Volt/var control chain. The voltage limit violations in low
voltage grid and the ICT challenge are solved by using concentrated reactive devices at the end of
low voltage feeders. Q-Autarkic customer plants relieve grids from the load-related reactive power. The optimal arrangement of the compensation devices is determined by a series of simulations. They are conducted in a common model of medium and low voltage grids. Results show that the best
performance is achieved by placing compensation devices at the secondary side of the supplying
transformer. The Volt/var control chain consists of two Volt/var secondary controls; one at medium
voltage level (which also controls the TSO-DSO reactive power exchange), the other at the customer
plant level. Keywords: volt/var control; distribution grid; photovoltaic; L(U)-control; Q-autarky; smart grid Behaviour of Distribution Grids with the Highest PV
Share Using the Volt/Var Control Chain Strategy
Daniel-Leon Schultis *
and Albana Ilo
TU Wi
I
tit t
f E
S
t
d El
t i
l D i
1040 Vi
A
t i
lb
il @t
i
t Daniel-Leon Schultis *
and Albana Ilo
TU Wien—Institute of Energy Systems and Electrical Drives, 1040 Vienna, Austria; albana.ilo@tuwien.ac.a
* Correspondence: daniel-leon.schultis@tuwien.ac.at; Tel.: +43-1-58801-370132 Daniel-Leon Schultis *
and Albana Ilo www.mdpi.com/journal/energies energies energies energies www.mdpi.com/journal/energies 1. Introduction Nowadays, climate change has become apparent, not only for scientists but also for everybody [1]. Reducing emissions through the use of renewable sustainable resources while maintaining a reliable
and secure electricity supply is becoming increasingly imperative. In this context, the large-scale
implementation of Distributed Generation (DG) holds a considerable potential [2]. European utilities supply 260 million customers, of which more than 99% are connected at the Low
Voltage (LV) level [3]. The use of inverters available in DGs, e.g., rooftop photovoltaic (PV), to control
the voltage in distribution networks [4] has introduced various local control strategies such as cos
ϕ (P), Q(U), and so on [5–7] that provoke uncontrolled reactive power flows on the superordinate
grids. Implementing PVs on the rooftop of each customer requires the coordination of millions of local
controllers needed to operate the power system reliably and securely. A flood of data exchange is
necessary that poses serious challenges to ICT, cyber security and data privacy [8–10]. The optimal volt/var management or control is one of the most essential processes for utilities
to maintain reliable voltages and to keep the power factor close to one. Currently, they use reactive
devices (RDs) to reduce the amount of reactive power flowing through transmission lines and to
maintain sufficient reactive power capability in transmission systems [11]. In medium voltage (MV)
grids, capacitor banks are mostly used to support the voltage [12]. The uncontrolled reactive power
flow in high voltage (HV) and MV levels provoked by the large scale implementation of local control
strategies in LV level upsets the current practices [13]. In the process of reactive power management are Energies 2019, 12, 3865; doi:10.3390/en12203865 2 of 23 Energies 2019, 12, 3865 already involved different actors, i.e., transmission (TSO) and distribution system operators (DSO) and
customers (as owners of PV inverters). Considering the perspective of individual actors or optimizing
individual functionalities may lead to suboptimal solutions and other, difficult to overcome, challenges. In contrast, the holistic architecture vision enables large-scale rollout of the new control paradigms
leading to optimal solutions [14]. Volt/var control chain strategy promises the solution of the problem
related to voltage control and reactive power management that is of utmost importance to utilities,
as they may favor the large scale integration of distributed generation. 2. Materials and Methods This work builds on the LINK-based holistic architecture and the associated strategy of volt/var
control chain, the fundamentals of which are outlined below. After that follows the model description
of the considered grids and of the simulated control setups. 1. Introduction y
y
g
g
g
The volt/var control chain strategy that comprises all grid levels, i.e., HV, MV, LV and Customer
Plant (CP) grid, located on the vertical axis (Y-axis) of the holistic model “The energy supply chain
net” [15] is introduced in [16]. It relies on the LINK-based holistic architecture [17], where the Secondary
Control (SC) is the crucial instrument to realize a sustainable and reliable grid operation of the whole
power system including CPs. The implementation of secondary control in CP level enables the full
reactive power (Q) compensation of the customer plants, leading to Q-self-sufficient or Q-Autarkic
customers [18]. The DSO owned reactive devices, e.g., L(U)-controlled coils, installed at LV feeders with
voltage limit violation potential, keep the voltage within the limits [19], while providing substantial
technical (reduced losses, Q-exchange and Distribution Transformer (DTR) loading [20]; and increased
hosting capacity [21]) and social (preserved data privacy and avoided discrimination [20]) benefits
compared to the cos ϕ (P)- and Q(U)-control strategies. Further on, the L(U)+Q-Autarky control
ensemble provides substantial technical benefits compared to the L(U)-control strategy [18]. All var-local control strategies, i.e., cosϕ (P), Q(U), L(U), etc., provoke uncontrolled reactive power
flows in the superordinate voltage level grids inclusive the HV grid. The reactive power margin
(RPM) decreases significantly if the DG production comes close to the demand or even exceeds it [22]. The power system approaches a voltage instable situation. This paper examines the effect of additional compensation devices (CDs) on the distribution grid
behaviour, when the volt/var control chain strategy that implies the L(U)+Q-autarky control ensemble
is used. Furthermore, the optimal link-grid size is investigated for the specific conditions. Section 2 gives an overview of the fundamentals of the volt/var control chain strategy,
the description of the customer plant and distribution grid model, and the definition of the simulated
control setups. Section 3 presents the investigation results concerning the distribution grid behaviour
and the effect of CD placement. Furthermore, a grid-link setup for practical implementation is
presented. Finally, in Section 4, some concluding remarks are summarized. 2.1. Fundamentals of Volt/Var Control Chain Strategy SC is the base instrument to realize the volt/var control chain strategy. It is one of the fundamental
components of each grid-link [17]. It refers to control actions that are calculated based on the grid-link
control area. It fulfils a predefined objective function by respecting the dynamic grid constraints on the
grid-link boundaries and the static constraints of electrical appliances (PQ-diagrams of generators,
transformer rating, etc.). Dynamic grid constraints are the reactive and active power exchange at
the grid-link boundaries that are agreed from the corresponding grid-link operators. The grid-link
contains SC-loops for both major entities of power systems—frequency and voltage. This paper
deals with the volt/var secondary control (VVSC) schemas. The generalized form of volt/var control
chain strategy [16] in the Y-axis of the holistic model “The energy supply chain net” that implies
the L(U)+Q-autarky control ensemble [18] is shown in Figure 1. All relevant links are drawn in
gold-coloured solid lines, while the neighbour grid-links are indicated by gold-dashed lines. Grid-links 3 of 23 Energies 2019, 12, 3865 are set upon three classical levels: CP, LV and MV level. The automation and communication path is
blue, while the power flow path is black. Energies 2019, 12, x FOR PEER REVIEW
3 of 23 Figure 1. The generalized form of the volt/var control chain strategy implying the L(U)+Q-autarky
o t ol e
e
ble
Figure 1. The generalized form of the volt/var control chain strategy implying the L(U)+Q-autarky
control ensemble. Figure 1. The generalized form of the volt/var control chain strategy implying the L(U)+Q-autarky
t
l
bl
Figure 1. The generalized form of the volt/var control chain strategy implying the L(U)+Q-autarky
control ensemble. RDs are used in power systems for two purposes: for voltage control (voltage control reactive
device – VCRD) or reactive power compensation (compensation device—CD). The corresponding
varPCs receive the set points U* and Q* as in Table 1
RDs are used in power systems for two purposes: for voltage control (voltage control reactive
device—VCRD) or reactive power compensation (compensation device—CD). The corresponding
varPCs receive the set-points U* and Q* as in Table 1. T bl 1 D
i
id
d f
lt
t
l
d
ti
Table 1. Devices considered for voltage-control and var-compensation. Table 1. Devices considered for voltage-control and var-compensation. Device
Purpose
Set-Point for varPC
RD
VCRD
Voltage control
U*
CD
Var compensation
Q*
Table 1. Devices considered for voltage-control and var-compensation. 2.1. Fundamentals of Volt/Var Control Chain Strategy Device
Purpose
Set-Point for varPC
RD
VCRD
Voltage control
U*
CD
Var compensation
Q* The main reason to introduce inductive devices on the low voltage grid is to keep the voltage
within the limits over the all-time horizon. L(U)s are VCRD, e.g., coils, connected at the end of LV
feeders, which may violate the upper voltage limit. The positioning of L(U)s at the LV feeder end
shows high effectiveness due to the prevalent high voltage sensitivity ( ∂U ∂Q
⁄
) [23]. Q-autarky is a
special mode of operation of the volt/var secondary control set up in the CP level (VVSCCP ). Neither
Q (𝑄
= 0 ) nor Q-data exchange with the LV grid is required when operating the distribution grid
with high PV-share. CPs are Q-self-sufficient. The main reason to introduce inductive devices on the low voltage grid is to keep the voltage
within the limits over the all-time horizon. L(U)s are VCRD, e.g., coils, connected at the end of LV
feeders, which may violate the upper voltage limit. The positioning of L(U)s at the LV feeder end
shows high effectiveness due to the prevalent high voltage sensitivity (∂U/∂Q) [23]. Q-autarky is a
special mode of operation of the volt/var secondary control set up in the CP level (VVSCCP). Neither Q
(QLV
CP = 0) nor Q-data exchange with the LV grid is required when operating the distribution grid with
high PV-share. CPs are Q-self-sufficient. 2.2. Model Description The customer plant model and the distribution grid model comprising MV_ and LV_link-grids that
are used to analyse the behaviour of distribution grids with different control setups are presented below. 2.1. Fundamentals of Volt/Var Control Chain Strategy VVSCLV calculates in real time: (d)
the var set-points for the Volt/var secondary controls VVSCMV,LV
ngb
of all neighbour MV_ or
LV_grid-links, while respecting the var constraint varCnsHV
MV at the border to the HV_link-grid. VVSCLV calculates in real time: (a)
the voltage and var set-points for the primary controls varPCLV
RD of all RDs included in the
LV_link-grid; (a)
the voltage and var set-points for the primary controls varPCLV
RD of all RDs included in the
LV_link-grid; (b)
the var set-points for the primary controls varPCLV
DG/DSt of all DGs and DSts connected to the
LV_link-grid;
LV CP (c)
the var set-points for the Volt/var secondary controls VVSCLV,CP
ngb
of all neighbour LV_ or
CP_grid-links, while respecting the var constraint varCnsMV
LV at the border to the MV_link-grid. (d)
VVSCCP calculates in real time (e)
the var set-point for the primary control varPCCP
inv of the PV-inverter connected to CP_link-grid;
while respecting the var constraint varCnsLV
CP at the border to the LV_link-grid. (e)
the var set-point for the primary control varPCCP
inv of the PV-inverter connected to CP_link-grid;
while respecting the var constraint varCnsLV
CP at the border to the LV_link-grid. In the generalized form of the VVC chain discussed above, grid-links are set up based on the
classical splitting method of the power system structure into HV, MV and LV levels. But, by definition
(the link-grid size is variable and is defined from the area, where the secondary-control is set up),
the link-grid size is variable. It may apply not only to the classical grid parts but also to a part of the
grid, which may include one or more voltage levels together, e.g., MV and LV level [17]. In the following is analysed the effect of the CDs on the distribution grid behaviour, when the
Volt/var control chain strategy that implies the L(U)+Q-autarky control ensemble is used to control the
voltage and the reactive power flow in distribution grids. It is supposed that the distribution grid is
operated by one DSO. The optimal link-grid size for these specific conditions is investigated. The basic
principle is keeping the number of secondary and primary control units as low as possible to avoid
complex automation schemes. 2.1. Fundamentals of Volt/Var Control Chain Strategy The following generalized equation is introduced for the first time to compactly represent the
VVC chain in the Y-axis:
The following generalized equation is introduced for the first time to compactly represent the
VVC chain in the Y-axis: MV
Y-axis = {VVSCMV൫voltPCOLTC
MV , varPCRD
MV, varPCDG/DSt
MV
, VVSCngb
MV,LV, varCnsMV
HV൯,
CLV൫varPCRD
LV, varPCDG/DSt
LV
, VVSCngb
LV,CP, varCnsLV
MV൯, VVSCCP൫varPCinv
CP, varCnsCP
LV = 0൯}
(1)
VVCY−axis= {VVSC MV
voltPCMV
OLTC, varPCMV
RD , varPCMV
DG/DSt, VVSCMV,LV
ngb
, varCnsHV
MV
,
VVSCLV
varPCLV
RD, varPCLV
DG/DSt, VVSCLV,CP
ngb
, varCnsMV
LV
, VVSCCP
varPCCP
inv, varCnsLV
CP= 0
(1) (1)(1) where VVSC
calculates in real time
(a) the voltage set-point for the primary
where VVSCMV calculates in real time where VVSC
calculates in real time
(a) the voltage set-point for the primary
where VVSCMV calculates in real time where VVSC
calculates in real time
(a) the voltage set-point for the primary control voltPCOLTC
MV of the supplying transformer and other
where VVSCMV calculates in real time e VVSC
calculates in real time
the voltage set-point for the primary control voltPCOLTC
MV of the supplying transformer and other
re VVSCMV calculates in real time transformers included in the MV_link-grid (e.g., 34.5 kV/11 kV, etc.) that have On-Load-Tap-
Changer (OLTC);
(b) the var set-points for the primary controls varPCRD
MV of all RDs included in the MV_link-grid;
(c)
the var set points for the primary controls varPCMV
of all DGs and Distributed Storages
(a)
the voltage set-point for the primary control voltPCMV
OLTC of the supplying transformer and
other transformers included in the MV_link-grid (e.g., 34.5 kV/11 kV, etc.)
that have
On-Load-Tap-Changer (OLTC); transformers included in the MV_link-grid (e.g., 34.5 kV/11 kV, etc.) that have On-Load-Tap-
Changer (OLTC);
(b) the var set-points for the primary controls varPCRD
MV of all RDs included in the MV_link-grid;
(c)
the var set points for the primary controls varPCMV
of all DGs and Distributed Storages
(a)
the voltage set-point for the primary control voltPCMV
OLTC of the supplying transformer and
other transformers included in the MV_link-grid (e.g., 34.5 kV/11 kV, etc.)
that have
On-Load-Tap-Changer (OLTC); ) the var set-points for the primary controls varPCDG/DSt of all DGs and Distributed Storag
(DSt) connected to the MV_link-grid;
b)
the var set-points for the primary controls varPCMV
RD of all RDs included in the MV_link-grid var set-points for the primary controls varPCDG/DSt of all DGs and Distributed Storages
St) connected to the MV_link-grid;
e var set-points for the primary controls varPCMV
RD of all RDs included in the MV_link-grid; 4 of 23 Energies 2019, 12, 3865 (c)
the var set-points for the primary controls varPCMV
DG/DSt of all DGs and Distributed Storages (DSt)
connected to the MV_link-grid; (d)
the var set-points for the Volt/var secondary controls VVSCMV,LV
ngb
of all neighbour MV_ or
LV_grid-links, while respecting the var constraint varCnsHV
MV at the border to the HV_link-grid. 2.2.1. Customer Plant Model The reactive power contribution
PV y te
i
dete
i ed by the a
lied
o t ol
t ate y a
de
ibed i
Se tio
2 3 It i
where f P,load
t
and f Q,load
t
are the active and reactive power load profile factors at time-point t; f P,PV
t
is
the active power production profile factor at time-point t; and Pload
peak = 1.368 kW is the peak active
l
d 𝑃𝑙𝑜𝑎𝑑,𝑖,𝑡= 𝑃𝑛𝑜𝑚,𝑡· ቀ𝐶𝑡
· ൫𝑈𝑖,𝑡𝑈𝑛𝑜𝑚
⁄
൯+ 𝐶𝑡
· ൫𝑈𝑖,𝑡𝑈𝑛𝑜𝑚
⁄
൯+ 𝐶𝑡ቁ
The load voltage dependency is modelled with a ZIP model according to: 𝑎𝑑,𝑖,𝑡
𝑃
= 𝑄𝑛𝑜𝑚,𝑡
𝑙𝑜𝑎𝑑· ቀ𝐶𝑡
𝑄,𝑍· ൫𝑈𝑖,𝑡𝑈𝑛𝑜𝑚
𝐿𝑉
⁄
൯
2 + 𝐶𝑡
𝑄,𝐼· ൫𝑈𝑖,𝑡𝑈𝑛𝑜𝑚
𝐿𝑉
⁄
൯+ 𝐶𝑡
𝑄,𝑃ቁ
(4b)
ூ , 𝐶௧
, and 𝐶௧
ொ, , 𝐶௧
ொ,ூ , 𝐶௧
ொ, are the active and reactive power ZIP
nt t. ZIP coefficients and load profiles are given for the considered 24h time
load and production profiles shown in Figure 2b are sampled into ∆t = 15min
PCP
load,i,t = Pload
nom,t·
CP,Z
t
·
Ui,t/ULV
nom
2 + CP,I
t ·
Ui,t/ULV
nom
+ CP,P
t
(4a)
QCP
load,i,t = Qload
nom,t·
CQ,Z
t
·
Ui,t/ULV
nom
2 + CQ,I
t
·
Ui,t/ULV
nom
+ CQ,P
t
(4b) (4a) (4b) izon in [26,27]. The load and production profiles shown in Figure 2b are sampled into ∆t = 15min
e-steps, resulting in N = 24h/∆t = 96 load-flow simulations per scenario. 2. Distribution Grid Models
MV and LV levels are modelled and simulated in a common model. For the sake of simplicity,
where CP,Z
t
, CP,I
t , CP,P
t
and CQ,Z
t
, CQ,I
t
, CQ,P
t
are the active and reactive power ZIP coefficients at
time-point t. ZIP coefficients and load profiles are given for the considered 24 h time horizon in [26,27]. The load and production profiles shown in Figure 2b are sampled into ∆t = 15 min time-steps, resulting
in N = 24 h/∆t = 96 load-flow simulations per scenario. 2.2.1. Customer Plant Model The profiles determine
the active and reactive power consumption of loads for nominal grid voltage (Pload
nom,t and Qload
nom,t) and
the active power production of the PV-system, as in: ,௧= 𝑓௧
𝑃
(3b)
𝑃௩,,௧
= 𝑓௧
,· 𝑃,
(3c)
ve and reactive power load profile factors at time-point t;
le factor at time-point t; and 𝑃
ௗ= 1.368 kW is the peak
is calculated based on the maximum 15-minutes mean
Pload
nom,t = f P,load
t
·Pload
peak,
(3a)
Qload
nom,t = f Q,load
t
·Pload
peak
(3b)
PCP
inv,i,t = f P,PV
t
·PCP
PV,r
(3c) (3a) (3b) (3c) p
(
ue of the active power flow measured throughout 2016 at the secondary side of the DTR of the
LV_link-grid described in Section 2.2.2 [24]) of each CP’s load. The reactive power contribution
PV-systems is determined by the applied control strategy as described in Section 2.3. It is
resting to note that nowadays the load has changed the behaviour in terms of reactive power. The
d behaves capacitive in the evening because the residential customers have mainly turned to LED
hting [26]. The load voltage dependency is modelled with a ZIP model according to:
𝑃𝑙
𝑑𝑖𝑡
𝐶𝑃
= 𝑃
𝑡
𝑙𝑜𝑎𝑑· ቀ𝐶𝑡
𝑃,𝑍· ൫𝑈𝑖𝑡𝑈𝐿𝑉
⁄
൯
2 + 𝐶𝑡
𝑃,𝐼· ൫𝑈𝑖𝑡𝑈𝐿𝑉
⁄
൯+ 𝐶𝑡
𝑃,𝑃ቁ
(4a)
where f P,load
t
and f Q,load
t
are the active and reactive power load profile factors at time-point t; f P,PV
t
is
the active power production profile factor at time-point t; and Pload
peak = 1.368 kW is the peak active
power demand (the value of Pload
peak is calculated based on the maximum 15-minutes mean value of the
active power flow measured throughout 2016 at the secondary side of the DTR of the real LV_link-grid
described in Section 2.2.2 [24]) of each CP’s load. The reactive power contribution of PV-systems is
determined by the applied control strategy as described in Section 2.3. It is interesting to note that
nowadays the load has changed the behaviour in terms of reactive power. The load behaves capacitive
in the evening because the residential customers have mainly turned to LED lighting [26]. p
ue of the active power flow measured throughout 2016 at the secondary side of the DTR of the
LV_link-grid described in Section 2.2.2 [24]) of each CP’s load. 2.2.1. Customer Plant Model Figure 2a shows the used customer plant structure. It has two components: the load or
power consumption and the electricity production. For each customer plant i and time-point t, it is
characterized by the active and reactive power consumption and production of the internal loads
(PCP
load,i,t and QCP
load,i,t) and the PV-system ( PCP
inv,i,t and QCP
inv,i,t), respectively. The active PLV
CP,i,t and reactive
power QLV
CP,i,t flows from the CP i to LV_link-grid at time-point t are given by: PLV
CP,i,t = PCP
inv,i,t −PCP
load,i,t,
(2a)
QLV
CP,i,t = QCP
inv,i,t −QCP
load,i,t
(2b) (2a) (2b) 5 of 23
23 Energies 2019, 12, 3865
i
2019 12
FOR PEER (a)
(b)
Figure 2. Customer plant model: (a) structure; (b) load and production profiles. Figure 2. Customer plant model: (a) structure; (b) load and production profiles. (b) (a) (b) (a) ure 2. Customer plant model: (a) structure; (b) load and production profiles. Figure 2. Customer plant model: (a) structure; (b) load and production profiles. Figure 2b shows the load and production profiles represented by solid and dashed lines,
pectively. Active and reactive power are coloured red and blue, respectively. The critical time-
nt tcrit, where the maximal PV production occurs, is marked as a black vertical line. The profiles
Each CP is connected to a boundary link node (BLiN) of the corresponding LV_link-grid
with the actual Ui,t and nominal voltage ULV
nom, and includes a PV-system with a module-rating
of PCP
PV,r = 5 kW [24] and an inverter-rating of SCP
inv,r = PCP
PV,r/0.9 [25]. ermine the active and reactive power consumption of loads for nominal grid voltage (𝑃𝑛𝑜𝑚,𝑡
𝑙𝑜𝑎𝑑 and
𝑚,𝑡
𝑑 ) and the active power production of the PV-system, as in:
𝑃𝑛𝑜𝑚,𝑡
𝑙𝑜𝑎𝑑= 𝑓𝑡
𝑃,𝑙𝑜𝑎𝑑· 𝑃𝑝𝑒𝑎𝑘
𝑙𝑜𝑎𝑑,
(3a)
ௗ
𝑓ொௗ
ௗ
b
,
Figure 2b shows the load and production profiles represented by solid and dashed lines,
respectively. Active and reactive power are coloured red and blue, respectively. The critical time-point
tcrit, where the maximal PV production occurs, is marked as a black vertical line. y are described separately below.
2.2.2. Distribution Grid Models Low Voltage Grid
Figure 3 shows a simplified one-line diagram of the used LV_link-grid model. MV and LV levels are modelled and simulated in a common model. For the sake of simplicity,
they are described separately below. 6 of 23 Energies 2019, 12, 3865 (A) Low Voltage Grid
E
i
2019 12
FOR PEER REVIEW Figure 3 shows a simplified one-line diagram of the used LV_link-grid model. nergies 2019, 12, x FOR PEER REVIEW
Energies 2019, 12, x FOR PEER REVIEW e 3 shows a simplified one-line diagram of the used LV_link-grid model. 12, x FOR PEER REVIEW
, 12, x FOR PEER REVIEW Figure 3. Simplified one-line diagram of the LV_link-grid model. Figure 3. Simplified one-line diagram of the LV_link-grid model. Figure 3. Simplified one-line diagram of the LV_link-grid model. Figure 3. Simplified one-line diagram of the LV_link-grid model. Figure 3. Simplified one-line diagram of the LV_link-grid model. Figure 3. Simplified one-line diagram of the LV_link-grid model. It is a real rural grid with four feeders with a minimum and maximum length of 565 m and 1.63
km, respectively. In this link-grid with a cable share of about 59% and a nominal voltage of 𝑈
=
0.4 kV are connected 61 residential customers. It is connected to the MV_link-grid through a 21
kV/0.42 kV, 400 kVA DTR with its tap changer fixed in mid-position. The detailed LV_link-grid
model data (instead of the 160 kVA DTR given in the mentioned reference, a 400 kVA one with a
rated primary and secondary voltage of 21 kV and 0.42 kV, respectively, and a short circuit voltage
of 3.7% with a resistive part of 1% is used, because of the high PV share. The tap changer is fixed in
its mid-position) is given in [28]. Figure 3 shows with red crosses the connection points of the L(U)s. It is a real rural grid with four feeders with a minimum and maximum length of 565 m and
1.63 km, respectively. In this link-grid with a cable share of about 59% and a nominal voltage of
ULV
nom = 0.4 kV are connected 61 residential customers. It is connected to the MV_link-grid through a
21 kV/0.42 kV, 400 kVA DTR with its tap changer fixed in mid-position. (B) Medium Voltage Grid
(B) Medium Voltage Grid
(B) Medium Voltage Grid
Fi
4 h
i Figure 4 shows a simplified one-line diagrams of the used MV_link-grids. Both MV_link-grids
are identical, except for the conductor type: Figures 4a,b represent the cases with cable and overhead
conductors in MV level, respectively. To each MV_link-grid are connected 32 LV_link-grids and two
PV-systems, each with a module and inverter rating of PPV,r
MV = 1 MW and Sinv,r
MV = 1 MVA,
respectively. The active power production 𝑃𝑖𝑛𝑣,𝑡
ெ of each PV-system connected to the MV_link-grid
at time-point t is determined by:
Figure 4 shows a simplified one-line diagrams of the used MV_link-grids. Both MV_link-grids
are identical, except for the conductor type: Figure 4a,b represent the cases with cable and overhead
conductors in MV level, respectively. To each MV_link-grid are connected 32 LV_link-grids and two
PV-systems, each with a module and inverter rating of PMV
PV,r= 1 MW and SMV
inv,r= 1 MVA, respectively. The active power production PMV
inv,t of each PV-system connected to the MV_link-grid at time-point t is
determined by:
Figure 4 shows a simplified one-line diagrams of the used MV_link-grids. Both MV_link-grids
are identical, except for the conductor type: Figures 4a,b represent the cases with cable and overhead
conductors in MV level, respectively. To each MV_link-grid are connected 32 LV_link-grids and two
PV-systems, each with a module and inverter rating of PPV,r
MV = 1 MW and Sinv,r
MV = 1 MVA,
respectively. The active power production 𝑃𝑖𝑛𝑣,𝑡
ெ of each PV-system connected to the MV_link-grid
at time-point t is determined by: 𝑃௩,௧
ெ= 𝑓௧
,· 𝑃,
ெ
(5)
PMV
inv,t = f P,PV
t
·PMV
PV,r
(5)
𝑃௩,௧
ெ= 𝑓௧
,· 𝑃,
ெ
(5) (5)
(5)
(5) ௩,௧
𝑓௧
,
( )
while the reactive power contribution is determined by the applied control strategy as described in
Section 2.3. The MV feeder length is 24 km and slack voltage is set to the nominal value of 110 kV. The detailed data of the MV link-grid models is given in Appendix A
while the reactive power contribution is determined by the applied control strategy as described in
Section 2.3. The MV feeder length is 24 km and slack voltage is set to the nominal value of 110 kV. The detailed data of the MV_link-grid models is given in Appendix A. while the reactive power contribution is determined by the applied control strategy as described in
Section 2.3. (B) Medium Voltage Grid
(B) Medium Voltage Grid
(B) Medium Voltage Grid
Fi
4 h
i The MV feeder length is 24 km and slack voltage is set to the nominal value of 110 kV. The detailed data of the MV_link-grid models is given in Appendix A. (a)
(b)
Figure 4. Simplified one-line diagrams of the MV_link-grid models with different conductor types:
(a) cable; (b) overhead line. (a)
(b)
Figure 4. Simplified one-line diagrams of the MV_link-grid models with different conductor types:
(a) cable; (b) overhead line. Figure 4. Simplified one-line diagrams of the MV_link-grid models with different conductor types:
(a) cable; (b) overhead line. (a)
(a) (a)
(b)
(b) (b)
(b) (b)
Figure 4. Simplified one-line diagrams of the MV_link-grid models with different conductor types:
(a) cable; (b) overhead line
Figure 4. Simplified one-line diagrams of the MV_link-grid models with different conductor types:
(a) cable; (b) overhead line. Figure 4. Simplified one-line diagrams of the MV_link-grid models with different conductor types:
(a) cable; (b) overhead line. (b)
Figure 4. Simplified one-line diagrams of the MV_link-grid models with different conductor types:
(a) cable; (b) overhead line
Figure 4. Simplified one-line diagrams of the MV_link-grid models with different conductor types:
(a) cable; (b) overhead line. Figure 4. Simplified one-line diagrams of the MV_link-grid models with different conductor types:
(a) cable; (b) overhead line. (b)
Figure 4. Simplified one-line diagrams of the MV_link-grid models with different conductor types:
(a) cable; (b) overhead line
Figure 4. Simplified one-line diagrams of the MV_link-grid models with different conductor types:
(a) cable; (b) overhead line. Figure 4. Simplified one-line diagrams of the MV_link-grid models with different conductor types:
(a) cable; (b) overhead line. y are described separately below.
2.2.2. Distribution Grid Models The detailed LV_link-grid
model data (instead of the 160 kVA DTR given in the mentioned reference, a 400 kVA one with a
rated primary and secondary voltage of 21 kV and 0.42 kV, respectively, and a short circuit voltage of
3.7% with a resistive part of 1% is used, because of the high PV share. The tap changer is fixed in its
mid-position) is given in [28]. Figure 3 shows with red crosses the connection points of the L(U)s. It is a real rural grid with four feeders with a minimum and maximum length of 565 m and 1.63
km, respectively. In this link-grid with a cable share of about 59% and a nominal voltage of 𝑈
=
0.4 kV are connected 61 residential customers. It is connected to the MV_link-grid through a 21
kV/0.42 kV, 400 kVA DTR with its tap changer fixed in mid-position. The detailed LV_link-grid
model data (instead of the 160 kVA DTR given in the mentioned reference, a 400 kVA one with a
rated primary and secondary voltage of 21 kV and 0.42 kV, respectively, and a short circuit voltage
of 3.7% with a resistive part of 1% is used, because of the high PV share. The tap changer is fixed in
its mid-position) is given in [28]. Figure 3 shows with red crosses the connection points of the L(U)s. (B) M di
V lt
G id (a) cable; (b) overhead line.
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2.3. Simulated Control Setups
2.3. Simulated Control Setups 2.3. Simulated Control Setups
The use of the reactive power to control the voltage in LV grids leads to an uncontrolled reactive
power flow up to the HV grid reducing its RPM This uncontrolled reactive power flow can be
The use of the reactive power to control the voltage in LV grids leads to an uncontrolled reactive
power flow up to the HV grid, reducing its RPM. This uncontrolled reactive power flow can be
The use of the reactive power to control the voltage in LV grids leads to an uncontrolled reactive
power flow up to the HV grid, reducing its RPM. This uncontrolled reactive power flow can be 7 of 23
ration
Here, Energies 2019, 12, 3865
coloured dotted lin
balancing. The latter practically compensated by CDs connected at different points of the grid. Therefore, a series of
simulations is performed to investigate the grid behaviour for various placements of CDs when the
VVC chain strategy is used. The grid-link setups are set assuming that the same DSO owns and operates
the MV and LV grids. They are derived from the generalized VVC chain strategy shown in Figure 1. The five identified cases are depicted in Figures 5–9. For each case is derived a specific equation
from the generalized form presented in Equation (1) that describes all elements involved in the VVC
chain, Equations (6)–(10). It is supposed that neither DSts nor DGs are connected to the LV_link-grids. One of the basic principles in setting up the grid-links is the minimization of the number of secondary
and primary control units to keep the CapEx and OpEx as low as possible. Therefore, no VVSC is
provided for the LV_grid-link, since only L(U) local controls (varLCLV
L(U)) are connected at the end of
some laterals: coordination is not relevant. The LV_grid-link is shown in gold-coloured dotted lines
because its existence must be discussed also in terms of load-generation balancing. The latter is not
within the scope of this paper. A grid-link is set up in the MV level. Here, the VVSC is important
to coordinate the Q-contribution of DGs, RDs and the neighbour grid-links with the voltage at the
secondary side or OLTC position of the supplying transformer (STR) while respecting all constraints
and optimizing the network performance at the same time. (a) cable; (b) overhead line.
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2.3. Simulated Control Setups
2.3. Simulated Control Setups with the voltage at the secondary side or OLTC position of the supplying transformer (STR) while
respecting all constraints and optimizing the network performance at the same time. All simulations are performed using both distribution grid models. The STR tap is fixed in its
mid-position so that the impact of CD placement on distribution grid behaviour can be clearly
analysed. 1. Control setup: Without any var control (no control)
Figure 5 shows the simplified form of the VVC chain strategy representing the setup without
any var control. In this form, the VVC chain in the Y-axis is presented by the following generalized
equation:
VVCY-axis= {VVSCMV൫varPCDG
MV൯}
(6)
Usually, VVSCMV sends the var set-points QDG
MV* to all DGs connected to the MV_link-grid. In
our simulations, all DGs, i.e., PV-systems, inject into the grid with a power factor of one, QDG
MV* = 0 . PV-systems in CP level inject with a power factor of one as well. Therefore, the LV grids supply
reactive power to the loads connected at the CP level. Figure 5. Simplified form of the VVC chain strategy representing the setup without any var control. In this control setup, reactive power is exchanged between all three levels: between HV_ and
MV_link-grid, MV_ and LV_link-grids, and LV_link-grids and CPs. 2. Control setup: L(U)-control and CP_Q-autarky (no CDs)
Figure 6 depicts the simplified form of the VVC chain strategy representing the setup with L(U)-
control and CP_Q-autarky. In this form, the VVC chain in the Y-axis is presented by the following
equation:
The VVSCMV sends the var set-points QDG
MV*= 0 to all PV-systems connected to the MV_link-
grid. To alleviate upper voltage limit violations, varLCL(U)
LV are set at the ends of the violated LV
VVCY-axis= {VVSCMV൫varPCDG
MV൯, varLCL(U)
LV , VVSCCP൫varPCinv
CP, varCnsCP
LV= 0൯}
(7)
Figure 5. Simplified form of the VVC chain strategy representing the setup without any var control. Energies 2019, 12, x FOR PEER REVIEW
8 of 23
feeders (see Figure 3). Each VVSCCP sends the required var set-point Qinv
CP* to the corresponding PV-
system to achieve Q-autarky, i.e., full reactive power compensation in CP level, satisfying the var
constraint varCnsCP
LV = 0 at all times [18]. Figure 6. Simplified form of the VVC chain strategy representing the setup with L(U)-control and
CP Q autarky
Figure 6. Simplified form of the VVC chain strategy representing the setup with L(U)-control and
CP_Q-autarky. (a) cable; (b) overhead line.
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2.3. Simulated Control Setups
2.3. Simulated Control Setups Therefore, the reactive power is exchanged only between HV_ and MV_link-grid. 5. Control setup: L(U)-control, CP_Q-Autarky and CDs at the DTRs’ LV-busbars ( CDLV
DTR )
Here, CD positioning is moved from the MV- to the LV-bus bars of DTRs, compensating the
reactive power required by LV_link-grid, Figure 9. The VVC chain in the Y-axis is presented by:
VVCY-axis= {VVSCMV൫varPCDG
MV, varPCCD
LV, varCnsLV
MV= 0൯, varPCL(U)
LV , VVSCCP൫varPCinv
CP, varCnsCP
LV= 0൯ The VVC chain Figure 8. Simplified form of the VVC chain strategy representing the setup with L(U)-control, CP_Q-
k
d
h
b
b
Figure 8. Simplified form of the VVC chain strategy representing the setup with L(U)-control,
CP_Q-autarky and CDs at the DTRs’ MV-busbars. VCY-axis= {VVSCMV൫varPCDG
MV, varPCCD
LV, varCnsLV
MV= 0൯, varPCL(U)
LV , VVSCCP൫varPCinv
CP, varCnsCP
LV autarky and CDs at the DTRs MV busbars. Therefore, the reactive power is exchanged only between HV_ and MV_link-g
5. Control setup: L(U)-control, CP_Q-Autarky and CDs at the DTRs’ LV-busbars ( CD
Here, CD positioning is moved from the MV- to the LV-bus bars of DTRs,
reactive power required by LV_link-grid, Figure 9. The VVC chain in the Y-axis is
VVCY-axis= {VVSCMV൫varPCDG
MV, varPCCD
LV, varCnsLV
MV= 0൯, varPCL(U)
LV , VVSCCP൫varP au a
y a
a
e
u
a
Therefore, the reactive power is exchanged only between HV_ and MV_link-grid. 5. Control setup: L(U)-control, CP_Q-Autarky and CDs at the DTRs’ LV-busbars ( CDLV
DTR )
Here, CD positioning is moved from the MV- to the LV-bus bars of DTRs, compensating the
reactive power required by LV_link-grid, Figure 9. The VVC chain in the Y-axis is presented by:
VVCY-axis= {VVSCMV൫varPCDG
MV, varPCCD
LV, varCnsLV
MV= 0൯, varPCL(U)
LV , VVSCCP൫varPCinv
CP, varCnsCP
LV= 0൯
Figure 9. Simplified form of the VVC chain strategy representing the setup with L(U)-control, CP_Q-
t
k
d CD
t th DTR ’ LV b
b
Figure 9. Simplified form of the VVC chain strategy representing the setup with L(U)-control,
CP_Q-autarky and CDs at the DTRs’ LV-busbars. Figure 9. Simplified form of the VVC chain strategy representing the setup with L(U)-control, CP_Q-
t
k
d CD
t th DTR ’ LV b
b
Figure 9. Simplified form of the VVC chain strategy representing the setup with L(U)-control,
CP_Q-autarky and CDs at the DTRs’ LV-busbars. y
Also in this case, the reactive power is exchanged only between HV_ and MV_link-grid. 3. (a) cable; (b) overhead line.
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2.3. Simulated Control Setups
2.3. Simulated Control Setups Figure 5 Simplified form of the VVC chain strategy representing the setup without any var control
Figure 5. Simplified form of the VVC chain strategy representing the setup without any var control. Energies 2019, 12, x FOR PEER REVIEW
8 of 23
feeders (see Figure 3). Each VVSCCP sends the required var set-point Qinv
CP* to the corresponding PV-
system to achieve Q-autarky, i.e., full reactive power compensation in CP level, satisfying the var
constraint varCnsCP
LV = 0 at all times [18]. power compen eve Q-autarky,
LV Figure 5 Simplified form of the VVC chain strategy representing the setup without any var control
Figure 5. Simplified form of the VVC chain strategy representing the setup without any var control. raint varCnsCP = 0 at all times [18]. In this control setup, reactive power is exchanged between all three levels: between HV_ and
MV_link-grid, MV_ and LV_link-grids, and LV_link-grids and CPs. 2. Control setup: L(U)-control and CP_Q-autarky (no CDs)
Figure 6 depicts the simplified form of the VVC chain strategy representing the setup with L(U)-
control and CP_Q-autarky. In this form, the VVC chain in the Y-axis is presented by the following
equation:
The VVSCMV sends the var set-points QDG
MV*= 0 to all PV-systems connected to the MV_link-
grid. To alleviate upper voltage limit violations, varLCL(U)
LV are set at the ends of the violated LV
VVCY-axis= {VVSCMV൫varPCDG
MV൯, varLCL(U)
LV , VVSCCP൫varPCinv
CP, varCnsCP
LV= 0൯}
(7)
Figure 6. Simplified form of the VVC chain strategy representing the setup with L(U)-control and
CP Q-autarky
Figure 6. Simplified form of the VVC chain strategy representing the setup with L(U)-control and
CP_Q-autarky. . To alleviate upper voltage limit violations, varLCL(U) are set at the ends of the violated L
Figure 6. Simplified form of the VVC chain strategy representing the setup with L(U)-control and
CP Q autarky
Figure 6. Simplified form of the VVC chain strategy representing the setup with L(U)-control and
CP_Q-autarky. Energies 2019, 12, 3865
VVCY-axis= {VVSCM 8 of 23
nsCP
LV= 0൯} 8 of 23
nsCP
LV= 0൯} Figure 7. Simplified form of the VVC chain strategy representing the setup with L(U)-control, CP_Q-
autarky and a CD at the STR MV busbar
Figure 7. Simplified form of the VVC chain strategy representing the setup with L(U)-control,
CP_Q-autarky and a CD at the STR MV-busbar. (a) cable; (b) overhead line.
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2.3. Simulated Control Setups
2.3. Simulated Control Setups Energies 2019, 12, x FOR PEER REVIEW
9 of 23
Energies 2019, 12, x FOR PEER REVIEW
9 of 23 Figure 7. Simplified form of the VVC chain strategy representing the setup with L(U)-control, CP_Q-
autarky and a CD at the STR MV-busbar. For the simulations, the CD is parametrized to respect the constraint varCnsMV
HV= 0 . Therefore,
the reactive power is exchanged only between MV_ and LV_link-grids. 4. Control setup: L(U)-control, CP_Q-Autarky and CD at the DTRs’ MV-busbars ( CDMV
DTR )
In this case, CD positioning is moved from the MV-bus bar of the STR to the MV-bus bars of the
DTRs, compensating the reactive power required by LV_link-grids, Figure 8. The VVC chain in the
Y-axis is presented by the following equation:
VVCY-axis= {VVSCMV൫varPCDG
MV, varPCCD
MV, varCnsLV
MV= 0൯, varLCL(U)
LV , VVSCCP൫varPCinv
CP, varCnsCP
LV= 0൯}
Figure 7. Simplified form of the VVC chain strategy representing the setup with L(U)-control,
CP_Q-autarky and a CD at the STR MV-busbar. Energies 2019, 12, x FOR PEER REVIEW
9 of 23
Figure 8. Simplified form of the VVC chain strategy representing the setup with L(U)-control, CP_Q-
autarky and CDs at the DTRs’ MV-busbars. Figure 8. Simplified form of the VVC chain strategy representing the setup with L(U)-control,
CP_Q-autarky and CDs at the DTRs’ MV-busbars. Energies 2019, 12, x FOR PEER REVIEW
9 of 23
Figure 8. Simplified form of the VVC chain strategy representing the setup with L(U)-control, CP_Q-
autarky and CDs at the DTRs’ MV-busbars. Therefore, the reactive power is exchanged only between HV_ and MV_link-grid. 5. Control setup: L(U)-control, CP_Q-Autarky and CDs at the DTRs’ LV-busbars ( CDLV
DTR )
Here, CD positioning is moved from the MV- to the LV-bus bars of DTRs, compensating the
reactive power required by LV_link-grid, Figure 9. The VVC chain in the Y-axis is presented by:
VVCY-axis= {VVSCMV൫varPCDG
MV, varPCCD
LV, varCnsLV
MV= 0൯, varPCL(U)
LV , VVSCCP൫varPCinv
CP, varCnsCP
LV= 0൯} Figure 7. Simplified form of the VVC chain strategy representing the setup with L(U)-control, CP_Q-
t
k
d
CD t th STR MV b
b
Figure 7. Simplified form of the VVC chain strategy representing the setup with L(U)-control,
CP_Q-autarky and a CD at the STR MV-busbar. gies 2019, 12, x FOR PEER REVIEW
9 o autarky and a CD at the STR MV busbar. (a) cable; (b) overhead line.
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2.3. Simulated Control Setups
2.3. Simulated Control Setups For the simulations, the CD is parametrized to respect the constraint varCnsMV
HV= 0 . Therefore,
the reactive power is exchanged only between MV_ and LV_link-grids. 4. Control setup: L(U)-control, CP_Q-Autarky and CD at the DTRs’ MV-busbars ( CDMV
DTR )
In this case, CD positioning is moved from the MV-bus bar of the STR to the MV-bus bars of the
DTRs, compensating the reactive power required by LV_link-grids, Figure 8. The VVC chain in the
Y-axis is presented by the following equation:
VVCY-axis= {VVSCMV൫varPCDG
MV, varPCCD
MV, varCnsLV
MV= 0൯, varLCL(U)
LV , VVSCCP൫varPCinv
CP, varCnsCP
LV= 0൯
Figure 8. Simplified form of the VVC chain strategy representing the setup with L(U)-control, CP_Q-
Figure 8. Simplified form of the VVC chain strategy representing the setup with L(U)-control,
CP_Q-autarky and CDs at the DTRs’ MV-busbars. Figure 8. Simplified form of the VVC chain strategy representing the setup with L(U)-control, CP_Q-
autarky and CDs at the DTRs’ MV-busbars. Therefore, the reactive power is exchanged only between HV_ and MV_link-grid. 5. Control setup: L(U)-control, CP_Q-Autarky and CDs at the DTRs’ LV-busbars ( CDLV
DTR )
Here, CD positioning is moved from the MV- to the LV-bus bars of DTRs, compensating the
reactive power required by LV_link-grid, Figure 9. The VVC chain in the Y-axis is presented by:
VVCY-axis= {VVSCMV൫varPCDG
MV, varPCCD
LV, varCnsLV
MV= 0൯, varPCL(U)
LV , VVSCCP൫varPCinv
CP, varCnsCP
LV= 0൯ autarky and a CD at the STR MV busbar. For the simulations, the CD is parametrized to respect the constraint varCnsMV
HV= 0 . Therefore,
the reactive power is exchanged only between MV_ and LV_link-grids. 4. Control setup: L(U)-control, CP_Q-Autarky and CD at the DTRs’ MV-busbars ( CDMV
DTR )
In this case, CD positioning is moved from the MV-bus bar of the STR to the MV-bus bars of the
DTRs, compensating the reactive power required by LV_link-grids, Figure 8. The VVC chain in the
Y-axis is presented by the following equation:
VVCY-axis= {VVSCMV൫varPCDG
MV, varPCCD
MV, varCnsLV
MV= 0൯, varLCL(U)
LV , VVSCCP൫varPCinv
CP, varCnsCP
LV= 0൯
Figure 8. Simplified form of the VVC chain strategy representing the setup with L(U)-control, CP_Q-
Figure 8. Simplified form of the VVC chain strategy representing the setup with L(U)-control,
CP_Q-autarky and CDs at the DTRs’ MV-busbars. Figure 8. Simplified form of the VVC chain strategy representing the setup with L(U)-control, CP_Q-
autarky and CDs at the DTRs’ MV-busbars. (a) cable; (b) overhead line.
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2.3. Simulated Control Setups
2.3. Simulated Control Setups The VVSCMV sends the var set-points QMV∗
DG = 0 to all PV-systems connected to the MV_lin
To alleviate upper voltage limit violations, varLCLV
L(U) are set at the ends of the violated LV
CP
CP The VVSCMV sends the var set-points QMV∗
DG = 0 to all PV-systems connected to the MV_link-grid. To alleviate upper voltage limit violations, varLCLV
L(U) are set at the ends of the violated LV feeders
(see Figure 3). Each VVSCCP sends the required var set-point QCP∗
inv to the corresponding PV-system g
L(U)
(see Figure 3). Each VVSCCP sends the required var set-point QCP∗
inv to the corresponding PV-system
to achieve Q-autarky, i.e., full reactive power compensation in CP level, satisfying the var constraint
varCnsLV
CP = 0 at all times [18]. CP
In this control setup, reactive power is exchanged between two levels: between HV_ and
MV_link-grid and between MV_ and LV_link-grids. 3. Control setup: L(U)-control, CP_Q-Autarky and CD at the STR MV-busbar (CDSTR
MV ) This control setup is derived from the second one and supplemented with a CD connected to the
STR MV-bus bar, Figure 7. The VVC chain in the Y-axis is presented as follows: VVCY−axis= {VVSC MV
varPCMV
CD , varPCMV
DG, varCnsHV
MV= 0
, varLCLV
L(U), VVSCCP
varPCCP
inv, varCnsLV
CP= 0
(8) VCY−axis= {VVSC MV
varPCMV
CD , varPCMV
DG, varCnsHV
MV= 0
, varLCLV
L(U), VVSCCP
varPCCP
inv, varCnsLV
CP= 0
(8 (8) For the simulations, the CD is parametrized to respect the constraint varCnsHV
MV= 0. Therefore,
the reactive power is exchanged only between MV_ and LV_link-grids. 4. Control setup: L(U)-control, CP_Q-Autarky and CD at the DTRs’ MV-busbars (CDDTR
MV ) In this case, CD positioning is moved from the MV-bus bar of the STR to the MV-bus bars of the
DTRs, compensating the reactive power required by LV_link-grids, Figure 8. The VVC chain in the
Y-axis is presented by the following equation: VVCY−axis= {VVSC MV
varPCMV
DG, varPCMV
CD , varCnsMV
LV = 0
, varLCLV
L(U), VVSCCP
varPCCP
inv, varCnsLV
CP= 0
(9) VVCY−axis= {VVSC MV
varPCMV
DG, varPCMV
CD , varCnsMV
LV = 0
, varLCLV
L(U), VVSCCP
varPCCP
inv, varCnsLV
CP= 0
(9 (9) Therefore, the reactive power is exchanged only between HV_ and MV_link-grid. Therefore, the reactive power is exchanged only between HV_ and MV_link-grid. (a) cable; (b) overhead line.
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2.3. Simulated Control Setups
2.3. Simulated Control Setups Results and Discussion
All simulations are performed using both distribution grid models. The STR tap is fixed in its
mid-position so that the impact of CD placement on distribution grid behaviour can be clearly analysed. 1. Control setup: Without any var control (no control) 3. Results and Discussion
1. Control setup: Without any var control (no control) The behaviour of both distribution grids is described below for the simulated control setups;
thereby, the effect of CD placement is analysed in detail. Based on these results, the optimal setup of
Figure 5 shows the simplified form of the VVC chain strategy representing the setup without any
var control. In this form, the VVC chain in the Y-axis is presented by the following generalized equation: he VVC chain strategy representing the setup with L(U)-control, CP_Q-
cussed. VVCY−axis= {VVSC MV
varPCMV
DG
o
(6) he VVC chain strategy representing the set
ussed. VVCY−axis= {VVSC MV
varPCMV
DG
o (6) Energies 2019, 12, 3865 9 of 23 Usually, VVSCMV sends the var set-points QMV∗
DG to all DGs connected to the MV_link-grid. In our
simulations, all DGs, i.e., PV-systems, inject into the grid with a power factor of one, QMV∗
DG
= 0. PV-systems in CP level inject with a power factor of one as well. Therefore, the LV grids supply reactive
power to the loads connected at the CP level. In this control setup, reactive power is exchanged between all three levels: between HV_ and
MV_link-grid, MV_ and LV_link-grids, and LV_link-grids and CPs. 2. Control setup: L(U)-control and CP_Q-autarky (no CDs) . Control setup: L(U)-control and CP_Q-autarky (no CDs) 2. Control setup: L(U)-control and CP_Q-autarky (no CDs) Figure 6 depicts the simplified form of the VVC chain strategy representing the setup with
L(U)-control and CP_Q-autarky. In this form, the VVC chain in the Y-axis is presented by the following
equation: VVCY−axis= {VVSC MV
varPCMV
DG
, varLCLV
L(U), VVSCCP
varPCCP
inv, varCnsLV
CP= 0
(7) (7) The VVSCMV sends the var set-points QMV∗
DG = 0 to all PV-systems connected to the MV_link-grid. To alleviate upper voltage limit violations, varLCLV
L(U) are set at the ends of the violated LV feeders
(see Figure 3). Each VVSCCP sends the required var set-point QCP∗
inv to the corresponding PV-system
to achieve Q-autarky, i.e., full reactive power compensation in CP level, satisfying the var constraint
varCnsLV
CP = 0 at all times [18]. 3.1. Behaviour of Distribution Grids As explained in Section 2.2.2, all simulations are performed in the common model of MV and LV
levels. Simulation results over the 24 h time horizon are shown graphically in Figures 10 and 12 for
the cable and overhead line structure, respectively. Whereas in Tables 2 and 3results at tcrit are listed. Simulations are made for different control setups; results are drown in different colours as follows:
“no control“ in dashed blackline; “no CDs” in purple; “CDSTR
MV ” in green; “CDDTR
MV ” in ocra yellow and
“CDDTR
LV ” in red solid line. (a) cable; (b) overhead line.
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2.3. Simulated Control Setups
2.3. Simulated Control Setups Control setup L(U) control CP Q Autarky and CDs at the DTRs’ LV busbars (CDDTR) Therefore, the reactive power is exchanged only between HV_ and MV_link-grid. 5. Control setup: L(U)-control, CP_Q-Autarky and CDs at the DTRs’ LV-busbars (CDDTR
LV ) 5. Control setup: L(U)-control, CP_Q-Autarky and CDs at the DTRs’ LV-busbars (CDDTR
LV ) Here, CD positioning is moved from the MV- to the LV-bus bars of DTRs, compensating the
reactive power required by LV_link-grid, Figure 9. The VVC chain in the Y-axis is presented by: VVCY−axis= {VVSC MV
varPCMV
DG, varPCLV
CD, varCnsMV
LV = 0
, varPCLV
L(U), VVSCCP
varPCCP
inv, varCnsLV
CP= 0
(10) VCY−axis= {VVSC MV
varPCMV
DG, varPCLV
CD, varCnsMV
LV = 0
, varPCLV
L(U), VVSCCP
varPCCP
inv, varCnsLV
CP= 0
(10 Also in this case, the reactive power is exchanged only between HV_ and MV_link-grid. Also in this case, the reactive power is exchanged only between HV_ and MV_link-grid. 10 of 23 Energies 2019, 12, 3865 10 of 23 3. Results and Discussion The behaviour of both distribution grids is described below for the simulated control setups;
thereby, the effect of CD placement is analysed in detail. Based on these results, the optimal setup of
the volt/var control chain is discussed. 3.1. Behaviour of Distribution Grids The behaviour of distribution grids is analysed using various parameters as: a)
the total reactive power consumption of all L(U)s included in the LV_link-grids, QL(U)
tot,t; (a)
the total reactive power consumption of all L(U)s included in the LV_link-grids, QL(U)
tot,t b)
the total reactive power contribution of all CDs included in MV_ or LV_link-grids, QCD
tot,t; (b)
the total reactive power contribution of all CDs included in MV_ or LV_link-grids, QCD
tot,t;
(c)
the reactive power exchange between HV_ and MV_link-grid, QHV
MV,t, at the STR primary side; (c)
the reactive power exchange between HV_ and MV_link-grid, QHV
MV,t, at the STR primary ,
(d)
the active power losses of the distribution grid, Ploss
t
, including losses of transformers, cables and
overhead lines; (d)
the active power losses of the distribution grid, Ploss
t
, including losses of transformers, cables and
overhead lines; (e)
the STR loading, LoadingSTR
t
; e)
the STR loading, LoadingSTR
t
; (f)
the mean loading of all DTRs, LoadingDTRs
t
, which is calculated as in (f)
the mean loading of all DTRs, LoadingDTRs
t
, which is calculated as in g
gt
LoadingDTRs
t
=
P32
k=1 LoadingDTR
k,t
32
(11) LoadingDTRs
t
=
P32
k=1 LoadingDTR
k,t
32
(11) (11) where LoadingDTR
k,t
is the loading of the DTR k at time-point t, and 32 is the number of DTRs;
(g)
the voltage limit violation index, VIt, which is calculated as in where LoadingDTR
k,t
is the loading of the DTR k at time-point t, and 32 is the number of DTRs; (g)
the voltage limit violation index, VIt, which is calculated as in g)
the voltage limit violation index, VIt, which is calculated as in (g)
the voltage limit violation index, VIt, which is calculated as in VIt =
Pmt
j=1
Uupper
u,t
−Uupper
lim
ULV
nom
+
Pnt
j=1
Ulower
lim −Ulower
v,t
ULV
nom
(12) (12) where mt is the number of LV_link-grid nodes that violate the upper voltage limit at time-point t;
nt is the number of LV_link-grid nodes that violate the lower voltage limit at time-point t; Uupper
u,t
is the voltage of the LV_link-grid node u with upper voltage limit violation at time-point t;
Ulower
v,t
is the voltage of the LV_link-grid node v with lower voltage limit violation at time-point t;
Uupper
lim = 1.1·ULV
nom= 0.44 kV is the upper voltage limit; and Ulower
lim = 0.9·ULV
nom= 0.36 kV is the lower
voltage limit. 3.1. Behaviour of Distribution Grids Only LV_link-grid nodes are considered because the simulations show that no
voltage limit violations appear in the MV_link-grid. where mt is the number of LV_link-grid nodes that violate the upper voltage limit at time-point t;
h
b
f
l
k
d
d
h
l
h l
l
l
upper where mt is the number of LV_link-grid nodes that violate the upper voltage limit at time-point t;
nt is the number of LV_link-grid nodes that violate the lower voltage limit at time-point t; Uupper
u,t
is the voltage of the LV_link-grid node u with upper voltage limit violation at time-point t;
Ulower
v,t
is the voltage of the LV_link-grid node v with lower voltage limit violation at time-point t;
Uupper
lim = 1.1·ULV
nom= 0.44 kV is the upper voltage limit; and Ulower
lim = 0.9·ULV
nom= 0.36 kV is the lower
voltage limit. Only LV_link-grid nodes are considered because the simulations show that no
voltage limit violations appear in the MV_link-grid. Table 2. Behaviour of the distribution grid with cable conductors in MV level at tcrit for different
control setups. Control
Setup
QL(U)
tot,tcrit
(Mvar)
QCD
tot,tcrit
(Mvar)
QHV
MV,tcrit
(Mvar)
Ploss
tcrit
(MW)
LoadingSTR
tcrit
(%)
LoadingDTRs
tcrit
(%)
No control
0.00
0.00
−0.70
0.94
52.44
64.40
No CDs
2.07
0.00
−2.65
1.17
53.28
67.47
CDSTR
MV
2.63
−3.17
0.00
1.23
50.95
67.74
CDDTR
MV
3.03
−3.67
0.11
1.25
50.81
68.11
CDDTR
LV
3.60
−4.24
0.11
1.33
50.35
60.74 Table 2. Behaviour of the distribution grid with cable conductors in MV level at tcrit for different
control setups. 11 of 23 Energies 2019, 12, 3865 Table 3. Behaviour of the distribution grid with overhead conductors in MV level at tcrit for different
control setups. Table 3. Behaviour of the distribution grid with overhead conductors in MV level at tcrit for different
control setups. Control
Setup
QL(U)
tot,tcrit
(Mvar)
QCD
tot,tcrit
(Mvar)
QHV
MV,tcrit
(Mvar)
Ploss
tcrit
(MW)
LoadingSTR
tcrit
(%)
LoadingDTRs
tcrit
(%)
No control
0.00
0.00
−1.97
1.22
51.85
63.53
No CDs
2.11
0.00
−4.04
1.52
54.03
67.61
CDSTR
MV
2.79
−4.66
0.00
1.60
48.93
67.90
CDDTR
MV
3.80
−4.52
−1.08
1.63
49.04
69.42
CDDTR
LV
4.40
−5.11
−1.06
1.72
48.50
58.49 Simulations show that the currents through the transformers, cables and overhead lines never
exceed their thermal ratings. The behaviour of distribution grids strongly depends on the PV injections
and the placement of CDs. 3.1.1. 3.1. Behaviour of Distribution Grids Distribution Grid with Cable Conductors in MV Level 3.1.1. Distribution Grid with Cable Conductors in MV Level Figure 10 shows the voltage profiles of the MV_link-grid with cable conductors and the backmost
LV_link-grid for the critical time-point tcrit. Without any var control, the upper voltage limit is violated with a voltage limit violation index of
35.51. In the case of “no CDs”, “CDSTR
MV ” and “CDDTR
MV ”, no voltage limit violations appear, but if CDs
are installed at the DTRs’ LV-busbars, the upper voltage limit is violated with a voltage limit violation
index of 0.04. Energies 2019, 12, x FOR PEER REVIEW
11 of 23 Figure 10. Voltage profile of the MV_link-grid with cable conductors and the backmost LV_link-grid
at t = tcrit . Figure 10. Voltage profile of the MV_link-grid with cable conductors and the backmost LV_link-grid at
t = tcrit. Figure 10. Voltage profile of the MV_link-grid with cable conductors and the backmost LV_link-grid
at t = tcrit . Figure 10. Voltage profile of the MV_link-grid with cable conductors and the backmost LV_link-grid at
t = tcrit. Figure 11 shows the behaviour of the distribution grid with cable conductors in MV level over
the 24 hour time horizon for all control setups. Figure 11a shows the total reactive power
consumption of all L(U)s connected at the distribution grid. When “no control” is applied, no L(U)s
are installed and as a result, there is no Q-consumption. The maximum total Q-consumption of L(U)s
is reached at tcrit for all cases. The lowest value of 2.07 Mvar is achieved when no CDs are applied,
while the highest one of 3.60 Mvar is reached when CDs are installed at the secondary sides of DTRs. Figure 11 shows the behaviour of the distribution grid with cable conductors in MV level over the
24 h time horizon for all control setups. Figure 11a shows the total reactive power consumption of all
L(U)s connected at the distribution grid. When “no control” is applied, no L(U)s are installed and as a
result, there is no Q-consumption. The maximum total Q-consumption of L(U)s is reached at tcrit for all
cases. The lowest value of 2.07 Mvar is achieved when no CDs are applied, while the highest one of
3.60 Mvar is reached when CDs are installed at the secondary sides of DTRs. 3.1.1. Distribution Grid with Cable Conductors in MV Level Energies 2019, 12, 3865
is reached at tcrit fo
while the highest o 12 of 23
applied,
f DTRs 12 of 23
applied,
f DTRs (a)
(b)
(c)
(d)
Energies 2019, 12, x FOR PEER REVIEW
12 of 23
Figure 11. Cont. (e)
(f)
Figure 11. Behaviour of the distribution grid with cable conductors in MV level for a 24 hour time
horizon and different control strategies: (a) Q-consumption of L(U)s; (b) Q-contribution of CDs; (c) Q-
exchange between HV_ and MV_link-grid; (d) active power losses; (e) STR loading; (f) mean DTR
Figure 11. Behaviour of the distribution grid with cable conductors in MV level for a 24 h time horizon
and different control strategies: (a) Q-consumption of L(U)s; (b) Q-contribution of CDs; (c) Q-exchange
between HV_ and MV_link-grid; (d) active power losses; (e) STR loading; (f) mean DTR loading. (b) (a) (b) (a) Energies 2019, 12, x FOR PEER REVIEW (d)
12 of 23
11. Cont. (c) (d) (c)
Figure 1
(e) (f) (f) (e) Figure 11. Behaviour of the distribution grid with cable conductors in MV level for a 24 hour time
horizon and different control strategies: (a) Q-consumption of L(U)s; (b) Q-contribution of CDs; (c) Q-
exchange between HV_ and MV_link-grid; (d) active power losses; (e) STR loading; (f) mean DTR
Figure 11. Behaviour of the distribution grid with cable conductors in MV level for a 24 h time horizon
and different control strategies: (a) Q-consumption of L(U)s; (b) Q-contribution of CDs; (c) Q-exchange
between HV_ and MV_link-grid; (d) active power losses; (e) STR loading; (f) mean DTR loading. loading. Figure 11b shows the reactive power contribution of the CDs. In the cases of “no control” and
“no CDs”, no CDs are installed, thus no Q-contribution is expected. In the other cases, the maximum
Q-contribution of CDs appears at tcrit . The CDMV
STR behaves inductive in time periods 0:00 to 9:20
a.m. and 03:00 to 12:00 p.m. to compensate the capacitive power produced by the cable. As the PV
injection increases from 9:00 a.m., the L(U)s included in LV_link-grid begin to consume inductive
power to eliminate the upper voltage limit violations. To compensate the additionally required
inductive power, the reactive power production of CDMV
STR changes from inductive to capacitive,
reaching the maximum Q-injection of 3.17 Mvar. 3.1.1. Distribution Grid with Cable Conductors in MV Level When CDs are installed in distribution substation,
they behave purely capacitive with a maximum Q-injection of 3.67 and 4.24 Mvar for the cases
Figure 11b shows the reactive power contribution of the CDs. In the cases of “no control” and
“no CDs”, no CDs are installed, thus no Q-contribution is expected. In the other cases, the maximum
Q-contribution of CDs appears at tcrit. The CDSTR
MV behaves inductive in time periods 0:00 to 9:20 a.m. and 03:00 to 12:00 p.m. to compensate the capacitive power produced by the cable. As the PV injection
increases from 9:00 a.m., the L(U)s included in LV_link-grid begin to consume inductive power to
eliminate the upper voltage limit violations. To compensate the additionally required inductive
power, the reactive power production of CDSTR
MV changes from inductive to capacitive, reaching the
maximum Q-injection of 3.17 Mvar. When CDs are installed in distribution substation, they behave
purely capacitive with a maximum Q-injection of 3.67 and 4.24 Mvar for the cases “CDDTR
MV ” and
“CDDTR
LV ”, respectively. loading. Figure 11b shows the reactive power contribution of the CDs. In the cases of “no control” and
“no CDs”, no CDs are installed, thus no Q-contribution is expected. In the other cases, the maximum
Q-contribution of CDs appears at tcrit . The CDMV
STR behaves inductive in time periods 0:00 to 9:20
a.m. and 03:00 to 12:00 p.m. to compensate the capacitive power produced by the cable. As the PV
injection increases from 9:00 a.m., the L(U)s included in LV_link-grid begin to consume inductive
power to eliminate the upper voltage limit violations. To compensate the additionally required
inductive power, the reactive power production of CDMV
STR changes from inductive to capacitive,
reaching the maximum Q-injection of 3.17 Mvar. When CDs are installed in distribution substation,
they behave purely capacitive with a maximum Q-injection of 3.67 and 4.24 Mvar for the cases
Figure 11b shows the reactive power contribution of the CDs. In the cases of “no control” and
“no CDs”, no CDs are installed, thus no Q-contribution is expected. In the other cases, the maximum
Q-contribution of CDs appears at tcrit. The CDSTR
MV behaves inductive in time periods 0:00 to 9:20 a.m. and 03:00 to 12:00 p.m. to compensate the capacitive power produced by the cable. 3.1.1. Distribution Grid with Cable Conductors in MV Level As the PV injection
increases from 9:00 a.m., the L(U)s included in LV_link-grid begin to consume inductive power to
eliminate the upper voltage limit violations. To compensate the additionally required inductive
power, the reactive power production of CDSTR
MV changes from inductive to capacitive, reaching the
maximum Q-injection of 3.17 Mvar. When CDs are installed in distribution substation, they behave
purely capacitive with a maximum Q-injection of 3.67 and 4.24 Mvar for the cases “CDDTR
MV ” and
“CDDTR
LV ”, respectively. Energies 2019, 12, 3865 13 of 23 Figure 11c shows the reactive power exchange between HV_ and MV_link-grid. In the control
setups “no control” and “no CDs”, the MV_link-grid draws reactive power from the HV_link-grid
between approx. 9:30 a.m. and 3:00 p.m. In the remaining time horizon, the MV_link-grid injects
reactive power into the HV_link-grid. In the case “no control”, two peaks of QHV
MV,t are identified: one
at tcrit, where the MV_link-grid draws 0.70 Mvar from the HV_link-grid; and the other at 10 p.m.,
where the MV_link-grid injects 0.80 Mvar into the HV_link-grid. This behaviour is caused by the
capacitive nature of the load in the evening and the cable structure of the MV_link-grid. In the case of
“no CDs”, the MV_link-grid draws the maximum reactive power of 2.65 Mvar from the HV_link-grid
at tcrit. If the CD is installed at the STR MV-busbar, no reactive power is exchanged between HV_ and
MV_link-grid over the all-time horizon. In the control setups “CDDTR
MV ” and “CDDTR
LV ”, the MV_link-grid
injects reactive power into HV_link-grid over the all-time horizon, showing their minimum Q-injection
of 0.11 Mvar at tcrit. Energies 2019, 12, x FOR PEER REVIEW
13 of 23
d h
h
l
f h
d
b
d I
ll
l Figure 11d shows the active power losses of the distribution grid. In all cases, losses increase
drastically between approx. 8:00 a.m. and 4:30 p.m. due to the PV injection. The maximum values
appear at tcrit, where the control setup “CDDTR
LV ” provokes the highest grid losses of 1.33 MW. Figure 11d shows the active power losses of the distribution grid. In all cases, losses increase
drastically between approx. 8:00 a.m. and 4:30 p.m. due to the PV injection. The maximum values
appear at tcrit , where the control setup “CDLV
DTR ” provokes the highest grid losses of 1.33 MW. 3.1.1. Distribution Grid with Cable Conductors in MV Level Fi
11 f h
h STR
d
DTR l
di
i
l
I
ll
STR
d
DTR Figure 11e,f show the STR and mean DTR loading, respectively. In all cases, STR and mean DTR
loading increase drastically between approx. 8:00 a.m. and 4:30 p.m. due to PV injections, reaching
their maximum values at tcrit. For the STR loading, the highest value of 53.28% is reached for the
control setup “no CDs”; while the lowest one of 50.35% is reached for the “CDDTR
LV ” case. For the mean
DTR loading, the highest value of 68.11% is reached for the control setup “CDDTR
MV ”; while the lowest
one of 60.74% is reached for the “CDDTR
LV ” case. Figure 11e,f show the STR and mean DTR loading, respectively. In all cases, STR and mean DTR
loading increase drastically between approx. 8:00 a.m. and 4:30 p.m. due to PV injections, reaching
their maximum values at tcrit . For the STR loading, the highest value of 53.28% is reached for the
control setup “no CDs”; while the lowest one of 50.35% is reached for the “CDLV
DTR ” case. For the
mean DTR loading, the highest value of 68.11% is reached for the control setup “CDMV
DTR ”; while the
lowest one of 60.74% is reached for the “CDLV
DTR ” case. 3.1.2. Distribution Grid with Overhead Conductors in MV Level
3.1.2. Distribution Grid with Overhead Conductors in MV Level Figure 12 shows the voltage profiles of the MV_link-grid with overhead line conductors and the
backmost LV_link-grid for the critical time-point tcrit. Figure 12 shows the voltage profiles of the MV_link-grid with overhead line conductors and the
backmost LV_link-grid for the critical time-point tcrit . Figure 12 shows the voltage profiles of the MV_link-grid with overhead line conductors and the
backmost LV_link-grid for the critical time-point tcrit. Figure 12 shows the voltage profiles of the MV_link-grid with overhead line conductors and the
backmost LV_link-grid for the critical time-point tcrit . Figure 12. Voltage profile of the MV_link-grid with overhead line conductors and the backmost
LV_link-grid at t = tcrit . Figure 12. Voltage profile of the MV_link-grid with overhead line conductors and the backmost
LV_link-grid at t = tcrit. Figure 12. Voltage profile of the MV_link-grid with overhead line conductors and the backmost
LV_link-grid at t = tcrit . Figure 12. Voltage profile of the MV_link-grid with overhead line conductors and the backmost
LV_link-grid at t = tcrit. Without any var control, the upper voltage limit is violated with a voltage limit violation index
of 58.38. In the case of “no CDs” and “CDMV
STR ”, no voltage limit violations appear, but if CDs are
installed at distribution substation, the upper voltage limit is violated with a voltage limit violation
index of 1.79 and 7.73, respectively, for the cases “CDMV
DTR ” and “CDLV
DTR ”. Without any var control, the upper voltage limit is violated with a voltage limit violation index of
58.38. In the case of “no CDs” and “CDSTR
MV ”, no voltage limit violations appear, but if CDs are installed
at distribution substation, the upper voltage limit is violated with a voltage limit violation index of
1.79 and 7.73, respectively, for the cases “CDDTR
MV ” and “CDDTR
LV ”. 14 of 23 Energies 2019, 12, 3865 Figure 13 shows the behaviour of the distribution grid with overhead line conductors in MV
level over the 24 h time horizon for all control setups. Figure 13a shows the total reactive power
consumption of all L(U)s connected at the distribution grid. When “no control” is applied, no L(U)s
are installed and as a result, there is no Q-consumption. The maximum total Q-consumption of L(U)s
is reached at tcrit for all cases. 3.1.2. Distribution Grid with Overhead Conductors in MV Level
3.1.2. Distribution Grid with Overhead Conductors in MV Level The lowest value of 2.11 Mvar is achieved when no CDs are applied,
while the highest one of 4.40 Mvar is reached when CDs are installed at the secondary sides of DTRs. LV_link-grid at t = tcrit . Without any var control, the upper voltage limit is violated with a voltage limit violation index
of 58.38. In the case of “no CDs” and “CDMV
STR ”, no voltage limit violations appear, but if CDs are
installed at distribution substation, the upper voltage limit is violated with a voltage limit violation
index of 1.79 and 7.73, respectively, for the cases “CDMV
DTR ” and “CDLV
DTR ”. while the highest one of 4.40 Mvar is reached when CDs are installed at the secondary sides of DTRs. (a)
(b)
Figure 13. Cont. Energies 2019, 12, x FOR PEER REVIEW
14 of 23
(c)
(d)
(e)
(f)
Figure 13. Behaviour of the distribution grid with overhead line conductors in MV level for a 24 hour
time horizon and different control strategies: (a) Q-consumption of L(U)s; (b) Q-contribution of CDs;
(c) Q-exchange between HV_ and MV_link-grid; (d) active power losses; (e) STR loading; (f) mean
DTR loading. Figure 13 shows the behaviour of the distribution grid with overhead line conductors in MV
level over the 24 hour time horizon for all control setups. Figure 13a shows the total reactive power
Figure 13. Behaviour of the distribution grid with overhead line conductors in MV level for a 24 h
time horizon and different control strategies: (a) Q-consumption of L(U)s; (b) Q-contribution of CDs;
(c) Q-exchange between HV_ and MV_link-grid; (d) active power losses; (e) STR loading; (f) mean
DTR loading. Figure 13b shows the reactive power contribution of the CDs. In the cases of “no control” and
“no CDs”, no CDs are installed, thus no Q-contribution is expected. If CDs are installed, they behave
l
iti
hi
th i
i
Q
t ib ti
t t
Th l
t
l
f 4 52 M
i (a)
Energies 2019, 12, x FOR PEER REVIEW (b)
14 of 23 (b) (a)
R REV 13. Cont. Figure
(c) (d) (c) (f) (e) (f) (e) Figure 13. 3.1.2. Distribution Grid with Overhead Conductors in MV Level
3.1.2. Distribution Grid with Overhead Conductors in MV Level Behaviour of the distribution grid with overhead line conductors in MV level for a 24 hour
time horizon and different control strategies: (a) Q-consumption of L(U)s; (b) Q-contribution of CDs;
(c) Q-exchange between HV_ and MV_link-grid; (d) active power losses; (e) STR loading; (f) mean
DTR loading. Figure 13. Behaviour of the distribution grid with overhead line conductors in MV level for a 24 h
time horizon and different control strategies: (a) Q-consumption of L(U)s; (b) Q-contribution of CDs;
(c) Q-exchange between HV_ and MV_link-grid; (d) active power losses; (e) STR loading; (f) mean
DTR loading. Figure 13 shows the behaviour of the distribution grid with overhead line conductors in MV
level over the 24 hour time horizon for all control setups. Figure 13a shows the total reactive power
consumption of all L(U)s connected at the distribution grid. When “no control” is applied, no L(U)s
Figure 13b shows the reactive power contribution of the CDs. In the cases of “no control” and
“no CDs”, no CDs are installed, thus no Q-contribution is expected. If CDs are installed, they behave
purely capacitive, reaching their maximum Q-contribution at tcrit. The lowest value of 4.52 Mvar is Energies 2019, 12, 3865 15 of 23 achieved when CDs are installed at the DTRs’ primary sides, while the highest one of 5.11 Mvar is
reached when they are installed at the DTRs’ secondary sides. Figure 13c shows the reactive power exchange between HV_ and MV_link-grid. In the control
setup “no control”, the MV_link-grid injects reactive power into the HV_link-grid between approx. 9:00 p.m. and 11:15 p.m. In the remaining time horizon, the MV_link-grid draws reactive power
from the HV_link-grid; two peaks of QHV
MV,t are identified: one at tcrit, where the MV_link-grid draws
1.97 Mvar from the HV_link-grid; and the other at 10 p.m., where the MV_link-grid injects 66 kvar into
the HV_link-grid. This behaviour is caused by the capacitive nature of the load in the evening. If the CD
is installed at the STR MV-busbar, no reactive power is exchanged between HV_ and MV_link-grid over
the all-time horizon. In all other cases, the MV_link-grid draws reactive power from the HV_link-grid
over the all-time horizon, with the maximum Q-exchange appearing at tcrit. In the case of “no CDs”,
the MV_link-grid draws the maximum reactive power of 4.04 Mvar from the HV_link-grid. Figure 13d shows the active power losses of the distribution grid. 3.1.2. Distribution Grid with Overhead Conductors in MV Level
3.1.2. Distribution Grid with Overhead Conductors in MV Level In all cases, losses increase
drastically between approx. 8:00 a.m. and 4:30 p.m. due to the PV injection. The maximum values
appear at tcrit, where the control setup “CDDTR
LV ” provokes the highest grid losses of 1.72 MW. Figure 13e,f show the STR and mean DTR loading, respectively. In all cases, STR and mean DTR
loading increase drastically between approx. 8:00 a.m. and 4:30 p.m. due to PV injections, reaching
their maximum values at tcrit. For the STR loading, the highest value of 54.03% is reached for the
control setup “no CDs”; while the lowest one of 48.50% is reached for the “CDDTR
LV ” case. For the mean
DTR loading, the highest value of 69.42% is reached for the control setup “CDDTR
MV ”; while the lowest
one of 58.49% is reached for the “CDDTR
LV ” case. 3.1.3. Effect of CD Placement of
CDs
(-)
Cable
CDSTR
MV
0.0000
6.5051
34.4286
17.9049
23.9071
1
CDDTR
MV
0.0000
6.6000
34.0946
18.5570
23.9116
32
CDDTR
LV
0.0016
7.2863
33.8003
19.0885
23.5122
32
Overhead
CDSTR
MV
0.0000
8.3320
32.5502
17.4605
23.9019
1
CDDTR
MV
0.1484
8.4739
32.3078
17.4682
24.1743
32
CDDTR
LV
0.8307
8.8536
31.9763
17.3888
22.3732
32 The CD placement at the STR MV-bus bar supports the elimination of voltage limit violations in
all cases, while the contrary is noticed when the CDs are placed on the DTR level. The placement of
CDs on the MV side of DTRs provokes violations of the upper voltage limit in the case of overhead
conductor type, VIavg = 0.1484. Meanwhile, when CDs are installed at the LV side of DTRs, limit
violations appear in both cases, cable and overhead, with a VIavg of 0.0016 and 0.8307, respectively. The CD placement at the STR MV-bus bar supports the elimination of voltage limit violations in
all cases, while the contrary is noticed when the CDs are placed on the DTR level. The placement of
CDs on the MV side of DTRs provokes violations of the upper voltage limit in the case of overhead
conductor type, VIavg = 0.1484. Meanwhile, when CDs are installed at the LV side of DTRs, limit
violations appear in both cases, cable and overhead, with a VIavg of 0.0016 and 0.8307, respectively. The CD placement at the STR MV-bus bar supports the elimination of voltage limit violations in
all cases, while the contrary is noticed when the CDs are placed on the DTR level. The placement of
CDs on the MV side of DTRs provokes violations of the upper voltage limit in the case of overhead
conductor type, VIavg = 0.1484. Meanwhile, when CDs are installed at the LV side of DTRs, limit
violations appear in both cases, cable and overhead, with a VIavg of 0.0016 and 0.8307, respectively. 3.1.3. Effect of CD Placement Table 4 shows the relevant criteria used for the evaluation of different CD locations within the
distribution grid with cable or overhead conductors in MV level. The energy loss, the average STR and
DTR loadings, the average voltage limit violation index, and the energy exchange between MV_ and
HV_link-grid are calculated according to Equations (A1)–(A5), Appendix B. Furthermore, one of the
criteria applies to the number of compensation devices to be installed in each case. Table 4. Criteria used for the evaluation of different CD locations within the distribution grid with
cable or overhead conductors in MV level. Table 4. Criteria used for the evaluation of different CD locations within the distribution grid with
cable or overhead conductors in MV level. Conductor
Type in
MV Level
Control
Setup
VIavg
(-)
Eloss
(MWh)
EHV
MV
(MWh)
LoadingSTR
avg
(%)
LoadingDTRs
avg
(%)
No. of
CDs
(-)
Cable
CDSTR
MV
0.0000
6.5051
34.4286
17.9049
23.9071
1
CDDTR
MV
0.0000
6.6000
34.0946
18.5570
23.9116
32
CDDTR
LV
0.0016
7.2863
33.8003
19.0885
23.5122
32
Overhead
CDSTR
MV
0.0000
8.3320
32.5502
17.4605
23.9019
1
CDDTR
MV
0.1484
8.4739
32.3078
17.4682
24.1743
32
CDDTR
LV
0.8307
8.8536
31.9763
17.3888
22.3732
32
The CD placement at the STR MV-bus bar supports the elimination of voltage limit violations in
all cases, while the contrary is noticed when the CDs are placed on the DTR level. The placement of
CDs on the MV side of DTRs provokes violations of the upper voltage limit in the case of overhead
conductor type, VIavg = 0.1484. Meanwhile, when CDs are installed at the LV side of DTRs, limit
violations appear in both cases, cable and overhead, with a VIavg of 0.0016 and 0.8307, respectively. Regarding the active power loss over the all-time horizon, i.e., the active energy loss, a clear
trend is observed for both conductor types in MV level: while the CD placement at the STR MV-bus
bar causes the lowest energy losses of 6.5051 and 8.3320 MWh for cable and overhead conductors in Table 4. Criteria used for the evaluation of different CD locations within the distribution grid with
cable or overhead conductors in MV level. Conductor
Type in
MV Level
Control
Setup
VIavg
(-)
Eloss
(MWh)
EHV
MV
(MWh)
LoadingSTR
avg
(%)
LoadingDTRs
avg
(%)
No. 3.1.3. Effect of CD Placement g
Regarding the active power loss over the all-time horizon, i.e., the active energy loss, a clear
trend is observed for both conductor types in MV level: while the CD placement at the STR MV-bus
bar causes the lowest energy losses of 6.5051 and 8.3320 MWh for cable and overhead conductors in Regarding the active power loss over the all-time horizon, i.e., the active energy loss, a clear
trend is observed for both conductor types in MV level: while the CD placement at the STR MV-bus
bar causes the lowest energy losses of 6.5051 and 8.3320 MWh for cable and overhead conductors in Regarding the active power loss over the all-time horizon, i.e., the active energy loss, a clear
trend is observed for both conductor types in MV level: while the CD placement at the STR MV-bus
bar causes the lowest energy losses of 6.5051 and 8.3320 MWh for cable and overhead conductors in Energies 2019, 12, 3865 16 of 23 MV level, respectively, the CD placement at DTRs’ LV-bus bars causes the highest ones of 7.2863 and
8.8536 MWh. level, respectively, the CD placement at DTRs’ LV-bus bars causes the highest ones of 7.2863 and
36 MWh. These results show that the compensation of the reactive power in distribution substation
significantly deteriorates the effectiveness of L(U)-control, leading to very high losses and voltage limit
violations in LV level. The amount of active power flowing from MV_ to HV_link-grid over the all-time horizon,
i.e., active energy exchange, depends on the placement of the CDs, Figure 14. Their placement in the
supplying substation supports the maximum active energy exchange in all cases. Energies 2019, 12, x FOR PEER REVIEW
16 of 23 (a)
(b)
Figure 14. Active energy exchange between MV_and HV_link-grid for different conductor types in
MV level: (a) cable; (b) overhead line. Figure 14. Active energy exchange between MV_and HV_link-grid for different conductor types in
MV level: (a) cable; (b) overhead line. (b) (a) (b) (a) Figure 14. Active energy exchange between MV_and HV_link-grid for different conductor types in
MV level: (a) cable; (b) overhead line. Figure 14. Active energy exchange between MV_and HV_link-grid for different conductor types in
MV level: (a) cable; (b) overhead line. Anyhow, due to the reduced active energy exchange, Figure 14, the lowest STR loading value
results for this control setup. 3.1.3. Effect of CD Placement The highest average STR loading of 19.0885% for the grid with cable
conductors in MV level appears when CDs are installed at the DTRs’ LV-bus bars. In the case of
overhead conductor type, the highest value of 17.4682% is reached when CDs are installed at MV-
bus bars of the DTRs. The number of CDs to install is very different. When placed at the STR MV-side, one CD per bus
bar is required (or two CDs in double bus bar configurations), while the placement in the distribution
The STR and DTR loading depends on the active and reactive power flows. In our simulations the
CD set on the MV-bus bar of the supplying substation completely compensates the reactive power
exchange at all times. Thus, the average STR loading is exclusively provoked by the active power flow,
achieving the minimum value of 17.9049% for the grid with cable conductors in MV level. In the case
of overhead conductor type, the lowest value of 17.3888% is reached when the CDs are placed at the
LV side of the DTRs. In this case, active and reactive energy flow through the STR, because the CDs
compensate the reactive power in DTR level. q
(
g
)
p
substations normally requires as many CD devices as there are DTRs in place; which are in our case
32 CDs. Table 4. Criteria used for the evaluation of different CD locations within the distribution grid with
cable or overhead conductors in MV level. Anyhow, due to the reduced active energy exchange, Figure 14, the lowest STR loading value
results for this control setup. The highest average STR loading of 19.0885% for the grid with cable
conductors in MV level appears when CDs are installed at the DTRs’ LV-bus bars. In the case of
overhead conductor type, the highest value of 17.4682% is reached when CDs are installed at MV-bus
bars of the DTRs. Conductor
Type in MV
Level
Control
Setup
VIavg
(-)
Eloss
(MWh)
EMV
HV
(MWh)
Loadingavg
STR
(%)
Loadingavg
DTRs
(%)
No. of
CDs
(-)
CDMV
STR
0.0000
6.5051
34.4286
17.9049
23.9071
1
The number of CDs to install is very different. 3.1.3. Effect of CD Placement When placed at the STR MV-side, one CD per bus
bar is required (or two CDs in double bus bar configurations), while the placement in the distribution
substations normally requires as many CD devices as there are DTRs in place; which are in our case
32 CDs. Cable
MV
CDMV
DTR
0.0000
6.6000
34.0946
18.5570
23.9
DTR
Figure 15 depicts a qualitative representation of the results given in Table 4. Cable
CDLV
DTR
0.0016
7.2863
33.8003
19.0885
23.5122
32
Overhead
CDMV
STR
0.0000
8.3320
32.5502
17.4605
23.9019
1
CDMV
DTR
0.1484
8.4739
32.3078
17.4682
24.1743
32
CDDTR
0 8307
8 8536
31 9763
17 3888
22 3732
32
The effectiveness of the solution depends on the size of the surface of the pentagon. The smaller
the surface of the latter, the more effective is the solution. Results show that the distribution grid
performs best when CDs are placed at the MV-bus bar of the STR. 17 of 23 Energies 2019, 12, 3865
Figure 15 dep
on a distribution (a)
(b)
Figure 15. Qualitative representation of the criteria used for the evaluation of various CD placements
on a distribution grid with different conductor types in MV level: (a) cable; (b) overhead line. The effectiveness of the solution depends on the size of the surface of the pentagon. The smaller
e surface of the latter, the more effective is the solution. Results show that the distribution grid
rforms best when CDs are placed at the MV-bus bar of the STR. 2. Discussion
Results have shown that the VVC chain strategy supports the integration of rooftop PVs on a
ge scale. Figure 16 shows the most suitable setup of the VVC chain for a distribution grid with the
ghest PV share operated by one DSO. The VVC chain is designed with a minimum number of
condary and primary control units to reduce the associated investments and operating costs. erived from Equation (1), the VVC chain in the Y-axis is presented by:
Two grid-link types, i.e., MV_ and CP_grid-link, are designed in this case. 3.1.3. Effect of CD Placement For the LV level, no
id-link is designed for four reasons:
MV_ and LV_link-grids have the same operator and as a result they do not have external
interfaces between each other [16];
No reactive power is exchanged between LV_link-grids and CPs because of the Q-autarky of the
latter;
VCY-axis={VVSCMV൫voltPCOLTC
MV , varPCRD
MV, varPCDG
MV, VVSCngb
MV, varCnsMV
HV൯, varLCL(U)
LV ,
VSCCP൫varPCinv
CP, varCnsCP
LV = 0൯}. (13) (a)
The effectiveness of the solution depends on t
he surface of the latter, the more effective is the
performs best when CDs are placed at the MV-bus
.2. Discussion
Results have shown that the VVC chain strat
arge scale. Figure 16 shows the most suitable setu
highest PV share operated by one DSO. The VVC
econdary and primary control units to reduce
Derived from Equation (1), the VVC chain in the Y
Two grid-link types, i.e., MV_ and CP_grid-li
grid-link is designed for four reasons:
a) MV_ and LV_link-grids have the same ope
VVCY-axis={VVSCMV൫voltPCOLTC
MV , varPCRD
MV, varPC
VVSCCP൫varPCinv
CP, varCnsCP
LV = 0൯}. (b)
he size of the surface of the pentagon. The smaller
solution. Results show that the distribution grid
bar of the STR. egy supports the integration of rooftop PVs on a
p of the VVC chain for a distribution grid with the
C chain is designed with a minimum number of
the associated investments and operating costs. -axis is presented by:
nk, are designed in this case. For the LV level, no
rator and as a result they do not have external
DG
MV, VVSCngb
MV, varCnsMV
HV൯, varLCL(U)
LV ,
(13) e latter, the mor
hen CDs are plac gy supp
of the V
h i
i (b)
hey d (a)
-grid Figure 15. Qualitative representation of the criteria used for the evaluation of various CD placements
on a distribution grid with different conductor types in MV level: (a) cable; (b) overhead line. g
p
y
interfaces between each other [16];
No reactive power is exchanged between LV_link-grids and CPs because of the Q-autarky of th
l 3.2. Discussion
latter;
(c)
No distribu For the LV level,
no grid-link is designed for four reasons: 18 of 23 Energies 2019, 12, 3865 (a)
MV_ and LV_link-grids have the same operator and as a result they do not have external interfaces
between each other [16]; (a)
MV_ and LV_link-grids have the same operator and as a result they do not have external interfaces
between each other [16]; (b)
No reactive power is exchanged between LV_link-grids and CPs because of the Q-autarky of
the latter; (b)
No reactive power is exchanged between LV_link-grids and CPs because of the Q-autarky of
the latter; No distributed energy resources are foreseen to deliver reactive power to the LV_link-grids; (c)
No distributed energy resources are foreseen to deliver reactive power to the LV_link-grids; (d)
At each LV feeder with voltage limit violation potential is installed one locally con At each LV feeder with voltage limit violation potential is installed one locally controlled L(U). The MV_grid-link includes a VVSCMV that coordinates the Q-contribution of DGs, RDs and
the neighbour grid-links with the voltage at the secondary side or OLTC position of the STR while
respecting the reactive power constraints, varCnsHV
MV, on the HV-MV intersection points and optimizing
the network performance at the same time. The HV-MV intersection points correspond in many cases
with TSO-DSO intersection points. varCnsHV
MV is dynamic and therefore needs to be discussed and
defined through real-time TSO and DSO cooperation in order to achieve an optimal solution, in both,
transmission and distribution grids. Simulation results have shown that the uncontrolled reactive power flow provoked by the locally
controlled units, varLCLV
L(U), included in LV_link-grid is best compensated by the CD installed at the
MV-bus bar of the STR. varLCLV
L(U) s, installed at the end of each LV lateral with voltage limit violation
potential, keep the voltage below the upper limit during the PV production period. The CP_grid-links have a VVSCCP
varPCCP
inv, varCnsLV
CP= 0
that takes care to fully compensate
the reactive power of the customer plant at all times. The interaction between the LV_link-grid and
CPs in terms of reactive power is not existing and therefore no exchange of information between the
DSO and the customers is required. Thus, the ICT challenge for the volt/var control is resolved at the
LV level. The CPs inject or obtain exclusively active power into or from the LV_link-grid. 3.2. Discussion
latter;
(c)
No distribu The VVSCMV is practically realized in real time in the frame of the industrial project
Central Volt/var control in Presence of Distributed Generation (ZUQDE, Salzburg, Austria) [29,30]. The distribution state estimator was realized in a MV grid of European type with a symmetrical
balanced behaviour. The VVSCMV
voltPCMV
OLTC, varPCMV
DG, cosϕCnsHV
MV
was successfully realized in
closed loop. This project has indicated that the implementation of the proposed VVC chain strategy
has great potential to be realized on an industrial scale. 3.2. Discussion
latter;
(c)
No distribu Results have shown that the VVC chain strategy supports the integration of rooftop PVs on a large
scale. Figure 16 shows the most suitable setup of the VVC chain for a distribution grid with the highest
PV share operated by one DSO. The VVC chain is designed with a minimum number of secondary
and primary control units to reduce the associated investments and operating costs. Derived from
Equation (1), the VVC chain in the Y-axis is presented by:
(d) At each LV feeder with voltage limit violation potential is installed one locally controlled L(U). The MV_grid-link includes a VVSCMV that coordinates the Q-contribution of DGs, RDs and the
neighbour grid-links with the voltage at the secondary side or OLTC position of the STR while
respecting the reactive power constraints, varCnsMV
HV , on the HV-MV intersection points and
optimizing the network performance at the same time The HV-MV intersection points correspond VVCY−axis= {VVSC MV
voltPCMV
OLTC, varPCMV
RD , varPCMV
DG, VVSCMV
ngb, varCnsHV
MV
, varLCLV
L(U),
VVSCCP
varPCCP
inv, varCnsLV
CP= 0
o
. (13)
many cases with TSO-DSO intersection points. varCnsMV
HV is dynamic and therefore needs to be
ussed and defined through real-time TSO and DSO cooperation in order to achieve an optimal
tion, in both, transmission and distribution grids. (13)
o be
mal Figure 16. The most suitable setup of the VVC chain for a distribution grid with the highest PV share
operated by one DSO. Figure 16. The most suitable setup of the VVC chain for a distribution grid with the highest PV share
operated by one DSO. Figure 16. The most suitable setup of the VVC chain for a distribution grid with the highest PV share
operated by one DSO
Figure 16. The most suitable setup of the VVC chain for a distribution grid with the highest PV share
operated by one DSO. p
y
Two grid-link types, i.e., MV_ and CP_grid-link, are designed in this case. For the LV level,
no grid-link is designed for four reasons: p
y
Two grid-link types, i.e., MV_ and CP_grid-link, are designed in this case. Appendix A Figure A1 shows the simplified one-line diagrams of the used MV_link-grid models. The YNyn6,
110 kV/20 kV, 18.5 MVA STR has a short-circuit voltage of 10.12% with a resistive part of 0.45%, core
losses of 28.2 kW and an open-circuit current of 0.19%. Both MV_link-grids connect two PV-systems,
each with a module-rating of PMV
PV,r = 1 MW, and an inverter-rating of SMV
inv,r = 1 MVA. Energies 2019, 12, x FOR PEER REVIEW
19 of 23 (a)
(b)
Figure A1. Detailed one-line diagrams of the MV_link-grid models with different conductor types:
(a) cable; (b) overhead line. Figure A1. Detailed one-line diagrams of the MV_link-grid models with different conductor types:
(a) cable; (b) overhead line. (a) (a) (b) Figure A1. Detailed one-line diagrams of the MV_link-grid models with different conductor types:
(a) cable; (b) overhead line. Figure A1. Detailed one-line diagrams of the MV_link-grid models with different conductor types:
(a) cable; (b) overhead line. Figure A1a shows the cable MV_link-grid. The cable segments have a resistance of 0.206 Ω/km,
a reactance of 0.1222 Ω/km, a capacitance of 254 nF/km, and a limiting current of 419 A. Figure A1b
shows the overhead line MV_link-grid. The line segments have a resistance of 0.358 Ω/km, a reactance
of 0.376 Ω/km, a capacitance of 9.6 nF/km, and a limiting current of 350 A. Figure A1a shows the cable MV_link-grid. The cable segments have a resistance of 0.206 Ω/km,
a reactance of 0.1222 Ω/km, a capacitance of 254 nF/km, and a limiting current of 419 A. Figure A1b
shows the overhead line MV_link-grid. The line segments have a resistance of 0.358 Ω/km, a reactance
of 0.376 Ω/km, a capacitance of 9.6 nF/km, and a limiting current of 350 A. 4. Conclusions Due to the current trends in distribution grids, i.e., implementation of distributed generation
with local volt/var control, the local voltage increases, the process of reactive power management
throughout the power grid becomes very difficult and the information and communications technology
(ICT) related challenge follows up. Therefore, solving the problem of voltage control and reactive
power management is of utmost importance to utilities, as they may favor the large scale integration of
distributed generation. Results of this investigation have shown that the VVC chain strategy, which roots on LINK-based
holistic architecture, supports the integration of rooftop PVs on a large scale. The inclusion of the
L(U)+CP_Q-autarky control ensemble in the control chain eliminates the violation of the upper voltage
limit at low voltage level, as well as the ICT challenges and social problems. The VVC chain is designed
with a minimum number of secondary and primary control units to reduce the associated investments
and operating costs. It consists of two volt/var secondary controls; one at medium voltage level (which
also controls the TSO-DSO reactive power exchange), the other at the customer plant level. MV and LV
grids have the best performance in terms of losses, loading of distribution and supplying transformers,
number of installed compensation devices and active power production, when the compensation
device is placed at the MV bus bar of supplying transformer. p
pp y
g
One part of the VVC chain, VVSCMV, is industrially realized in real time in another project. Nevertheless, the industrial implementation of the entire VVC chain is the next step to prove the
practical relevance of the results of this study. 19 of 23 Energies 2019, 12, 3865 Author Contributions: methodology, D.-L.S.; conceptualization A.I.; writing—original draft preparation, D.-L.S.;
writing—review and editing, A.I. Funding: This research received no external funding. Acknowledgments: The authors acknowledge the TU Wien University Library for financial support throug
Open Access Funding Programme. Acknowledgments: The authors acknowledge the TU Wien University Library for financial support through its
Open Access Funding Programme. Conflicts of Interest: The authors declare no conflict of interest. flicts of Interest: The authors declare no conflict of interest. Appendix B
Appendix B Different criteria are used to evaluate the simulated control setups in both distribution grid
Different criteria are used to evaluate the simulated control setups in both distribution grid models. l Different criteria are used to evaluate the simulated control setups in both distribution grid
models. A i
l
Th
l
Ploss
d
f h
l
d fl
Different criteria are used to evaluate the simulated control setups in both distribution grid models. Active power loss—The active power loss Ploss
t
at time-point t is direct output of the load-flow
i
l ti
d i
l d
th
ti
l
f th STR
d ll
h
d li
bl
d DTR Different criteria are used to evaluate the simulated control setups in both distribution grid
models. Active power loss—The active power loss Pt
loss at time-point t is direct output of the load-flow
simulations and includes the active power losses of the STR and all overhead lines, cables and DTRs. Different criteria are used to evaluate the simulated control setups in both distribution grid models. Active power loss—The active power loss Ploss
t
at time-point t is direct output of the load-flow
simulations and includes the active power losses of the STR and all overhead lines, cables and DTRs. The active energy loss Eloss over the all-time horizon is: l-time horizon is:
Eloss = ∆t · ∑
Pt
loss
∀t
. (B1)
Eloss = ∆t·
X
∀t Ploss
t
. (A1) (B1)
(A1) ∆t = 15 min is the time-step used for the simulations. ∆t = 15 min is the time-step used for the simulations. where ∆t 15 min is the time step used for the simulations. Average STR loading—The STR loading Loadingt
STR at time-point t is a direct output of the load-
flow simulations The average STR loading LoadingSTR over the all time horizon is:
Average STR loading—The STR loading LoadingSTR
t
at time-point t is a direct output of the load-flow
simulations. The average STR loading LoadingSTR
avg over the all-time horizon is: avg
Loadingavg
STR =
∑
Loadingt
STR
∀t
N
,
(B2)
LoadingSTR
avg =
P
∀t LoadingSTR
t
N
,
(A2) (B2)
(A2) E
i
2019 12 3865
20 f 23 Energies 2019, 12, 3865 20 of 23 where N = 96 is the number of conducted load-flow simulations per control setup and distribution
grid model. Appendix B
Appendix B Average DTR loading—The mean loading of all DTRs LoadingDTRs
t
at time-point t is calculated
according to Equation (11). The average DTRs’ loading LoadingDTRs
avg
over the all-time horizon is: LoadingDTRs
avg
=
P
∀t LoadingDTRs
t
N
(A3) (A3) Average voltage limit violation index—The voltage limit violation index VIt at time-point t is
calculated according to Equation (12). The average voltage limit violation index over the all-time
horizon is:
P
VI Average voltage limit violation index—The voltage limit violation index VIt at time-point t is
calculated according to Equation (12). The average voltage limit violation index over the all-time
horizon is: VIavg =
P
∀t VIt
N
(A4) (A4) Active energy exchange—The active power flow from MV_ to HV_link-grid PHV
MV,t at the STR primary
side at time-point t is direct output of the load-flow simulations. The total active energy exchange EHV
MV
between MV_ and HV_link-grid over the all-time horizon is: EHV
MV = ∆t·
X
∀t PHV
MV,t
(A5) (A5) Table A2 lists the nomenclature of all variables used for calculations. Table A2 lists the nomenclature of all variables used for calculations. Table A2. Nomenclature of all variables used for calculations. CP,Z
t
, CP,I
t , CP,P
t
Active power ZIP coefficients for time-point t. CQ,Z
t
, CQ,I
t
, CQ,P
t
Reactive power ZIP coefficients for time-point t. EHV
MV
Active energy exchange between MV_ and HV_link-grid over the all-time horizon. Eloss
Active energy loss over the all-time horizon. f P,load
t
Active power load profile factor at time-point t. f Q,load
t
Reactive power load profile factor at time-point t. f P,PV
t
Active power production profile factor at time-point t. LoadingDTR
k,t
Loading of the DTR k at time-point t. LoadingDTRs
t
Mean loading of all DTRs at time-point t. LoadingDTRs
avg
The average DTRs’ loading over the all-time horizon. LoadingSTR
t
The STR loading at time-point t. LoadingSTR
avg
The average STR loading over the all-time horizon. mt
Number of LV_link-grid nodes that violate the upper voltage limit at time-point t. nt
Number of LV_link-grid nodes that violate the lower voltage limit at time-point t. N
Number of conducted load-flow simulations per control setup and distribution grid model. PCP
inv,i,t
Active power production of the PV-system of the CP i at time-point t. PCP
load,i,t
Active power consumption of the loads of the CP i at time-point t. PCP
PV,r
Module-rating of the PV-system of each CP. PLV
CP,i,t
Active power flow from the CP i to LV_link-grid at time-point t. Pload
nom,t
Active power consumption of each CP’s load for nominal grid voltage at time-point t. Pload
peak
Peak active power demand of each CP’s load. PMV
inv,t
Active power production of each PV-system connected to the MV_link-grid at time-point t. PMV
PV,r
Module-rating of each PV-system connected to the MV_link-grid. Ploss
t
Active power losses of the distribution grid at time-point t. PHV
MV,t
Active power flow from the MV_ to HV_link-grid at time-point t. QCP
inv,i,t
Reactive power production of the PV-system of the CP i at time-point t. QCP
load,i,t
Reactive power consumption of the loads of the CP i at time-point t. QLV
CP,i,t
Reactive power flow from the CP i to LV_link-grid at time-point t. Qload
nom,t
Reactive power consumption of each CP’s load for nominal grid voltage at time-point t. QL(U)
tot,t
Total reactive power consumption of all L(U)s included in the LV_link-grids at time-point t. Appendix C Table A1 lists all the abbreviations and the corresponding full forms used in the paper. Table A1. Abbreviations and corresponding full forms. BLiN
Boundary link node
MV
Medium voltage
BPN
Boundary producer node
OLTC
On load tap changer
BSN
Boundary storage node
OpEx
Operational expenditures
CapEx
Capital expenditures
PC
Primary control
CD
Compensation device
PV
Photovoltaic
CP
Customer plant
RD
Reactive device
DG
Distributed generation
RPM
Reactive power margin
DSO
Distribution system operator
SC
Secondary control
DSt
Distributed storage
STR
Supplying transformer
DTR
Distribution transformer
TSO
Transmission system operator
HV
High voltage
VCRD
Voltage control reactive device
ICT
Information and communications
technology
VVC
Volt/var control
LC
Local control
VVSC
Volt/var secondary control
LV
Low voltage
cos ϕCnsHV
MV
cos ϕ constraint at the border to the
HV_link-grid
varPCLV
DG/DSt
Primary controls of DGs and DSts
connected to the LV_link-grids
varCnsHV
MV
Var constraint at the border to the
HV_link-grid
varPCLV
RD
Primary controls of RDs included in the
LV_link-grids
varCnsMV
LV
Var constraint at the border to the
MV_link-grid
varPCCP
inv
Primary controls of PV-inverters
connected to CP_link-grids
varCnsLV
CP
Var constraint at the border to the
LV_link-grid
voltPCMV
OLTC
Primary controls of the STR or other
transformers with OLTC included in the
MV_link-grid
varLCLV
L(U)
Local controls of L(U)s included in the
LV_link-grids
VVSCMV
VVSC of MV_grid-link
varPCMV
CD
Primary controls of CDs included in the
MV_link-grid
VVSCLV
VVSC of LV_grid-link
varPCMV
DG/DSt
Primary controls of DGs and DSts
connected to the MV_link-grid
VVSCCP
VVSC of CP_grid-link
varPCMV
RD
Primary controls of RDs included in the
MV_link-grid
VVSCMV,LV
ngb
VVSC of neighbour MV_ or
LV_grid-links
varPCLV
CD
Primary controls of CDs included in the
LV_link-grids
VVSCLV,CP
ngb
VVSC of neighbour LV_ or
CP_grid-links Table A1. Abbreviations and corresponding full forms. Table A1. Abbreviations and corresponding full forms. 21 of 23 Energies 2019, 12, 3865 Table A2 lists the nomenclature of all variables used for calculations. QCD
tot,t
Total reactive power contribution of all CDs included in MV_ or LV_link-grids at time-point t. QHV
MV,t
Reactive power flow from the MV_ to HV_link-grid at time-point t. SCP
inv,r
Inverter-rating of the PV-system of each CP. SMV
inv,r
Inverter-rating of each PV-system connected to the MV_link-grid. Ui,t
Actual voltage at the BLiN of the CP i at time-point t. ULV
nom
Nominal voltage of LV_link-grids. Uupper
u,t
Voltage of the LV_link-grid node u with upper voltage limit violation at time-point t. Ulower
v,t
Voltage of the LV_link-grid node v with lower voltage limit violation at time-point t. Uupper
lim
Upper voltage limit. Ulower
lim
Lower voltage limit. VIt
Voltage limit violation index at time-point t. VIavg
Average voltage limit violation index over the all-time horizon. tcrit
Critical time-point, where the maximal PV production occurs. ∆t
Ti
t
d t
l th l
d
d
d
ti
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Reference genes for normalising gene expression data in collagenase-induced rat intracerebral haemorrhage
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PUBLISHED VERSION Cook, Naomi Louise; Kleinig, Timothy John; Van Den Heuvel, Corinna; Vink, Robert
Reference genes for normalising gene expression data in collagenase-induced rat intracerebral
haemorrhage
BMC Molecular Biology, 2010; 11:7 Cook, Naomi Louise; Kleinig, Timothy John; Van Den Heuvel, Corinna; Vink, Robert
Reference genes for normalising gene expression data in collagenase-induced rat intracerebral
haemorrhage
BMC Molecular Biology, 2010; 11:7 © 2010 Cook et al; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution
License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited. The electronic version of this article is the complete one and can be found online at:
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to make derivative works;
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Under the following conditions: Attribution
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any of these conditions can be waived if the authors gives permission. Tuesday, 29 October 2013 PERMISSIONS
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Anyone is free:
to copy, distribute, and display the work;
to make derivative works;
to make commercial use of the work;
Under the following conditions: Attribution
the original author must be given credit;
for any reuse or distribution, it must be made clear to others what the license terms
of this work are;
any of these conditions can be waived if the authors gives permission. Tuesday, 29 October 2013 RESEARCH ARTICLE Open Access Open Access © 2010 Cook 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. Reference genes for normalising gene expression
data in collagenase-induced rat intracerebral
haemorrhage Naomi L Cook, Timothy J Kleinig, Corinna van den Heuvel, Robert Vink* Naomi L Cook, Timothy J Kleinig, Corinna van den Heuvel, Robert Vink* Abstract Background: The mechanisms of brain injury following intracerebral haemorrhage (ICH) are incompletely
understood. Gene expression studies using quantitative real-time RT-PCR following ICH have increased our
understanding of these mechanisms, however the inconsistent results observed may be related to inappropriate
reference gene selection. Reference genes should be stably expressed across different experimental conditions,
however, transcript levels of common reference genes have been shown to vary considerably. Reference gene
panels have therefore been proposed to overcome this potential confounder. Results: The present study evaluated the stability of seven candidate reference genes in the striatum and overlying
cortex of collagenase-induced ICH in rodents at survival times of 5 and 24 hours. Transcript levels of the candidate
reference genes were quantified and ranked in order of stability using geNorm. When our gene of interest,
transient receptor potential melastatin 2 (TRPM2), was normalised against each reference gene individually, TRPM2
mRNA levels were highly variable. When normalised to the four most stable reference genes selected for accurate
normalisation of data, we found no significant difference between ICH and vehicle rats. Conclusion: The panel of reference genes identified in the present study will enable more accurate normalisation
of gene expression data in the acute phase of experimental ICH. due to its wider dynamic range of quantification, higher
sensitivity and precision, and a decreased risk of con-
tamination compared to gel-based PCR [4-6]. Data
obtained from real-time RT-PCR assays require a reli-
able method of normalisation to correct for such factors
as differences in quantity and quality of RNA samples
and for efficiency of the reverse transcription reaction
[7,8]. Several normalisation strategies exist, the most
common being the use of one or more endogenously
expressed reference genes [9]. A reference gene should
be expressed at a stable level in different tissue types
and be unaffected by the experimental condition under
investigation [10]. However, several of the most com-
monly used reference genes, such as glyceraldehyde-3-
phosphate dehydrogenase (GAPDH) and b-actin, have
been shown to have variable expression patterns and
thus to be unsuitable for normalising real-time RT-PCR
data in certain conditions [11-14]. The use of an inap-
propriate reference gene for normalisation may lead to
incorrect
data
interpretation
[15]. In
contrast, * Correspondence: robert.vink@adelaide.edu.au
Discipline of Anatomy and Pathology, School of Medical Sciences, The
University of Adelaide, Adelaide SA 5005, Australia Anyone is free: http://hdl.handle.net/2440/59375 http://hdl.handle.net/2440/59375 Cook et al. BMC Molecular Biology 2010, 11:7
http://www.biomedcentral.com/1471-2199/11/7 Background Intracerebral haemorrhage (ICH) accounts for around
10 - 15% of all strokes worldwide, with a higher propor-
tion in Asian countries. Compared with ischaemic
stroke, it causes disproportionate mortality and morbid-
ity [1] and research efforts to understand its pathophy-
siology are accordingly of high importance [2]. Brain
injury following ICH is a dynamic process, due both to
rebleeding and to downstream injury pathways activated
by the blood clot [3]. However, no therapies addressing
these secondary injury pathways have been proven
beneficial. One approach to understanding these injury pathways
is to study gene expression in animal models using
quantitative real-time reverse transcription polymerase
chain reaction (real-time RT-PCR). Real-time RT-PCR is
the method of choice for quantifying mRNA transcripts Cook et al. BMC Molecular Biology 2010, 11:7
http://www.biomedcentral.com/1471-2199/11/7 Page 2 of 11 nm and 280 nm using a UV spectrophotometer. All
samples had A260:A280 ratios of between 2.1 and 2.2,
and A260:A230 ratios of between 2.2 and 2.6. RNA
integrity was assessed using automated micro-capillary
electrophoresis in the Agilent Bioanalyzer. An RNA
Integrity Number (RIN) (reviewed in [36]) was assigned
to each sample by the Agilent Bioanalyzer Expert 2100
software. All RIN values obtained were in the range of
8.6 - 9.4, representing high quality RNA with minimal
degradation. nm and 280 nm using a UV spectrophotometer. All
samples had A260:A280 ratios of between 2.1 and 2.2,
and A260:A230 ratios of between 2.2 and 2.6. RNA
integrity was assessed using automated micro-capillary
electrophoresis in the Agilent Bioanalyzer. An RNA
Integrity Number (RIN) (reviewed in [36]) was assigned
to each sample by the Agilent Bioanalyzer Expert 2100
software. All RIN values obtained were in the range of
8.6 - 9.4, representing high quality RNA with minimal
degradation. normalisation to the geometric mean of the expression
of multiple reference genes [16] is considered to be a
reliable and conservative approach [17]. Previous studies employing quantitative real-time RT-
PCR in rat ICH models have utilised a single reference
gene for normalisation, usually without including a
reference gene validation protocol [18-22]. Therefore, as
an example, we demonstrate the impact of different
reference gene selection methods on determining the
transcript levels of transient receptor potential melasta-
tin 2 (TRPM2) channels following ICH. TRPM2 consists
of a calcium-permeable channel fused to a protein
kinase domain [23], and is highly expressed in the brain
and immune cells [24,25]. Genes The relative standard curve method [30] was used to
calculate TRPM2 mRNA level in the perihematomal
region of ICH and vehicle rats with survival times of 5 h
and 24 h, relative to each of the seven reference genes
individually. Figure 1a shows TRPM2 mRNA level at 5
h post-ICH. Large variations were observed depending
on which reference gene was used for normalisation. At
the 24 hour time point, when TRPM2 data were nor-
malised to GUSB only, a significant (p < 0.01) 1.85-fold
increase in mean TRPM2 mRNA level was observed in
the collagenase ICH rats compared to saline vehicles
(Figure 1b). A significant (p < 0.05) 1.4-fold increase
was found in TRPM2 mRNA level when data were nor-
malised to HPRT only. When each of the other refer-
ence genes was used individually for normalisation,
there were no significant differences. Given the discre-
pancy in these results and the large variation between
samples, we proceeded with a reference gene evaluation
study to determine the most stable reference genes in
the collagenase model of ICH. Background It has been implicated in cell
death pathways relating to oxidative stress [26,27] and
ischaemia [28] and its mRNA level has been shown to
increase in a time-dependent manner following transient
middle cerebral artery occlusion in rats [29]. No studies
to date have quantified the mRNA level of TRPM2 fol-
lowing ICH. Reference Gene Stability The geNorm v3.5 application was used to determine the
most stable reference genes out of the seven candidate
genes tested. We were interested to see whether the sta-
bility of the different reference genes was influenced by
the different survival times of the collagenase ICH rats Results RNA Concentration and Integrity TRPM2 mRNA Level Normalised to Individual Reference
Genes TRPM2 mRNA Level Normalised to Individual Reference
Genes Real-time RT-PCR Primer pairs and reaction conditions were optimised
using the standard cDNA pool prior to amplifying
unknown cDNA samples. Melting curve analysis was
consistent with a single reaction product for each gene,
and product size was confirmed by 2% agarose gel elec-
trophoresis stained with ethidium bromide and visua-
lised with UV light (not shown). After validation of
primer specificity, real-time PCR was carried out for
unknown collagenase ICH and saline vehicle cDNA
samples and serial dilutions from the cDNA pool. Reac-
tion efficiencies were calculated automatically by the
Corbett Rotor-Gene 6 software. The efficiency of all
runs was between 95% and 105%. Minimum R2 values
of 0.985 were accepted for each run, however, most
runs had R2 values over 0.99. g
We aimed to identify the most appropriate reference
genes to normalise real-time RT-PCR data in the col-
lagenase model of ICH in rats. Accordingly, real-time
RT-PCR was used to measure transcript levels of seven
reference genes: GAPDH, b-2-microglobulin (B2MG),
RNA Polymerase II (POL2R), TATA Box Binding Pro-
tein (TBP), hypoxanthine guanine phosphoribosyltrans-
ferase (HPRT), succinate dehydrogenase complex,
subunit A (SDHA) and b-glucuronidase (GUSB), plus
our gene of interest, TRPM2. We initially normalised
TRPM2 data to individual reference genes using the
relative standard curve method [30]. Next, a reference
gene validation study was conducted using the geNorm
application [16] to rank reference genes in order of sta-
bility. Other software programs are also available to
assess the stability of candidate reference genes, includ-
ing Normfinder [31] and BestKeeper [32]. Although dif-
ferent algorithms are utilised in each application, studies
have reported fairly consistent reference gene rankings
between all three programs, particularly with regard to
identifying the least stable genes [33-35]. However, we
chose to use geNorm in the present study given that it
also determines the number of reference genes required
for accurate normalisation. The panel of stable reference
genes identified by geNorm was then applied to normal-
ise data regarding our gene of interest, TRPM2. The
results presented herein will help to provide a guideline
for selecting stable reference genes for normalising real-
time RT-PCR data in future studies. RNA Concentration and Integrity The concentration of extracted total RNA was quanti-
fied by measurement of the absorbance at 230 nm, 260 Cook et al. BMC Molecular Biology 2010, 11:7
http://www.biomedcentral.com/1471-2199/11/7 Page 3 of 11 Figure 1 Relative mRNA level of TRPM2 normalised to individual reference genes. TRPM2 mRNA level at (A) 5 hours and (B) 24 hours in
the perihematomal brain region (RBG) of collagenase-induced ICH rats compared to saline vehicles. Bars represent mean of triplicate
measurements from 5 animals per group ± SEM. Single asterisk denotes statistical significance (p < 0.05) between 24 h ICH and vehicle rats;
double asterisk denotes statistical significance (p < 0.01) between 24 h ICH and vehicle rats, as assessed by t-tests. Figure 1 Relative mRNA level of TRPM2 normalised to individual reference genes. TRPM2 mRNA level at (A) 5 hours and (B) 24 hours in
the perihematomal brain region (RBG) of collagenase-induced ICH rats compared to saline vehicles. Bars represent mean of triplicate
measurements from 5 animals per group ± SEM. Single asterisk denotes statistical significance (p < 0.05) between 24 h ICH and vehicle rats;
double asterisk denotes statistical significance (p < 0.01) between 24 h ICH and vehicle rats, as assessed by t-tests. In the combined group of all samples, B2MG and
GUSB were found to be the most stable reference genes,
while TBP was the least stable. Indeed, B2MG and
GUSB were the two most stable reference genes in 3
out of the 5 groups tested, while TBP was ranked as the
least stable gene in all 5 groups. Overall, HPRT dis-
played variable stability; it was ranked among the least
stable two genes in 3 out of 5 groups, but conversely
was among the top two stable genes in the other 2
groups. POL2R was never one of the two most stable
genes in any group, however, it was consistently ranked
as the third most stable reference gene in 4 out of 5
groups. SDHA and GAPDH exhibited reasonable stabi-
lity across the different groups. (5 hours and 24 hours), and whether the injury itself
could produce localised fluctuations in reference genes
between the perihematomal (RBG) and matching con-
tralateral (LBG) regions of the brain. RNA Concentration and Integrity way ANOVA revealed statistically significant differences
in relative mRNA level within the SDHA group. TRPM2 mRNA Level Normalised to Multiple Reference
Genes way ANOVA revealed statistically significant differences
in relative mRNA level within the SDHA group. The qBasePlus program was used to calculate the nor-
malised mRNA level of TRPM2 in the perihematomal
brain region at 5 h and 24 h post-ICH, relative to the
most stable reference genes for each time point as deter-
mined by geNorm (5 h: GAPDH, HPRT, POL2R and
SDHA; 24 h: B2MG, GUSB and POL2R). qBasePlus uti-
lises a modified version of the 2-ΔΔCt method of relative
expression analysis [37] that takes into account multiple
reference genes and gene-specific amplification efficien-
cies [38]. There was no significant difference in mean
TRPM2 transcript levels between collagenase ICH and
saline vehicle animals (Figure 5). RNA Concentration and Integrity es in order of stability as determined by geNorm Ranking of candidate reference genes in order of stability as determined by geNorm. The most stable reference genes are listed at
at the bottom. * indicates number of reference genes required for accurate normalisation of real-time RT-PCR data, revealed by pair
and LBG refer to the perihematomal and the contralateral region, respectively, of collagenase ICH and vehicle rats. ce genes in order of stability as determined by geNorm. The most stable reference genes are listed at the top and the least stable
mber of reference genes required for accurate normalisation of real-time RT-PCR data, revealed by pairwise variation analysis. RBG
atomal and the contralateral region, respectively, of collagenase ICH and vehicle rats. Figure 2 shows geNorm output charts from the com-
bined group of all collagenase-induced ICH and saline
vehicle rats. All of the candidate reference genes had
expression stability (M) values under the recommended
cut-off value of 1.5 [16]. The highest M value obtained
from all sample subsets tested was 0.867 (not shown). Figure 2a shows M values for each reference gene and
ranks genes in order of stability from left to right. geN-
orm does not discriminate between the two most stable
genes since it relies on pairwise correlations. In Figure
2b, pairwise variation (V) analysis determined that four
reference genes were optimal for accurate normalisation
in the collective group, indicated at the V3/4 step of the
chart. The ideal value of V (y-axis) is recommended to be
under 0.15 [16] and was achieved in all sample subsets. Given that all of our candidate reference genes had M
values well below 1.5 and that the pairwise variation
threshold was achieved even when all seven genes were
used for normalisation (V6/7 step in Figure 2b), we deter-
mined whether even the least stable genes would serve as
appropriate normalisation factors (i.e. meet the geNorm
M and V thresholds) when the three most stable genes
were removed from analysis. Accordingly, the values for
B2MG, GUSB and POL2R were removed from the geN-
orm input file and the stability of GAPDH, HPRT, SDHA
and TBP were re-assessed. When these four reference
genes were evaluated in the absence of the most stable
genes, the M values were under 1.5 (not shown), how-
ever, the threshold of 0.15 in the pairwise variation analy-
sis was not achieved (far right bar in Figure 2b). RNA Concentration and Integrity Therefore, we used
geNorm to ascertain the most stable reference genes in
the following five groups: a collective group of all col-
lagenase ICH and saline vehicle samples; 5 hour collage-
nase ICH and saline vehicle samples; 24 collagenase
ICH and all saline vehicle samples; RBG only from all
collagenase ICH and saline vehicle samples; LBG only
from all collagenase ICH and saline vehicle samples. The most stable reference genes for each group plus
recommended number of reference genes for accurate
normalisation are summarised in Table 1. (5 hours and 24 hours), and whether the injury itself
could produce localised fluctuations in reference genes
between the perihematomal (RBG) and matching con-
tralateral (LBG) regions of the brain. Therefore, we used
geNorm to ascertain the most stable reference genes in
the following five groups: a collective group of all col-
lagenase ICH and saline vehicle samples; 5 hour collage-
nase ICH and saline vehicle samples; 24 collagenase
ICH and all saline vehicle samples; RBG only from all
collagenase ICH and saline vehicle samples; LBG only
from all collagenase ICH and saline vehicle samples. The most stable reference genes for each group plus
recommended number of reference genes for accurate
normalisation are summarised in Table 1. Cook et al. BMC Molecular Biology 2010, 11:7
http://www.biomedcentral.com/1471-2199/11/7 Page 4 of 11 Table 1 Ranking of candidate reference genes in order of stability as determined by geNorm. All ICH and saline
vehicle rats
5 hour ICH and saline
vehicle rats
24 hour ICH and saline
vehicle rats
RBG of all ICH and saline
vehicle rats
LBG of all ICH and saline
vehicle rats
B2MG*/GUSB*
GAPDH*/HPRT*
B2MG*/GUSB*
HPRT*/SDHA*
B2MG*/GUSB*
POL2R*
POL2R*
POL2R*
GAPDH*
POL2R*
GAPDH*
SDHA*
GAPDH
B2MG*
SDHA*
SDHA
B2MG
SDHA
GUSB*
GAPDH
HPRT
GUSB
HPRT
POL2R
HPRT
TBP
TBP
TBP
TBP
TBP
Ranking of candidate reference genes in order of stability as determined by geNorm. The most stable reference genes are listed at the top and the least stable
at the bottom. * indicates number of reference genes required for accurate normalisation of real-time RT-PCR data, revealed by pairwise variation analysis. RBG
and LBG refer to the perihematomal and the contralateral region, respectively, of collagenase ICH and vehicle rats. Table 1 Ranking of candidate reference genes in order of stability as determined by geNorm. ndidate reference genes in order of stability as determined by geNorm. Discussion In this study we have evaluated reference genes for use
as real-time RT-PCR normalising factors in collagenase-
induced ICH. Based on our results, we conclude that
use of a single normalisation reference gene is poten-
tially hazardous, and suggest a panel of reference genes
for more accurate transcript quantification. Real-time RT-PCR is a robust and sensitive technique
for quantifying mRNA transcripts, and constitutes a
powerful tool for increasing our understanding of the
genomic response to ICH. It requires an appropriate
normalisation strategy to control for error, the most
common being the use of one or more endogenous
reference genes [39]. A reference gene should be
expressed at a stable level regardless of the experimental
context, however, the expression of commonly used
reference genes has been shown to vary considerably. Normalisation of real-time RT-PCR data using a single,
non-validated reference gene may lead to inaccurate bio-
logical conclusions, and previous studies have high-
lighted the need to validate reference genes for each
new experimental condition [7,9,15]. Figure 3 shows the average raw (i.e. not yet normal-
ised) Ct values of the collagenase ICH and saline vehicle
rats for each candidate reference gene. The raw Ct
values of GAPDH were fairly consistent between groups,
while HPRT exhibited more variation. The collagenase
ICH samples generally had more raw Ct variation than
the saline vehicle samples. In Figure 4, the raw Ct values
were normalised to the geometric mean of the four
most stable reference genes determined by geNorm
(B2MG, GUSB, POL2R and GAPDH), in order to obtain
the relative mRNA level of each reference gene. One- Our previous study [40] identified suitable reference
genes for normalising real-time RT-PCR data in the rat Cook et al. BMC Molecular Biology 2010, 11:7
http://www.biomedcentral.com/1471-2199/11/7 Page 5 of 11 Figure 2 Ranking of candidate reference genes in order of stability by geNorm. (A) Expression stability values (M) of candidate reference
genes in 5 h and 24 h collagenase-induced ICH and saline vehicle rats. geNorm ranks reference genes in order of least stable to most stable
(left to right) by stepwise exclusion of the least stable gene. (B) Pairwise variation (V) analysis determines the optimal number of reference genes
that should be used for accurate normalisation, with a threshold value of 0.15 [16]. In this case, the V3/4 step achieves the cut-off value, and
therefore, the appropriate number of reference genes for accurate normalisation is four. Discussion The far right bar represents pairwise variation when the
three most stable genes are removed from analysis and the stability of the remaining genes (GAPDH, HPRT, SDHA and TBP) are re-assessed. In
this case the V threshold is not met. Figure 2 Ranking of candidate reference genes in order of stability by geNorm. (A) Expression stability values (M) of candidate reference
genes in 5 h and 24 h collagenase-induced ICH and saline vehicle rats. geNorm ranks reference genes in order of least stable to most stable
(left to right) by stepwise exclusion of the least stable gene. (B) Pairwise variation (V) analysis determines the optimal number of reference genes
that should be used for accurate normalisation, with a threshold value of 0.15 [16]. In this case, the V3/4 step achieves the cut-off value, and
therefore, the appropriate number of reference genes for accurate normalisation is four. The far right bar represents pairwise variation when the
three most stable genes are removed from analysis and the stability of the remaining genes (GAPDH, HPRT, SDHA and TBP) are re-assessed. In
this case the V threshold is not met. cerebral cortex and hippocampus following traumatic
brain injury (TBI). The present study focuses on a dif-
ferent neurological disorder with a different animal
model, brain regions and survival times to our previous
study. Our results demonstrate that the panels of stable
reference genes in ICH are not the same as those identi-
fied in our previous TBI reference gene study [40], con-
sistent with the recommendation that reference gene
validation be carried out whenever a new experimental
variable is introduced [9]. Reference gene validation was thus a crucial prerequisite to gene expression analysis in
ICH, and the current study has identified the appropri-
ate panel of reference genes for further studies. Alternative normalisation strategies include the use of
exogenous standards and normalisation to total RNA. Several groups have reported reliable normalisation with
exogenous standards [41-43]. A known amount of exo-
genous standard can be incorporated into the RNA
extraction process, which will then be affected by the
same experimental error as the RNA of interest [44]. Cook et al. BMC Molecular Biology 2010, 11:7
http://www.biomedcentral.com/1471-2199/11/7 Page 6 of 11 Figure 3 Average real-time RT-PCR cycle threshold (Ct) values o
yet normalised) values from individual groups of collagenase ICH and
represents the 25th and 75th percentile. Discussion Intriguingly,
B2MG and GUSB were among the least stable genes in
the 5 hour group, while GAPDH and HPRT were
ranked 4th and 6th, respectively, at 24 hours post-ICH. These results clearly demonstrate that stability of a can-
didate reference gene at one time point does not neces-
sarily confer stability at another. Therefore, it is likely
that the use of a single reference gene would be inade-
quate for normalising real-time RT-PCR data from dif-
ferent survival times of ICH. This is relevant because, as
discussed, previous studies utilising real-time RT-PCR in
rodent models of ICH generally have used one reference
gene for normalisation without including a validation
protocol. For example, HPRT was one of the most vari-
ably expressed genes in our study, but has been used as
a single normalising factor in other studies quantifying
mRNAs of interest over a time course of ICH [21,22]. It
is possible that studies using a single, non-validated
reference gene for normalisation could fail to detect
small changes in the mRNA species of interest [14], or
erroneously conclude that changes have occurred [15]. Indeed, another study from our laboratory [40] demon-
strated that the mRNA level of our gene of interest var-
ied significantly when normalised to individual reference
genes, but was far more reliable when a panel of vali-
dated reference genes were used for normalisation. However, exogenous standards can be difficult to pro-
duce and are subject to degradation, and do not control
for differences in quality of RNA template [17]. Another
approach is to normalise to total RNA mass, which con-
sists predominantly of ribosomal RNA (rRNA) mole-
cules. However, it has been shown that rRNA content is
not always an accurate predictor of the mRNA fraction
[16]. Furthermore, normalisation to total RNA does not
control for errors introduced during reverse transcrip-
tion or PCR [44]. To our knowledge, this is the only study which has
validated reference genes for use as normalising factors
in the collagenase model of ICH in rats. We quantified
the transcript level of seven candidate reference genes
(B2MG, GUSB, GAPDH, HPRT, POL2R, SDHA and
TBP) and our gene of interest, TRPM2, in the perihe-
matomal and matching contralateral regions in rats 5
and 24 hours following collagenase-induced ICH, as well
as in saline vehicle controls. Discussion Bars represent mean of triplicate
measurements from 5 animals, ± SEM. importance of validating the stability of reference genes
to be used as normalising factors in real-time RT-PCR
studies. When analysing all ICH and saline vehicle samples
together, the most stable reference genes were B2MG
and GUSB, with four reference genes recommended as
the optimal number for accurate normalisation. The
inclusion of additional reference genes further decreases
the value of V, even when all seven genes are used for
normalisation (V6/7). However, once the threshold value
of 0.15 has been reached, additional reference genes do
not significantly increase the reliability of normalisation
[16]. Although the observed differences in reference
gene stability were subtle, this does not imply that any
reference gene combination would be appropriate. To
illustrate this point, removing the 3 most stable refer-
ence genes (B2MG, GUSB and POL2R) from our geN-
orm input file showed that GAPDH, HPRT, SDHA and
TBP assessed together do not meet the proposed cut-off
value of V (0.15). Therefore, the most stable four genes,
as determined by geNorm, should be used. Figure 5 Normalised mRNA level of TRPM2 relative to multiple
reference genes. TRPM2 mRNA level in the perihematomal brain
region (RBG) of collagenase-induced ICH animals was compared to
saline vehicle controls, at 5 h and 24 h survival times. The most
stable reference genes determined by geNorm were used for
normalisation in each group (5 h: GAPDH, HPRT, POL2R, SDHA; 24 h:
B2MG, GUSB, POL2R). Bars represent mean of triplicate
measurements from 5 animals, ± SEM. Figure 5 Normalised mRNA level of TRPM2 relative to multiple
reference genes. TRPM2 mRNA level in the perihematomal brain
region (RBG) of collagenase-induced ICH animals was compared to
saline vehicle controls, at 5 h and 24 h survival times. The most
stable reference genes determined by geNorm were used for
normalisation in each group (5 h: GAPDH, HPRT, POL2R, SDHA; 24 h:
B2MG, GUSB, POL2R). Bars represent mean of triplicate
measurements from 5 animals, ± SEM. y g
We also compared the stability of the candidate refer-
ence genes at individual time points following collage-
nase-induced ICH (5 and 24 hours). The most stable
genes at 5 hours (GAPDH and HPRT) were different to
those at 24 hours (B2MG and GUSB). Discussion Whiskers represent the maxim Figure 3 Average real-time RT-PCR cycle threshold (Ct) values of candidate reference genes. Data are displayed as average raw Ct (not
yet normalised) values from individual groups of collagenase ICH and saline vehicle rats. The line in the box indicates the median, while the box
represents the 25th and 75th percentile. Whiskers represent the maximum and minimum values. Figure 3 Average real-time RT-PCR cycle threshold (Ct) values of candidate reference genes. D Figure 3 Average real-time RT-PCR cycle threshold (Ct) values of candidate reference genes. Data are displayed as average raw Ct (not
yet normalised) values from individual groups of collagenase ICH and saline vehicle rats. The line in the box indicates the median, while the box
represents the 25th and 75th percentile. Whiskers represent the maximum and minimum values. Cook et al. BMC Molecular Biology 2010, 11:7
http://www.biomedcentral.com/1471-2199/11/7 Page 7 of 11 Figure 4 Normalised mRNA levels of candidate reference genes. The mRNA level of each group (5 hour and 24 hour collagenase ICH and
saline vehicle rats) was normalised to the geometric mean of the expression of the four most stable reference genes determined by geNorm
(B2MG, GUSB, POL2R and GAPDH). Each bar represents the normalised mean of triplicate measurements from 5 animals, ± SEM. Asterisk denotes
statistical significance (p < 0.05) between ICH 24 hour LBG and vehicle RBG samples by one-way ANOVA. Figure 4 Normalised mRNA levels of candidate reference genes. The mRNA level of each group (5 hour and 24 hour collagenase ICH and
saline vehicle rats) was normalised to the geometric mean of the expression of the four most stable reference genes determined by geNorm
(B2MG, GUSB, POL2R and GAPDH). Each bar represents the normalised mean of triplicate measurements from 5 animals, ± SEM. Asterisk denotes
statistical significance (p < 0.05) between ICH 24 hour LBG and vehicle RBG samples by one-way ANOVA. Cook et al. BMC Molecular Biology 2010, 11:7
http://www.biomedcentral.com/1471-2199/11/7 Page 8 of 11 Figure 5 Normalised mRNA level of TRPM2 relative to multiple
reference genes. TRPM2 mRNA level in the perihematomal brain
region (RBG) of collagenase-induced ICH animals was compared to
saline vehicle controls, at 5 h and 24 h survival times. The most
stable reference genes determined by geNorm were used for
normalisation in each group (5 h: GAPDH, HPRT, POL2R, SDHA; 24 h:
B2MG, GUSB, POL2R). RNA Extraction Total RNA was extracted from the left and right stria-
tum with overlying cortex (’LBG’ (basal ganglia) and
RBG, respectively) of collagenase ICH rats (n = 5 from
each time point) and saline vehicle rats (n = 5) using
the RNeasy Lipid Tissue kit (Qiagen, Doncaster, Austra-
lia) according to the manufacturer’s instructions. Fifty
mg tissue was used in each RNA extraction, which
included an on-column DNase treatment step (Qiagen). RNA was quantified by UV spectrometry using the
Nanophotometer (Implen, Australia) to measure absor-
bance at 230, 260 and 280 nm. RNA integrity was evalu-
ated using the Agilent Bioanalyzer RNA 6000 Nano
Chip (Series II) kit. Discussion Initially, data regarding
TRPM2 transcript level in the perihematomal region
were normalised to individual reference genes using the
relative standard curve method of analysis [30]. At the
24 h survival time point, when TRPM2 data were nor-
malised to either GUSB or HPRT, significant differences
were found. However, no differences were found when
normalised to other commonly-used reference genes. There was considerable variation between samples when
data were analysed in this way (Figure 1). However, with
normalisation to a reference gene panel far more consis-
tent results were obtained (Figure 5). Had we used
HPRT or GUSB as a single reference gene for normali-
sation, it is likely that we would have drawn erroneous
conclusions about our data. These results highlight the The candidate reference genes in the present study
also showed differences in stability between the perihe-
matomal brain and the uninjured contralateral region. Page 9 of 11 Cook et al. BMC Molecular Biology 2010, 11:7
http://www.biomedcentral.com/1471-2199/11/7 Cook et al. BMC Molecular Biology 2010, 11:7
http://www.biomedcentral.com/1471-2199/11/7 with bone wax and the scalp wound closed with wound
clips after irrigation with bupivacaine. As might be expected, differences in gene stability were
most marked in the perihaematomal region. A cascade
of pathological processes including excitotoxicity,
oedema and inflammation are initiated by ICH [18], all
of which may impact reference gene expression. Animals recovered in their home cage in a climate-
and light-controlled environment with free access to
food and water. Animals were killed by decapitation
under deep isoflurane anesthesia either at 5 or 24 hours
post-surgery. The brain was quickly extracted and a 4
mm thick slice taken of cortex and basal ganglia, incor-
porating the injection site at the centre and the bulk of
the perihematomal brain. This was bisected into right
and left hemispheres and immediately snap-frozen for
RNA extraction. Conclusions We have evaluated the expression stability of seven can-
didate reference genes following collagenase-induced
ICH in rats. We have identified B2MG, GUSB, POL2R
and GAPDH as an appropriate panel of reference genes
to be used in the acute phase (5 and 24 hours) of
experimental ICH. When analysed in subsets of these
samples (according to survival time and brain region),
variations in the stability of the reference genes were
revealed. Our gene of interest, TRPM2, was inconsis-
tently elevated following injury when normalised to indi-
vidual reference genes. However, when normalised to a
panel of the most stable genes, no significant differences
were seen. These results emphasise the importance of
identifying and validating suitable reference genes to
prevent erroneous conclusions. The results of the pre-
sent study will enable more accurate normalisation of
real-time RT-PCR data at 5 and 24 hour time points fol-
lowing collagenase-induced ICH. Collagenase-induced ICH Complementary DNA was synthesised using the Super-
Script III Reverse Transcription kit (Invitrogen, Mt
Waverley, Australia). Two μg total RNA was added to
250 ng random hexamers (Geneworks, Adelaide, Austra-
lia), 1 mM each dNTP (Invitrogen) and nuclease-free
water to 13 μL. Reactions were heated to 65°C for 5
minutes then immediately placed on ice for 1 minute. To each tube, 4.75 μL 5× First Strand Buffer, 1 μL
RNase OUT (Invitrogen), 0.02 M dithiothreitol and 200
units SuperScript III reverse transcriptase were added. Reactions containing nuclease-free water in place of
enzyme served as negative controls. Reactions were
incubated at 25°C for 5 minutes, 55°C for 60 minutes
and 70°C for 15 minutes. cDNA was diluted to 10 ng/
μL with nuclease-free water and stored at -20°C. Real-time PCR All animal protocols were approved by the Institute of
Medical and Veterinary Science and the University of
Adelaide Animal Ethics committees and were con-
formed to guidelines issued by the Australian National
Health and Medical Research Council. Adult male Sprague-Dawley rats (n = 15) weighing
300-340 g were used in the study, and obtained four
days prior to surgery to ameliorate any acute stress-
induced changes in gene expression. The study used col-
lagenase to induce intracerebral hemorrhage [45]. Briefly, animals were anesthetized using isoflurane (1.5 -
2.0%) in a 30:70 mix of oxygen and nitrogen via a nose
cone and placed in a Kopf stereotaxic frame. A rectal
thermometer was inserted and temperature maintained
between 36.5 - 37.5°C with a thermostatically-controlled
heat pad. The scalp was shaved and cleaned, and bupi-
vacaine instilled. A midline scalp incision was made, the
skull exposed and a burrhole drilled 0.7 mm anterior
and 3.0 mm lateral to bregma. Using a syringe driver
(Harvard Instruments), 0.2 U type VII bacterial collage-
nase (Sigma C0773) in 2 μL normal saline, or saline
alone (’vehicle’) was infused over 4 minutes, via a 30 G
needle, into the centre of the striatum (6.0 mm anterior
to bregma). The needle was left in place for five min-
utes, and then withdrawn slowly. The hole was sealed Acknowledgements NLC and TJK are grateful recipients of postgraduate scholarships from the
National Health and Medical Research Council of Australia. TJK’s scholarship
was co-awarded by the National Heart Foundation (Grant ID 465450). This
work was also supported, in part, by the Neurosurgical Research Foundation,
and by a Pfizer neurosciences research grant for TJK. μ
Real-time PCR Amplification was carried out in a Rotor-Gene 3000
(Corbett Research, Mortlake, Australia) with an initial
UDG incubation of 50°C for 2 minutes, initial denatura-
tion of 95°C for 2 minutes, followed by 40 cycles of: 95°
C for 15 sec denaturation, primer-specific annealing
temperature for 15 sec (see [40]), and 72°C for 15 sec
extension. TRPM2 annealing temperature was 60°C. Fluorescence data were collected during the extension
step of each cycle. Specificity of amplicons were verified
by melting curve analysis after 40 cycles (72°C to 95°C)
and 2% agarose gel electrophoresis stained with ethi-
dium bromide and visualised under UV light. All cDNA
samples were run in triplicate. Negative controls con-
taining water instead of cDNA were present in all runs. No-reverse transcriptase controls were included for each
gene to test for genomic DNA amplification. had been determined. Statistical analyses were carried
out using unpaired Student’s t-tests and one-way analy-
sis of variance (ANOVA) with Newman-Keuls post-hoc
tests. Data Analysis 4. Bustin SA: Absolute quantification of mRNA using real-time reverse
transcription polymerase chain reaction. Journal of Molecular
Endocrinology 2000, 25:169-193. 4. Bustin SA: Absolute quantification of mRNA using real-time reverse
transcription polymerase chain reaction. Journal of Molecular
Endocrinology 2000, 25:169-193. Standard curves made from serial dilutions of pooled
cDNA were used to calculate PCR efficiency (E) using
the formula: E (%) = (10[-1/slope] - 1) × 100. Raw values
of input RNA were determined for each sample from
the standard curve using the Corbett Rotor-Gene 6 soft-
ware. The relative standard curve method [30] was
applied to these raw quantities to calculate TRPM2
mRNA level relative to each of the seven reference
genes individually. The cycle threshold (Ct) of an indivi-
dual sample reflects the cycle at which a detectable
number of PCR products have accumulated above back-
ground fluorescence [47]. Ct values were calculated
from the standard curve, entered into the qBasePlus
software [38] and used to generate an input file for geN-
orm v3.5 [16]. geNorm determined the most stable
reference genes out of the panel of candidate genes
using expression stability analysis by pairwise correla-
tions. An expression stability measure, M, was assigned
to each gene, which was used to rank candidate refer-
ence genes in order of stability. The most stable refer-
ence genes were determined in the following groups: all
collagenase ICH and saline vehicle samples; 5 hour col-
lagenase ICH and saline vehicle samples; 24 collagenase
ICH and all saline vehicle samples; RBG only from all
collagenase ICH and saline vehicle samples; LBG only
from all collagenase ICH and saline vehicle samples. Normalised mRNA levels of each gene were calculated
using qBasePlus once the most stable reference genes 5. Klein D: Quantification using real-time PCR technology: applications and
limitations. Trends in Molecular Medicine 2002, 8(6):257-260. 5. Klein D: Quantification using real-time PCR technology: applications and
limitations. Trends in Molecular Medicine 2002, 8(6):257-260. 6. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R,
Nolan T, Pfaffl MW, Shipley GL, et al: The MIQE guidelines: minimum
information for publication of quantitative real-time PCR experiments. Clinical Chemistry 2009, 55(4):611-622. 7. Radonić A, Thulke S, Mackay IM, Landt O, Siegert W, Nitsche A: Guideline
to reference gene selection for quantitative real-time PCR. Biochemical
and Biophysical Research Communications 2004, 313:856-862. 7. Authors’ contributions NLC carried out the reference gene study, performed the statistical analyses
and drafted the manuscript. TJK carried out the animal surgery, and assisted
with statistical analyses and drafting the manuscript. CVDH supervised the
study, participated in study design and provided useful discussion. RV
participated in study design and coordination and provided useful
discussion. All authors read and approved the final manuscript. Received: 24 July 2009
Accepted: 20 January 2010 Published: 20 January 2010 Received: 24 July 2009
Accepted: 20 January 2010 Published: 20 January 2010 Received: 24 July 2009 μ
Real-time PCR The candidate reference genes used in this study were:
GAPDH, B2MG, POL2R, TBP, HPRT, SDHA and
GUSB. Primer sequences for reference genes have been
described elsewhere [40]. TRPM2 primer sequences
were (5’ - 3’): forward, GAAGGAAAGAGGGGGTGTG
and reverse, CATTGGTGATGGCGTTGTAG [46], with
a product size of 101 base pairs. Real-time RT-PCR
amplifications for reference genes and TRPM2 were car-
ried out using 10 μL 2× Invitrogen Platinum SYBR
Green SuperMix-UDG, 300 nM forward and reverse Page 10 of 11 Cook et al. BMC Molecular Biology 2010, 11:7
http://www.biomedcentral.com/1471-2199/11/7 primers (400 nM for POL2R and GAPDH), 1 μL cDNA
and nuclease-free water in a total volume of 20 μL. One
hundred mM MgCl2 (Invitrogen) was added to POL2R
reactions. A set of standards was included in each run,
comprising five-fold serial dilutions made from aliquots
of pooled cDNA, derived from an RNA pool of all sam-
ples. Serial dilutions contained the following amounts of
cDNA: 50 ng, 10 ng, 2 ng and 0.4 ng. The standard ser-
ies encompassed the unknown cDNA concentrations. Amplification was carried out in a Rotor-Gene 3000
(Corbett Research, Mortlake, Australia) with an initial
UDG incubation of 50°C for 2 minutes, initial denatura-
tion of 95°C for 2 minutes, followed by 40 cycles of: 95°
C for 15 sec denaturation, primer-specific annealing
temperature for 15 sec (see [40]), and 72°C for 15 sec
extension. TRPM2 annealing temperature was 60°C. Fluorescence data were collected during the extension
step of each cycle. Specificity of amplicons were verified
by melting curve analysis after 40 cycles (72°C to 95°C)
and 2% agarose gel electrophoresis stained with ethi-
dium bromide and visualised under UV light. All cDNA
samples were run in triplicate. Negative controls con-
taining water instead of cDNA were present in all runs. No-reverse transcriptase controls were included for each
gene to test for genomic DNA amplification. primers (400 nM for POL2R and GAPDH), 1 μL cDNA
and nuclease-free water in a total volume of 20 μL. One
hundred mM MgCl2 (Invitrogen) was added to POL2R
reactions. A set of standards was included in each run,
comprising five-fold serial dilutions made from aliquots
of pooled cDNA, derived from an RNA pool of all sam-
ples. Serial dilutions contained the following amounts of
cDNA: 50 ng, 10 ng, 2 ng and 0.4 ng. The standard ser-
ies encompassed the unknown cDNA concentrations. 1.
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Cite this article as: Cook et al.: Reference genes for normalising gene
expression data in collagenase-induced rat intracerebral haemorrhage. BMC Molecular Biology 2010 11:7. doi:10.1186/1471-2199-11-7
Cite this article as: Cook et al.: Reference genes for normalising gene
expression data in collagenase-induced rat intracerebral haemorrhage. BMC Molecular Biology 2010 11:7. doi:10.1186/1471-2199-11-7
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Your research papers will be:
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BioMedcentral Publish with BioMed Central and every
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Avelumab (anti–PD-L1) as first-line switch-maintenance or second-line therapy in patients with advanced gastric or gastroesophageal junction cancer: phase 1b results from the JAVELIN Solid Tumor trial
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Journal for immunotherapy of cancer
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RESEARCH ARTICLE Open Access Chung et al. Journal for ImmunoTherapy of Cancer (2019) 7:30
https://doi.org/10.1186/s40425-019-0508-1 Chung et al. Journal for ImmunoTherapy of Cancer (2019) 7:30
https://doi.org/10.1186/s40425-019-0508-1 (2019) 7:30 Chung et al. Journal for ImmunoTherapy of Cancer
https://doi.org/10.1186/s40425-019-0508-1 Avelumab (anti–PD-L1) as first-line
switch-maintenance or second-line therapy
in patients with advanced gastric or
gastroesophageal junction cancer:
phase 1b results from the JAVELIN Solid
Tumor trial ung1*, Hendrik-Tobias Arkenau2, Jeeyun Lee3, Sun Young Rha1, Do-Youn Oh4, Lucjan Wyrwicz5 Hyun Cheol Chung1*, Hendrik-Tobias Arkenau2, Jeeyun Lee3, Sun Young Rha1, Do-Youn Oh4, Lucjan Wyrwicz5,
Yoon-Koo Kang6, Keun-Wook Lee7, Jeffrey R. Infante8, Sung Sook Lee9, Margaret Kemeny10, Ulrich Keilholz11,
Bohuslav Melichar12, Alain Mita13, Ruth Plummer14, Denis Smith15, Arnold B. Gelb16, Huiling Xiong16, Janet Hong16,
Vikram Chand16,18 and Howard Safran17 © 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. * Correspondence: unchung8@yuhs.ac p
g
y
Prior presentation Interim analyses were presented at the American Society
of Clinical Oncology (ASCO) Gastrointestinal Cancers Symposium, San
Francisco, CA, 21-23 January 2016 (abstract 167) and the ASCO Annual
Meeting, Chicago, IL, 3-7 June 2016 (abstract 4009). Analyses reported in the
current manuscript were presented at the American Association for Cancer
Research Annual Meeting, Chicago, IL, 14-18 April 2018. 1Yonsei Cancer Center, Yonsei University College of Medicine, Yonsei University
Health System, Seoul 03722, South Korea
Full list of author information is available at the end of the article Abstract Background: We evaluated the antitumor activity and safety of avelumab, a human anti–PD-L1 IgG1 antibody, as
first-line switch-maintenance (1 L-mn) or second-line (2 L) treatment in patients with advanced gastric/gastroesophageal
cancer (GC/GEJC) previously treated with chemotherapy. Methods: In a phase 1b expansion cohort, patients without (1 L-mn) or with (2 L) disease progression following first-line
chemotherapy for advanced GC/GEJC received avelumab 10 mg/kg intravenously every 2 weeks. Endpoints included best
overall response, progression-free survival (PFS), overall survival (OS), and safety. (Continued on next page) (Continued on next page) * Correspondence: unchung8@yuhs.ac Background patients [9]. PD-L1 is a key therapeutic target for reactivat-
ing antitumor immune responses [10]. Additionally, PD-L1
is expressed in ≈30 to 60% of GC/GEJC specimens, with a
higher frequency seen in certain pathological and genomic
subtypes [11]. Immunotherapy with anti–PD-1 antibodies
has been associated with durable antitumor responses
in early-phase studies of patients with GC/GEJC
[10, 12, 13]. Background
Gastric cancer (GC) is an aggressive disease that repre-
sents the third leading cause of cancer-related death
worldwide [1]. Gastroesophageal junction cancer (GEJC)
has similar biology, prognosis, and treatment guidelines
as GC [2, 3]. First-line (1 L) standard of care for ad-
vanced inoperable GC/GEJC is based on combination
fluoropyrimidine and platinum treatment, with trastuzu-
mab added for HER2+ tumors. Second-line (2 L) options
include regimens based on irinotecan, taxanes, and/or
ramucirumab [2, 3]. However, cytotoxic regimens are
associated with cumulative toxicity that may restrict
long-term treatment, resulting in limited duration of re-
sponse and overall survival (OS). Maintenance therapy,
ie, continued treatment with an agent administered in
the 1 L induction regimen or sequential treatment with a
different agent until progression (switch maintenance),
has the potential to extend durations of response and
OS, particularly when an agent with a different mechan-
ism of action is employed, while avoiding potential addi-
tive toxicity associated with further chemotherapy or
combination treatment. As such, maintenance therapy
has become an established strategy for several advanced
tumors [4, 5]. Although the role of maintenance therapy
in treating GC/GEJC is less well defined, observational
and retrospective studies of maintenance fluoropyrimidine
treatment in advanced GC/GEJC have shown that this
approach is feasible and may improve progression-free
survival (PFS) compared with observation alone [6–8]. Avelumab is a human anti–PD-L1 monoclonal antibody
that has been approved in various countries for the treat-
ment of metastatic Merkel cell carcinoma and in the United
States and Canada for the treatment of advanced urothelial
carcinoma progressing after platinum-containing chemo-
therapy. In phase 1 and 2 studies across various advanced
cancers, avelumab has demonstrated a tolerable safety pro-
file and durable antitumor activity [14–16]. In preclinical
studies, avelumab activated both adaptive and innate im-
mune effector cells [17, 18], suggesting an additional mech-
anism of action compared with other approved anti–PD-1/
PD-L1 antibodies. To investigate the efficacy and safety of avelumab in
the treatment of advanced GC/GEJC, we enrolled a
cohort of patients in the phase 1 JAVELIN Solid Tumor
trial. Background Patients were enrolled following 1 L chemotherapy;
those without disease progression received avelumab as
1 L switch maintenance (1 L-mn subgroup), and those
with disease progression received avelumab as 2 L treat-
ment (2 L subgroup). To our knowledge, this is the first
study of an anti–PD-L1 agent administered as switch-
maintenance therapy in this disease. In recent years, much attention has been focused on
anticancer therapies that activate the immune response. In
a randomized phase 2 study of patients with advanced
GC/GEJC, switch-maintenance ipilimumab (anti–CTLA-4)
after 1 L chemotherapy did not improve immune-related
PFS or OS compared with best supportive care, which in-
cluded continued fluoropyrimidine chemotherapy in most © 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. Page 2 of 10 Chung et al. Journal for ImmunoTherapy of Cancer (2019) 7:30 (2019) 7:30 (Continued from previous page)
Results: Overall, 150 patients were enrolled (1 L-mn, n = 90; 2 L, n = 60) and median follow-up in the 1 L-mn and 2 L
subgroups was 36.0 and 33.7 months, respectively. The confirmed objective response rate was 6.7% in both subgroups
(95% CI, 2.5–13.9% and 1.8–16.2%, respectively), including complete responses in 2.2% of the 1 L-mn subgroup (n = 2). In
the 1 L-mn and 2 L subgroups, median duration of response was 21.4 months (95% CI, 4.0–not estimable) and 3.5 months
(95% CI, 2.8–8.3) and disease control rates were 56.7 and 28.3%, respectively. Median PFS in the 1 L-mn and 2 L subgroups
was 2.8 months (95% CI, 2.3–4.1) and 1.4 months (95% CI, 1.3–1.5), with 6-month PFS rates of 23.0% (95% CI, 14.7–32.4%)
and 7.9% (95% CI, 2.6–17.2%), and median OS was 11.1 months (95% CI, 8.9–13.7) and 6.6 months (95% CI, 5.4–9.4),
respectively. In the 1 L-mn subgroup, median OS measured from start of 1 L chemotherapy was 18.7 months
(95% CI, 15.4–20.6). Across both subgroups, 20.7% had an infusion-related reaction of any grade. Other common
treatment-related adverse events (TRAEs) of any grade included fatigue (10.0%) and nausea (6.7%). Treatment-related
serious adverse events occurred in 4.0% of patients. Overall, 8.7% had a grade ≥3 TRAE, including 1 treatment-related death. Conclusion: Avelumab showed clinical activity and an acceptable safety profile in patients with GC/GEJC. Trial registration: ClinicalTrials.gov NCT01772004; registered 21 January 2013. Keywords: Avelumab, Metastatic, Gastric, Esophagogastric junction, Adenocarcinoma, Maintenance Statistical methods Enrollment of 150 patients was planned for this cohort
based on the anticipated sample size required to estimate
and provide 95% Clopper-Pearson confidence intervals
(CIs) for potential objective response rates (ORR; propor-
tion of patients with a confirmed CR or PR; eg, 10% [5.7–
16.0%] for 15 responders or 20% [13.9–27.3%] for 30
responders). Safety and antitumor activity were analyzed
in all patients who received ≥1 dose of avelumab. Time-to-event endpoints (PFS, OS, duration of response,
and duration of follow-up) were estimated using the
Kaplan–Meier method and CIs for the median were
calculated using the Brookmeyer–Crowley method. Study design and patients JAVELIN Solid Tumor (NCT01772004) is an inter-
national, open-label, phase 1 trial. In the phase 1b, non-
randomized expansion cohort reported here, eligible Chung et al. Journal for ImmunoTherapy of Cancer (2019) 7:30 Chung et al. Journal for ImmunoTherapy of Cancer Page 3 of 10 patients
had
histologically
confirmed,
unresectable,
locally advanced or metastatic GC/GEJC, and previous
treatment with 1 L combination chemotherapy; patients
with prior neoadjuvant platinum-based doublet or triplet
chemotherapy who were not candidates for surgery were
also eligible. Patients should not have received >1 line of
prior treatment for metastatic disease, and patients with
prior checkpoint inhibitor or trastuzumab treatment
were ineligible (Additional file 1: Table S1). Patients in
the 2 L subgroup were not permitted to have received
anticancer treatment within 28 days before the start of
study treatment, whereas in the 1 L-mn subgroup,
patients were permitted to be enrolled within 28 days if
all toxicity from prior therapy had resolved to grade ≤1. A fresh or archival tumor specimen was required, but
patients were not preselected based on PD-L1 status
(ie, all-comer design). Patients were enrolled in accordance
with an approved protocol, international standards of good
clinical practice, and institutional safety monitoring, and
written informed consent was obtained. The study protocol
was approved by the institutional review board or
independent ethics committee at each center. cutoffs of ≥1% of tumor cells positive for partial or
complete membrane PD-L1 staining of any intensity. HER2
and microsatellite status were recorded retrospectively
from medical records when available. Procedures and assessments Patients received avelumab 10 mg/kg intravenously every
2 weeks until confirmed disease progression, unaccept-
able toxicity, or protocol-based criteria for withdrawal
[15]. Premedication
with
diphenhydramine
and
acetaminophen was required 30 to 60 min before all
avelumab infusions. Safety was assessed at each biweekly trial visit and in-
cluded assessment of adverse events (AEs), physical
examination, clinical laboratory tests (hematology, hep-
atic panels, and serum chemistry), and documentation of
concurrent medications. AEs and laboratory abnormal-
ities were classified and graded according to National
Cancer Institute Common Terminology Criteria for
Adverse Events version 4.0. A serious AE (SAE) was de-
fined as any untoward event that was life-threatening,
required hospitalization, resulted in disability, was a con-
genital anomaly, or resulted in death. Immune-related
AEs were identified using a prespecified list of Medical
Dictionary for Regulatory Activities terms plus medical
review. Clinical activity was assessed by investigators
using Response Evaluation Criteria in Solid Tumors
version
1.1. Radiographic
tumor
assessments
were
performed at baseline and every 6 weeks. In patients
achieving a partial response (PR) or complete response
(CR), a confirmatory CT or MRI scan was done ≥28 days
later (preferably at the scheduled 6-week interval). PD-L1 expression was assessed in tumor cells using a
proprietary immunohistochemistry assay (PD-L1 IHC
73-10; Dako, Carpinteria, CA), as described previously
[15, 19]; in this report, PD-L1 status was defined using Outcomes Primary endpoints for the entire JAVELIN Solid Tumor
trial were dose-limiting toxicities during the first 3 weeks
of treatment in the phase 1a dose-escalation part
(reported previously [20]) and confirmed best overall
response adjudicated by independent review in specified
efficacy expansion cohorts (not including the GC/GEJC
cohort reported here). Secondary endpoints assessed in
the current cohort included investigator-assessed best
overall response, duration of response, PFS, OS, safety,
and evaluation of PD-L1 expression [15]. Results
Patients Five patients remained
on avelumab treatment at data cutoff, all in the 1 L-mn
subgroup (5.6%). Results
Patients Between 13 February 2014 and 11 August 2015, 150
patients with histologically confirmed GC/GEJC were
enrolled, including 90 without disease progression after
1 L chemotherapy
(1 L-mn subgroup) and 60 with
progressive
disease
(2 L
subgroup),
per
investigator
assessment (Table 1). In the 1 L-mn subgroup, 27.8%
had achieved a PR with prior chemotherapy; in the 2 L
subgroup, prior responses were PR in 13.3% and CR in
1.7%. The median interval between end of prior chemo-
therapy and start of avelumab was 45 days (1 L-mn
subgroup) and 77 days (2 L subgroup). Across both
subgroups, 30.7% had PD-L1+ tumors. At data cutoff (30 September 2017), patients in the 1
L-mn and 2 L subgroups had received a median (range)
of 7 (1–79) and 4.5 (1–44) avelumab doses, and median
duration of treatment was 3.2 months (interquartile
range [IQR], 1.4–6.1) and 2.2 months (IQR, 1.4–5.2),
respectively. Median duration of follow-up was 36.0
months (IQR, 33.7–37.7) in the 1 L-mn subgroup and
33.7 months (IQR, 27.9–34.9) in the 2 L subgroup. In
both subgroups, the most common reason for treatment Chung et al. Results
Patients Journal for ImmunoTherapy of Cancer (2019) 7:30 Page 4 of 10 Table 1 Baseline characteristics in the first-line switch-maintenan
and second-line subgroups
Characteristics
1 L-mn subgroup
(n = 90)
2 L subgroup
(n = 60)
Median age (IQR), years
59 (52.0–67.0)
62.5 (51.5–66
Sex, n (%)
Male
68 (75.6)
46 (76.7)
Female
22 (24.4)
14 (23.3)
ECOG PS, n (%)
0
37 (41.1)
23 (38.3)
1
53 (58.9)
37 (61.7)
Geographic region, n (%)
North America
31 (34.4)
32 (53.3)
Asia
34 (37.8)
10 (16.7)
Europe
25 (27.8)
18 (30.0)
Race, n (%)
White
44 (48.9)
36 (60.0)
Asian
35 (38.9)
13 (21.7)
Black
4 (4.4)
4 (6.7)
Other
7 (7.8)
7 (11.7)
Histology, n (%)
Tubular
18 (20.0)
3 (5.0)
Signet ring
17 (18.9)
13 (21.7)
Mucinous
4 (4.4)
4 (6.7)
Papillary
1 (1.1)
0
Other/not specified
1 (1.1)
0
Unknown
37 (41.1)
39 (65.0)
PD-L1 expression status based on ≥1% cutoff on tumor cells, n (%)
PD-L1+
26 (28.9)
20 (33.3)
PD-L1−
51 (56.7)
25 (41.7)
Not evaluable
13 (14.4)
15 (25.0)
HER2 status, n (%)
HER2−
62 (68.9)
29 (48.3)
HER2+
4 (4.4)
5 (8.3)
Unknown
24 (26.7)
26 (43.3)
Microsatellite status, n (%)
Low
1 (1.1)
0
Stable
21 (23.3)
17 (28.3)
High
2 (2.2)
2 (3.3)
Unknown
66 (73.3)
41 (68.3)
Prior gastrectomy, n (%)
24 (26.7)
14 (23.3)
Metastatic disease status at study entry, n (%)
M0
5 (5.6)
2 (3.3)
M1
85 (94.4)
58 (96.7)
Tumor size at baselinea
Median (IQR), mm
33 (19–52)
44 (25–69.5)
Unknown, n (%)
1 (1.1)
1 (1.7) Table 1 Baseline characteristics in the first-line switch-maintenance
and second-line subgroups Table 1 Baseline characteristics in the first-line switch-maintenance
and second-line subgroups (Continued)
Characteristics
1 L-mn subgroup
(n = 90)
2 L subgroup
(n = 60)
Best response to prior anticancer therapy, n (%)
Complete response
0
1 (1.7)
Partial response
25 (27.8)
8 (13.3)
Stable disease
59 (65.6)
23 (38.3)
Progressive disease
0
22 (36.7)
Not evaluable or unknown
6 (6.7)
5 (8.3)
Prior anticancer therapy
(any setting), n (%)
90 (100)
60 (100)
Number of prior lines of anticancer therapy for metastatic or locally
advanced disease, n (%)
0
1 (1.1)
5 (8.3)
1
87 (96.7)
53 (88.3)
2
2 (2.2)
1 (1.7)
Unknown
0
1 (1.7)
Median prior lines (range)
1.0 (0–2)
1.0 (0–2)
Interval from end of prior chemotherapy to start of avelumab therapy
Median (IQR), days
45 (35–64)
77 (49–135)
Data missing, n (%)
8 (8.9)
15 (25.0)
aSum of the longest diameters of target lesions
Abbreviations: 1 L-mn first-line switch-maintenance, 2 L second line, ECOG PS
Eastern Cooperative Oncology Group performance status, IQR interquartile range Table 1 Baseline characteristics in the first-line switch-maintenance
and second-line subgroups (Continued) Interval from end of prior chemotherapy to start of avelumab therapy discontinuation was disease progression (1 L-mn, 75.6%; discontinuation was disease progression (1 L-mn, 75.6%;
2 L, 71.7%); other reasons were AE (13.3%, 10.0%), death
(3.3%, 8.3%), withdrawal of consent (1.1%, 6.7%), loss to
follow-up (0%, 1.7%), protocol noncompliance (1.1%, 0%),
and physician decision (0%, 1.7%). Antitumor activity: 1 L-mn subgroup y
g
p
The confirmed ORR (additional effect after the end of
chemotherapy) was 6.7% (n = 6; 95% CI, 2.5–13.9%)
(Additional file 1: Table S2). Notably, 2 patients (2.2%)
had a CR; both patients were Asian and had stable dis-
ease (SD) as best response to prior chemotherapy, and
PD-L1 status was positive in 1 patient and not evaluable
in the other. Four patients (4.4%) had a PR, which repre-
sented
additional
tumor
shrinkage
following
prior
chemotherapy; best response to prior chemotherapy in
these patients was PR (n = 2) and SD (n = 2) (Additional
file 1: Table S3). Forty-five patients (50.0%) had SD of
any duration as best response (disease control rate,
56.7%). Early and durable responses were observed
(Fig. 1a and Additional file 1: Figure S1A), with a median
time to response of 1.4 months (IQR, 1.3–4.1), a median
duration of response of 21.4 months (95% CI, 4.0–not
estimable), and an estimated 66.7% (95% CI, 19.5–
90.4%) of responses lasting ≥6 months. Responses were Chung et al. Journal for ImmunoTherapy of Cancer (2019) 7:30 Page 5 of 10 Chung et al. Journal for ImmunoTherapy of Cancer 200
180
160
140
120
100
80
60
40
20
-20
-40
-60
-80
-100
0
0
2
4
6
8
10
12
14
16
Time since treatment initiation (months)
Change in target lesions from baseline (%)
6
3
8
3
4
3
2
3
0
3
8
2
6
2
4
2
2
2
0
2
8
1
First occurence of new lesion
Patient off treatment
Complete response
Partial response
Stable disease
Progressive disease
Not evaluable
A
200
180
160
140
120
100
80
60
40
20
-20
-40
-60
-80
-100
0
0
2
4
6
8
10
12
14
16
Time since treatment initiation (months)
Change in target lesions from baseline (%)
6
3
8
3
4
3
2
3
0
3
8
2
6
2
4
2
2
2
0
2
8
1
First occurence of new lesion
Patient off treatment
Partial response
Stable disease
Progressive disease
Not evaluable
B
Fig. 1 Change in sum of target lesion diameters over time with avelumab in evaluable patients. a First-line switch-maintenance subgroup (n = 81). b Second-line subgroup (n = 52). Color coding is based on best overall response per Response Evaluation Criteria in Solid Tumors version 1.1. Antitumor activity: 1 L-mn subgroup Dotted
lines indicate the 3-month timepoint and changes in target lesion size of −30, 0, and +20% 200
180
160
140
120
100
80
60
40
20
-20
-40
-60
-80
-100
0
0
2
4
6
8
10
12
14
16
Time since treatment initiation (months)
Change in target lesions from baseline (%)
6
3
8
3
4
3
2
3
0
3
8
2
6
2
4
2
2
2
0
2
8
1
First occurence of new lesion
Patient off treatment
Complete response
Partial response
Stable disease
Progressive disease
Not evaluable
A Time since treatment initiation (months)
200
180
160
140
120
100
80
60
40
20
-20
-40
-60
-80
-100
0
0
2
4
6
8
10
12
14
16
Time since treatment initiation (months)
Change in target lesions from baseline (%)
6
3
8
3
4
3
2
3
0
3
8
2
6
2
4
2
2
2
0
2
8
1
First occurence of new lesion
Patient off treatment
Partial response
Stable disease
Progressive disease
Not evaluable
B B Fig. 1 Change in sum of target lesion diameters over time with avelumab in evaluable patients. a First-line switch-maintenance subgroup (n = 81). b Second-line subgroup (n = 52). Color coding is based on best overall response per Response Evaluation Criteria in Solid Tumors version 1.1. Dotted
lines indicate the 3-month timepoint and changes in target lesion size of −30, 0, and +20% Fig. 1 Change in sum of target lesion diameters over time with avelumab in evaluable patients. a First-line switch-maintenance subgroup (n = 81). b Second-line subgroup (n = 52). Color coding is based on best overall response per Response Evaluation Criteria in Solid Tumors version 1.1. Dotted
lines indicate the 3-month timepoint and changes in target lesion size of −30, 0, and +20% Duration of avelumab treatment in these 2 patients was
36.2 months
(ongoing)
and 23.5 months,
respectively. Another patient had SD before disease progression at
week 13 (due to a new lesion), followed by a 100%
reduction in target lesions sustained for >6 months. ongoing at data cutoff in 2 patients, including 1 patient
with a CR. In evaluable patients with PD-L1+ or PD-L1−
tumors, confirmed ORR was 7.7% (2/26; 95% CI, 0.9–
25.1%) vs 3.9% (2/51; 95% CI, 0.5–13.5%). Antitumor activity: 1 L-mn subgroup Of 81 patients
evaluable for change in size of target lesions, 13 (16.0%)
had shrinkage of ≥30% and 37 (45.7%) had shrinkage of
any level (Additional file 1: Figure S2A). No correlation
was seen between response to prior chemotherapy and
tumor shrinkage on avelumab. g
Median PFS and OS measured from start of avelumab
therapy (ie, not including prior chemotherapy) were 2.8
months (95% CI, 2.3–4.1) and 11.1 months (95% CI,
8.9–13.7), respectively. The 6-month and 12-month PFS
rates were 23.0% (95% CI, 14.7–32.4%) and 13.0% (95%
CI, 6.6–21.6%), respectively, and the 12-month OS rate
was 46.2% (95% CI, 35.6–56.1%) (Fig. 2a and b). In
patients from Asian and non-Asian countries, median
OS was 12.4 months (95% CI, 9.7–20.0) and 9.4 months Two patients had prolonged shrinkage in target
lesions with avelumab after documented progressive
disease (due to >30% increase in target lesion size vs
baseline at weeks 7–13 in 1 patient and a new lesion in
the
other
patient),
suggestive
of
pseudoprogression. Chung et al. Journal for ImmunoTherapy of Cancer (2019) 7:30 Chung et al. Antitumor activity: 1 L-mn subgroup Journal for ImmunoTherapy of Cancer Page 6 of 10 100
90
80
70
60
50
40
30
20
10
0
0
2
4
6
8
10
12
14
16
Time since treatment initiation (months)
Progression-free survival (%)
18
20
22
24
26
28
30
32
34
36
90
Number at risk
55
36
17
13
8
8
5
5
5
5
5
5
4
4
4
4
2
0
Median PFS: 2.8 months (95% CI, 2.3–4.1)
6-month PFS rate: 23.0% (95% CI, 14.7–32.4%)
12-month PFS rate: 13.0% (95% CI, 6.6–21.6%)
100
90
80
70
60
50
40
30
20
10
0
0
2
4
6
8
10
12
14
16
Time since treatment initiation (months)
Overall survival (%)
18
20
22
24
26
28
30
32
34
38
36
90
83
78
70
60
49
41
33
29
26
24
19
18
15
11
11
10
7
0
3
Number at risk
Median OS: 11.1 months (95% CI, 8.9–13.7)
12-month OS rate: 46.2% (95% CI, 35.6–56.1%)
100
90
80
70
60
50
40
30
20
10
0
0
2
4
6
8
10 12 14 16
Time since prior treatment initiation (months)
Overall survival (%)
18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48
85
Number at risk
85 85 83 75 72 63 56 49 44 36 30 26 22 22 18 16 15 14 12
9
6
4
2
0
Median OS: 18.7 months (95% CI, 15.4–20.6)
12-month OS rate: 74.1% (95% CI, 63.4–82.1%)
24-month OS rate: 31.7% (95% CI, 22.2–41.7%)
100
90
80
70
60
50
40
30
20
10
0
0
2
4
6
8
10
12
14
16
Time since treatment initiation (months)
Progression-free survival (%)
18
Median PFS: 1.4 months (95% CI, 1.3–1.5)
6-month PFS rate: 7.9% (95% CI, 2.6–17.2%)
12-month PFS rate: 2.0% (95% CI, 0.2–9.1%)
20
22
24
26
28
30
32
34
36
60
Number at risk
19
12
4
3
3
1
1
1
0
0
0
0
0
0
0
0
0
0
100
90
80
70
60
50
40
30
20
10
0
0
2
4
6
8
10
12
14
16
Time since treatment initiation (months)
Overall survival (%)
18
20
22
24
26
28
30
32
34
36
38
60
54
40
33
24
18
13
11
10
8
8
8
6
5
3
3
1
1
1
0
Number at risk
Median OS: 6.6 months (95% CI, 5.4–9.4)
12-month OS rate: 25.6% (95% CI, 14.9–37.6%)
A
C
B
D
E
Fig. Antitumor activity: 1 L-mn subgroup e OS in the 2 L subgroup (n = 60) 100
90
80
70
60
50
40
30
20
10
0
0
2
4
6
8
10
12
14
16
Time since treatment initiation (months)
Progression-free survival (%)
18
20
22
24
26
28
30
32
34
36
90
Number at risk
55
36
17
13
8
8
5
5
5
5
5
5
4
4
4
4
2
0
Median PFS: 2.8 months (95% CI, 2.3–4.1)
6-month PFS rate: 23.0% (95% CI, 14.7–32.4%)
12-month PFS rate: 13.0% (95% CI, 6.6–21.6%)
A 100
90
80
70
60
50
40
30
20
10
0
0
2
4
6
8
10 12 14 16
Time since prior treatment initiation (months)
Overall survival (%)
18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48
85
Number at risk
85 85 83 75 72 63 56 49 44 36 30 26 22 22 18 16 15 14 12
9
6
4
2
0
Median OS: 18.7 months (95% CI, 15.4–20.6)
12-month OS rate: 74.1% (95% CI, 63.4–82.1%)
24-month OS rate: 31.7% (95% CI, 22.2–41.7%)
C C A 90
55
36
17
13
8
8
5
5
5
5
5
5
4
4
4
4
2
0
100
90
80
70
60
50
40
30
20
10
0
0
2
4
6
8
10
12
14
16
Time since treatment initiation (months)
Overall survival (%)
18
20
22
24
26
28
30
32
34
38
36
90
83
78
70
60
49
41
33
29
26
24
19
18
15
11
11
10
7
0
3
Number at risk
Median OS: 11.1 months (95% CI, 8.9–13.7)
12-month OS rate: 46.2% (95% CI, 35.6–56.1%)
B 100
D 100
90
80
70
60
50
40
30
20
10
0
0
2
4
6
8
10
12
14
16
Time since treatment initiation (months)
Progression-free survival (%)
18
Median PFS: 1.4 months (95% CI, 1.3–1.5)
6-month PFS rate: 7.9% (95% CI, 2.6–17.2%)
12-month PFS rate: 2.0% (95% CI, 0.2–9.1%)
20
22
24
26
28
30
32
34
36
60
Number at risk
19
12
4
3
3
1
1
1
0
0
0
0
0
0
0
0
0
0
D B Median PFS: 1.4 months (95% CI, 1.3–1.5)
6-month PFS rate: 7.9% (95% CI, 2.6–17.2%)
12-month PFS rate: 2.0% (95% CI, 0.2–9.1%) 90
83
78
70
60
49
41
33
29
26
24
19
18
15
11
11
10
7
0
3
60
19
12
4
3
3
1
1
1
0
0
0
0
0
0
0
0
0
0
100
90
80
70
60
50
40
30
20
10
0
0
2
4
6
8
10
12
14
16
Time since treatment initiation (months)
Overall survival (%)
18
20
22
24
26
28
30
32
34
36
38
60
54
40
33
24
18
13
11
10
8
8
8
6
5
3
3
1
1
1
0
Number at risk
Median OS: 6.6 months (95% CI, 5.4–9.4)
12-month OS rate: 25.6% (95% CI, 14.9–37.6%)
E
Fig. Antitumor activity: 1 L-mn subgroup 2 Kaplan–Meier estimates of progression-free survival (PFS) and overall survival (OS). a PFS from start of avelumab in the first-line switch-
maintenance (1 L-mn) subgroup (n = 90). b OS from start of avelumab in the 1 L-mn subgroup (n = 90). c OS from start of 1 L chemotherapy in
the 1 L-mn subgroup (n = 90). d PFS in the second-line (2 L) subgroup (n = 60). Antitumor activity: 1 L-mn subgroup 2 Kaplan–Meier estimates of progression-free survival (PFS) and overall survival (OS). a PFS from start of avelumab in the first-line switch-
maintenance (1 L-mn) subgroup (n = 90). b OS from start of avelumab in the 1 L-mn subgroup (n = 90). c OS from start of 1 L chemotherapy in
the 1 L-mn subgroup (n = 90). d PFS in the second-line (2 L) subgroup (n = 60). e OS in the 2 L subgroup (n = 60) 100
E Antitumor activity: 2 L subgroup (95% CI, 7.4–13.7), respectively. Median OS measured
from start of 1 L chemotherapy was 18.7 months (95%
CI, 15.4–20.6) overall (Fig. 2c), and 20.6 months (95%
CI, 17.1–28.1) and 15.8 months (95% CI, 12.3–19.9) in
patients from Asian and non-Asian countries, respect-
ively. In PD-L1+ and PD-L1−subgroups, median PFS
was 3.0 (95% CI, 1.4–4.1) and 2.7 (95% CI, 1.4–3.6) months
(hazard ratio [HR], 0.844 [95% CI, 0.505–1.411]) and
median OS was 15.9 (95% CI, 11.4–20.7) and 10.4 (95% CI,
8.3–12.4) months (HR, 0.588 [95% CI, 0.342–1.009]),
respectively (Additional file 1: Figures S3A and S4A). The ORR was 6.7% (95% CI, 1.8–16.2%; PR in 4 patients),
and the disease control rate was 28.3% (13 patients [21.7%]
had SD as best response) (Additional file 1: Table S2). Me-
dian time to and duration of response were 2.0 months
(IQR, 1.3–2.7) and 3.5 months (95% CI, 2.8–8.3), respect-
ively. An estimated 25.0% (95% CI, 0.9–66.5%) of responses
lasted ≥6 months (Fig. 1b and Additional file 1: Figure S1B). Of 52 evaluable patients, 7 (13.5%) had target lesion shrink-
age of ≥30% and 16 (30.8%) had shrinkage of any level
(Additional file 1: Figure S2B). Median PFS was 1.4 months The ORR was 6.7% (95% CI, 1.8–16.2%; PR in 4 patients),
and the disease control rate was 28.3% (13 patients [21.7%]
had SD as best response) (Additional file 1: Table S2). Me-
dian time to and duration of response were 2.0 months
(IQR, 1.3–2.7) and 3.5 months (95% CI, 2.8–8.3), respect-
ively. An estimated 25.0% (95% CI, 0.9–66.5%) of responses
lasted ≥6 months (Fig. 1b and Additional file 1: Figure S1B). Of 52 evaluable patients, 7 (13.5%) had target lesion shrink-
age of ≥30% and 16 (30.8%) had shrinkage of any level
(Additional file 1: Figure S2B). Median PFS was 1.4 months Chung et al. Antitumor activity: 1 L-mn subgroup Journal for ImmunoTherapy of Cancer (2019) 7:30 Page 7 of 10 Table 2 Any-grade TRAEs occurring in ≥10% of patients or grade ≥3 in any patient and infusion-related reactions in the first-line
switch-maintenance or second-line subgroup
Patients, n (%)
1 L-mn subgroup (n = 90)
2 L subgroup (n = 60)
Any grade
Grade 3
Grade 4
Grade 5
Any grade
Grade 3
Grade 4
Grade 5
Any TRAEa
57 (63.3)
7 (7.8)
0
1 (1.1)
28 (46.7)
3 (5.0)
2 (3.3)
0
Fatigue
10 (11.1)
2 (2.2)
0
0
5 (8.3)
0
0
0
Decreased appetite
3 (3.3)
0
0
0
2 (3.3)
1 (1.7)
0
0
Asthenia
1 (1.1)
0
0
0
3 (5.0)
2 (3.3)
0
0
Colitis
2 (2.2)
1 (1.1)
0
0
0
0
0
0
Elevated amylase
2 (2.2)
0
0
0
1 (1.7)
1 (1.7)
0
0
Elevated lipase
2 (2.2)
1 (1.1)
0
0
1 (1.7)
0
1 (1.7)
0
Elevated γ-glutamyltransferase
2 (2.2)
1 (1.1)
0
0
0
0
0
0
Anemia
1 (1.1)
1 (1.1)
0
0
1 (1.7)
1 (1.7)
0
0
Decreased platelet count
1 (1.1)
1 (1.1)
0
0
1 (1.7)
0
1 (1.7)
0
Abdominal pain
1 (1.1)
1 (1.1)
0
0
0
0
0
0
Adrenal insufficiency
1 (1.1)
0
0
0
1 (1.7)
1 (1.7)
0
0
Autoimmune hepatitisb
1 (1.1)
0
0
1 (1.1)
0
0
0
0
Decreased hemoglobin
1 (1.1)
1 (1.1)
0
0
0
0
0
0
Hepatic failureb
1 (1.1)
0
0
1 (1.1)
0
0
0
0
Hyperglycemia
1 (1.1)
1 (1.1)
0
0
0
0
0
0
Hypokalemia
1 (1.1)
1 (1.1)
0
0
0
0
0
0
Peripheral motor neuropathy
1 (1.1)
1 (1.1)
0
0
0
0
0
0
Infusion-related reactionc
20 (22.2)
1 (1.0)
0
0
11 (18.3)
0
0
0
aThe incidence of treatment-related infusion-related reaction based on the single MedDRA preferred term is not listed
bOccurred in the same patient
cIncludes adverse events categorized as infusion-related reaction, drug hypersensitivity, or hypersensitivity reaction that occurred on the day of infusion or day
after infusion, in addition to signs and symptoms of infusion-related reaction that occurred on the same day of infusion and resolved within 2 days (including
adverse events classified by investigators as related or unrelated to treatment)
1 L-mn first-line switch-maintenance, 2 L second line, TRAE treatment-related adverse event Table 2 Any-grade TRAEs occurring in ≥10% of patients or grade ≥3 in any patient and infusion-related reactions in the first-line
switch-maintenance or second-line subgroup Table 2 Any-grade TRAEs occurring in ≥10% of patients or grade ≥3 in any patient and infusion-related reactions in the first-line
i h
i
d li
b Occurred in the same patient
cIncludes adverse events categorized as infusion-related reaction, drug hypersensitivity, or hypersensitivity reaction that occurred on the day of infusion or day
after infusion, in addition to signs and symptoms of infusion-related reaction that occurred on the same day of infusion and resolved within 2 days (including
adverse events classified by investigators as related or unrelated to treatment)
1 L-mn first-line switch-maintenance 2 L second line TRAE treatment-related adverse event y
g
)
1 L-mn first-line switch-maintenance, 2 L second line, TRAE treatment-related adverse event (6.0%), and pruritus (5.3%). Antitumor activity: 1 L-mn subgroup Thirteen patients (8.7%) had
a grade ≥3 TRAE (1 L-mn subgroup, 8 [8.9%]; 2 L
subgroup, 5 [8.3%]), most commonly fatigue, asthenia,
anemia, and elevated lipase (n = 2 each; 1.3%). One
patient (0.7%) had a grade 3 infusion-related reaction
(1 L-mn subgroup). Two patients (both 2 L subgroup) had
a grade 4 TRAE: elevated lipase (n = 1) and decreased
platelet count (n = 1). Overall, 81 patients (54.0%) had a
SAE, which was related to treatment in 6 patients (4.0%; 3
in each subgroup). One treatment-related death occurred
(1 L-mn subgroup) in a patient with peritoneal metastases
and ascites at study entry who developed grade 5
autoimmune hepatitis and hepatic failure. Twenty-three
patients (15.3%) had an immune-related AE, including
grade ≥3 in 3 patients (2.0%): colitis (grade 3, 1 L-mn),
autoimmune hepatitis/hepatic failure (grade 5, 1 L-mn),
and adrenal insufficiency (grade 3, 2 L). Avelumab was
permanently discontinued following a TRAE in 8 patients
(5.3% overall; 1 L-mn: 6 [6.7%]; 2 L: 2 [3.3%]), of whom 3
(2.0%)
discontinued
because
of
an
infusion-related
reaction. (95% CI, 1.3–1.5), and 6-month and 12-month PFS rates
were 7.9% (95% CI, 2.6–17.2%) and 2.0% (95% CI, 0.2–
9.1%), respectively (Fig. 2d). Median OS was 6.6 months
(95% CI, 5.4–9.4), and the 12-month OS rate was 25.6%
(95% CI, 14.9–37.6%) (Fig. 2e). In patients from Asian and
non-Asian countries, median OS was 8.3 months (95% CI,
2.0–10.4) and 6.3 months (95% CI, 4.3–9.4), respectively. No
significant difference was seen in PFS and OS based on
PD-L1 status (Additional file 1: Figures S3B and S4B). (95% CI, 1.3–1.5), and 6-month and 12-month PFS rates
were 7.9% (95% CI, 2.6–17.2%) and 2.0% (95% CI, 0.2–
9.1%), respectively (Fig. 2d). Median OS was 6.6 months
(95% CI, 5.4–9.4), and the 12-month OS rate was 25.6%
(95% CI, 14.9–37.6%) (Fig. 2e). In patients from Asian and
non-Asian countries, median OS was 8.3 months (95% CI,
2.0–10.4) and 6.3 months (95% CI, 4.3–9.4), respectively. No
significant difference was seen in PFS and OS based on
PD-L1 status (Additional file 1: Figures S3B and S4B). Occurred in the same patient
cIncludes adverse events categorized as infusion-related reaction, drug hypersensitivity, or hypersensitivity reaction that occurred on the day of infusion or day
after infusion, in addition to signs and symptoms of infusion-related reaction that occurred on the same day of infusion and resolved within 2 days (including
adverse events classified by investigators as related or unrelated to treatment)
1 L mn first line switch maintenance 2 L second line TRAE treatment related adverse event aThe incidence of treatment-related infusion-related reaction based on the single MedDRA preferred term is not listed
bOccurred in the same patient Discussion were initiated. In a randomized phase 3 trial of nivolumab
vs placebo as third-line or later treatment in Asian patients
with GC/GEJC (n = 493), the ORR was 11.2% vs 0%
(P<.0001), median PFS was 1.6 vs 1.5 months (P<.0001),
and median OS was 5.3 vs 4.1 months (P<.0001), respect-
ively [13]. However, to date no improvement in OS has
been shown in studies comparing single-agent checkpoint
inhibitors with chemotherapy, such as trials of 2 L pem-
brolizumab
vs
paclitaxel
(KEYNOTE-061)
[29]
and
third-line avelumab vs physician choice of chemotherapy
(JAVELIN Gastric 300) [30]. Results from phase 3 trials
assessing alternative anti–PD-1/PD-L1–based regimens in
the 1 L setting, such as switch-maintenance (sequential)
or combination (concurrent) approaches, are needed. In this single-arm phase 1b cohort of 150 patients with
previously
treated
advanced
GC/GEJC,
avelumab
showed evidence of durable antitumor activity as 1 L-mn
and 2 L therapy. The ORR was 6.7% in both subgroups,
although
median
durations
of
response
were
21.4
months in the 1 L-mn subgroup and 3.5 months in the 2
L subgroup. Remarkably, 2 patients (2.2%) in the 1 L-mn
subgroup had a CR after achieving only SD on prior
chemotherapy. Avelumab
showed
a
tolerable
safety
profile, including a low rate of grade ≥3 TRAEs (8.7%)
and immune-related AEs (any grade, 15.3%; grade ≥3,
2.0%), similar to observations in other tumor types [21]. Detailed
guidance
for
recognizing
and
managing
immune-related AEs with this class of agents have been
published by consensus groups [22, 23]. The incidence
of TRAEs of any grade was higher in the 1 L-mn sub-
group compared with the 2 L subgroup (63.3% vs
46.7%), which may be due to the longer treatment dur-
ation and shorter interval from end of prior chemother-
apy to start of avelumab in the 1 L-mn subgroup,
although the incidence of grade ≥3 TRAEs was similar in
both subgroups (8.9% vs 8.3%, respectively). pp
Available data indicate that the benefits seen with
anti–PD-1/PD-L1 antibodies in GC/GEJC may be lim-
ited to a small proportion of patients. Thus, predictive
biomarkers to identify subpopulations more likely to
respond to immunotherapy are a focus of ongoing
research [10]. In this study, clinical activity was seen
both in PD-L1+ and PD-L1−tumors including similar
ORR and PFS and a nonsignificant trend in the 1 L-mn
subgroup for longer OS in PD-L1+ tumors. Discussion It should be
noted that the PD-L1 assay used in this study differs
from those used in studies of other approved anti–PD-1/
PD-L1 antibodies. Also, in the present study, PD-L1
status was based solely on tumor cell expression,
whereas in studies of pembrolizumab in patients with
GC/GEJC, in which antitumor activity was associated
with PD-L1 expression, PD-L1 status was determined
based
on
expression
on
tumor
or
immune
cells
(combined positive score). In addition, responses to
pembrolizumab in patients with GC/GEJC have been
associated with microsatellite instability–high/mismatch
repair–deficient status and Epstein-Barr virus status [12,
31]. In the
current
trial,
microsatellite status
was
available for only a small number of patients and findings
were inconclusive. Assessment of novel biomarkers is
planned for future avelumab studies in GC/GEJC. g
p (
p
y)
Approximately 70% of patients achieve a response or
SD with standard 1 L chemotherapy [24, 25]; however,
duration of OS is usually short [2, 3]. In the 1 L-mn sub-
group, median PFS was 2.8 months (6-month rate,
23.0%), median OS measured from the start of avelumab
was 11.1 months (12-month rate, 46.2%), and median
OS measured from the start of prior chemotherapy was
18.7 months. Thus, the OS seen in the 1 L-mn subgroup,
which enrolled patients without disease progression fol-
lowing chemotherapy, is encouraging for this subgroup
of patients. Administering immunotherapy sequentially
after completion of 1 L chemotherapy may enhance the
immunostimulatory effects of chemotherapy while redu-
cing the toxicity that may result when anti–PD-1 anti-
bodies are administered in combination with other
agents (eg, chemotherapy or ipilimumab) [26, 27]. To
further assess this strategy, a randomized phase 3 trial is
comparing avelumab switch-maintenance treatment with
continuation of 1 L platinum-based chemotherapy in
patients with advanced GC/GEJC (JAVELIN Gastric 100;
NCT02625610). Conclusion The data in the present phase 1b study demonstrate that
avelumab administered as maintenance therapy (after
disease control with standard chemotherapy) has antitumor
activity and acceptable safety in patients with advanced
GC/GEJC, supporting further investigations of this
treatment approach. Several early-phase studies assessed anti–PD-1 monother-
apy in patients with chemotherapy-treated (later-line) GC/
GEJC outside of the maintenance setting [12, 13, 27, 28],
and median PFS and OS reported in non–PD-L1–selected
populations were 2.0 months and 5.5–6.2 months, respect-
ively. Survival data for avelumab (anti–PD-L1) in the 2 L
subgroup (median PFS and OS of 1.4 and 6.6 months,
respectively) appear consistent with these studies. Subse-
quently, phase 3 trials assessing later-line treatment with
anti–PD-1/PD-L1 monotherapy in advanced GC/GEJC Safety Across both subgroups, 85 of 150 patients (56.7%) had a
treatment-related AE (TRAE) of any grade, including 57
of 90 (63.3%) in the 1 L-mn subgroup and 28 of 60
(46.7%) in the 2 L subgroup. Patterns of TRAEs were
similar in both subgroups (Table 2 and Additional file 1:
Table S4). Overall, infusion-related reactions and related
symptoms
occurred
in
20.7%. Time
to
onset
of
infusion-related reaction was first or second infusion in
29 of 31 cases (93.5%). Other common TRAEs (any
grade in ≥5%) were fatigue (10.0%), nausea (6.7%), chills Chung et al. Journal for ImmunoTherapy of Cancer (2019) 7:30 Page 8 of 10 Page 8 of 10 Availability of data and materials For all new products or new indications approved in both the European
Union and the United States after January 1, 2014, Merck KGaA, Darmstadt,
Germany will share patient-level and study-level data after deidentification,
as well as redacted study protocols and clinical study reports from clinical tri-
als in patients. These data will be shared with qualified scientific and medical
researchers, upon researcher’s request, as necessary for conducting legitimate
research. Such requests must be submitted in writing to the company’s data
sharing portal. More information can be found at https://www.merckgroup. com/en/research/our-approach-to-research-and-development/healthcare/
clinical-trials/commitment-responsible-data-sharing.html. Where Merck KGaA
has a co-research, co-development or co-marketing/co-promotion agree-
ment or where the product has been out-licensed, it is recognized that the
responsibility for disclosure may be dependent on the agreement between
parties. Under these circumstances, Merck KGaA will endeavour to gain
agreement to share data in response to requests. AstraZeneca; stock and other ownership interests: Bristol-Myers Squibb. y
HTA, JL, SYR, DYO, LW, JRI, SSL, MK, DS, and HS declare that they have no
competing interests. HTA, JL, SYR, DYO, LW, JRI, SSL, MK, DS, and HS 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. Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations. Funding This trial was sponsored by Merck KGaA and is part of an alliance between
Merck KGaA and Pfizer, Inc., New York, NY, USA. Medical writing support was
provided by ClinicalThinking and was funded by Merck KGaA and Pfizer. HX: employment: EMD Serono Inc. (a business of Merck KGaA, Darmstadt,
Germany). JH: employment: EMD Serono Inc. (a business of Merck KGaA, Darmstadt,
Germany). VC: employment: EMD Serono Inc. (a business of Merck KGaA, Darmstadt,
Germany) when the study was performed; current employment at VC: employment: EMD Serono Inc. (a business of Merck KGaA, Darmstadt,
Germany) when the study was performed; current employment at
AstraZeneca; stock and other ownership interests: Bristol-Myers Squibb. HTA, JL, SYR, DYO, LW, JRI, SSL, MK, DS, and HS declare that they have no
competing interests. Abbreviations BM: honoraria: Bristol-Myers Squibb, Merck & Co, Merck KGaA, Novartis,
Roche; consulting or advisory role: Bristol-Myers Squibb, Merck & Co,
Merck KGaA, Novartis, Roche; travel, accommodations, expenses: Bristol-Myers
Squibb, Novartis, Roche. 1 L-mn: First-line switch-maintenance; 2 L: Second-line; AE: Adverse event;
CR: Complete response; ECOG PS: Eastern Cooperative Oncology Group
performance status; GC: Gastric cancer; GEJC: Gastroesophageal junction cancer;
HR: Hazard ratio; ORR: Objective response rate; OS: Overall survival;
PFS: Progression-free survival; PR: Partial response; SAE: Serious adverse event;
SD: Stable disease; TRAE: Treatment-related adverse event 1 L-mn: First-line switch-maintenance; 2 L: Second-line; AE: Adverse event;
CR: Complete response; ECOG PS: Eastern Cooperative Oncology Group
performance status; GC: Gastric cancer; GEJC: Gastroesophageal junction cancer;
HR: Hazard ratio; ORR: Objective response rate; OS: Overall survival; PFS: Progression-free survival; PR: Partial response; SAE: Serious adverse event;
SD: Stable disease; TRAE: Treatment-related adverse event p
RP: honoraria: Bristol-Myers Squibb, Roche/Genentech; consulting or advisory
role: AstraZeneca, BioMarin, Clovis Oncology, Karus Therapeutics, Mission
Therapeutics, Merck & Co, Novartis, Roche/Genentech, Tesaro, Vertex;
speakers bureau: Novartis; research funding: AstraZeneca/MedImmune, Clovis
Oncology, Eisai, Etherapeutics, Ipsen, Vertex; patents, royalties, other intellectual
property: named on patent for use of PARP inhibitor (rucaparib); travel,
accommodations, expenses: Merck & Co, Roche/Genentech. ABG: employment: EMD Serono Inc. (a business of Merck KGaA, Darmstadt,
Germany) when the study was performed; stock ownership: Halozyme
Therapeutics. Author details
1 1Yonsei Cancer Center, Yonsei University College of Medicine, Yonsei University
Health System, Seoul 03722, South Korea. 2Sarah Cannon Research Institute/
University College London, London, UK. 3Samsung Medical Center,
Sungkyunkwan University School of Medicine, Seoul, South Korea. 4Seoul
National University Hospital, Cancer Research Institute, Seoul National University
College of Medicine, Seoul, South Korea. 5Centrum Onkologii-Instytut im. M. Sklodowskiej Curie, Warszawa, Poland. 6Asan Medical Center, University of Ulsan
College of Medicine, Seoul, South Korea. 7Seoul National University Bundang
Hospital, Seoul National University College of Medicine, Seongnam, South
Korea. 8Sarah Cannon Research Institute/Tennessee Oncology, PLLC, Nashville,
USA. 9Inje University College of Medicine, Busan, South Korea. 10Queens Cancer
Center, Mt Sinai School of Medicine, New York, USA. 11Charité Comprehensive
Cancer Center, Charitéplatz 1, Berlin, Germany. 12Palacky University Medical
School and Teaching Hospital, I.P. Pavlova 6, Olomouc, Czech Republic. 13Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center,
Los Angeles, USA. 14Northern Centre for Cancer Care and Newcastle University,
Freeman Hospital, Newcastle upon Tyne, UK. 15Medical Oncology, Bordeaux
University Hospital, Bordeaux CEDEX, France. 16EMD Serono, Inc, Billerica, USA. 17Brown University, Providence, USA. 18Present address: AstraZeneca
Pharmaceuticals LP, Gaithersburg, USA. Authors’ contributions
P
i i
f
d
i Provision of study materials or patients: HCC, HTA, JL, SYR, DYO, LW, YKK,
KWL, JRI, SSL, MK, UK, BM, AM, RP, DS, HS. Collection and assembly of data:
all authors. Data analysis and interpretation: all authors. Manuscript writing:
all authors. Final approval of manuscript: all authors. Accountable for all
aspects of the work: all authors. Additional file Additional file 1: Table S1. Eligibility criteria. Table S2. Response to
avelumab in the 1 L-mn and 2 L subgroups. Table S3. Best response to
avelumab compared with best response to prior anticancer therapy in
the 1 L-mn subgroup. Table S4. Overall summary of safety. Figure S1. Additional file 1: Table S1. Eligibility criteria. Table S2. Response to
avelumab in the 1 L-mn and 2 L subgroups. Table S3. Best response to
avelumab compared with best response to prior anticancer therapy in
the 1 L-mn subgroup. Table S4. Overall summary of safety. Figure S1. Page 9 of 10 Page 9 of 10 Page 9 of 10 Chung et al. Journal for ImmunoTherapy of Cancer (2019) 7:30 Chung et al. Journal for ImmunoTherapy of Cancer KWL: research funding: AstraZeneca/MedImmune, Daiichi Sankyo, Five Prime
Therapeutics, Green Cross Corp, Macrogenics, Merck KGaA, Merck & Co, Ono
Pharmaceutical, Taiho Pharmaceutical. Time to and duration of response in responding patients. Figure S2. Best
change in sum of target lesion diameters from baseline with avelumab in
evaluable patients. Figure S3. Progression-free survival by PD-L1 expression
status (≥1% tumor cell cutoff) in evaluable patients. Figure S4. Overall
survival by PD-L1 expression status (≥1% tumor cell cutoff) in evaluable
patients. (DOCX 794 kb) Time to and duration of response in responding patients. Figure S2. Best
change in sum of target lesion diameters from baseline with avelumab in
evaluable patients. Figure S3. Progression-free survival by PD-L1 expression
status (≥1% tumor cell cutoff) in evaluable patients. Figure S4. Overall
survival by PD-L1 expression status (≥1% tumor cell cutoff) in evaluable
patients. (DOCX 794 kb) UK: honoraria: AstraZeneca, Bristol-Myers Squibb, Glycotope GmbH, Merck
KGaA, Merck & Co, Novartis, Pfizer, Roche/Genentech; consulting or advisory
role: AstraZeneca, Bristol-Myers Squibb, Merck Serono, Merck & Co, Pfizer;
speakers bureau: AstraZeneca, Bristol-Myers Squibb, Glycotope GmbH, Merck
Serono, MSD Oncology, Novartis; research funding: Pfizer; travel, accommodations,
expenses: AstraZeneca, Merck KGaA, Merck & Co. Acknowledgements The authors thank the patients and their families, investigators, coinvestigators,
and study teams at each of the participating centers and at Merck KGaA,
Darmstadt, Germany, and EMD Serono Research & Development Institute, Inc.,
Billerica, MA, USA (a business of Merck KGaA, Darmstadt, Germany). ABG: employment: EMD Serono Inc. (a business of Merck KGaA, Darmstadt,
Germany) when the study was performed; stock ownership: Halozyme
Therapeutics. 1.
Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, et al.
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NCCN Clinical Practice Guidelines in Oncology. Gastric Cancer. V2.2018.
https://www.nccn.org/professionals/physician_gls/pdf/gastric.pdf. Accessed
15 Oct 2018. Consent for publication
Not applicable. Received: 24 October 2018 Accepted: 13 January 2019 Received: 24 October 2018 Accepted: 13 January 2019 Ethics approval and consent to participate The trial was conducted in accordance with the ethics principles of the
Declaration of Helsinki and the International Council for Harmonisation
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Eli Lilly, GlaxoSmithKline, Merck & Co, Merck KGaA, Ono Pharmaceutical, Taiho
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15 Oct 2018. YKK: consulting or advisory role: Bristol-Myers Squibb, Eli Lilly, Ono Pharmaceutical,
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Metformin-Induced Mitochondrial Complex I Inhibition: Facts, Uncertainties, and Consequences
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Frontiers in endocrinology
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Metformin-Induced Mitochondrial
Complex I Inhibition: Facts,
Uncertainties, and Consequences INSERM, LBFA, Université Grenoble Alpes, Grenoble, France Metformin is the most widely prescribed drug to treat patients with type II diabetes, for
whom retrospective studies suggest that metformin may have anticancer properties. However, in experiments performed with isolated cells, authors have reported both
pro- and anti-apoptotic effects of metformin. The exact molecular mechanism of action
of metformin remains partly unknown despite its use for over 60 years and more
than 17,000 articles in PubMed. Among the various widely recognized or recently
proposed targets, it has been reported consistently that metformin is capable of inhibiting
mitochondrial respiratory chain Complex I. Since most of the effects of metformin
have been replicated by other inhibitors of Complex I, it has been suggested that
the mechanism of action of metformin involved the inhibition of Complex I. However,
compared to conventional Complex I inhibitors, the metformin-induced inhibition of
Complex I has unique characteristics. Among these, the most original one is that the
concentrations of metformin required to inhibit Complex I are lower in intact cells than
in isolated mitochondria. Experiments with isolated mitochondria or Complex I were
generally performed using millimolar concentrations of metformin, while plasma levels
remain in the micromolar range in both human and animal studies, highlighting that
metformin concentration is an important issue. In order to explain the effects in animals
based on observations in cells and mitochondria, some authors proposed a direct effect
of the drug on Complex I involving an accumulation of metformin inside the mitochondria
while others proposed an indirect effect (the drug no longer having to diffuse into the
mitochondria). This brief review attempts to: gather arguments for and against each
hypothesis concerning the mechanism by which metformin inhibits Complex I and to
highlight remaining questions about the toxicity mechanism of metformin for certain
cancer cells. Edited by:
Frederic Bost,
Centre National de la Recherche
Scientifique (CNRS), France Reviewed by:
Gayathri K. Natarajan,
Medical College of Wisconsin,
United States
Adam Mickiewicz,
University in Poznan, Poland *Correspondence:
Eric Fontaine
eric.fontaine@univ-grenoble-alpes.fr *Correspondence:
Eric Fontaine
eric.fontaine@univ-grenoble-alpes.fr Specialty section:
This article was submitted to
Cellular Endocrinology,
a section of the journal
Frontiers in Endocrinology
Received: 27 April 2018
Accepted: 27 November 2018
Published: 17 December 2018
Citation:
Fontaine E (2018) Metformin-Induced
Mitochondrial Complex I Inhibition:
Facts, Uncertainties, and
Consequences. Front. Endocrinol. 9:753. doi: 10.3389/fendo.2018.00753 Specialty section:
This article was submitted to
Cellular Endocrinology,
a section of the journal
Frontiers in Endocrinology Keywords: metformin, mitochondria, Complex I, pharmacokinetic, cell death, cancer, permeability transition Keywords: metformin, mitochondria, Complex I, pharmacokinetic, cell death, cancer, permeability transition MINI REVIEW
published: 17 December 2018
doi: 10.3389/fendo.2018.00753 MINI REVIEW published: 17 December 2018
doi: 10.3389/fendo.2018.00753 INTRODUCTION Received: 27 April 2018
Accepted: 27 November 2018
Published: 17 December 2018 Metformin is a drug with pleiotropic effects. It takes part in glucose homeostasis, mainly by
inhibiting liver glucose production (1). It also modifies the production of reactive oxygen species
and affects cell death processes (2, 3). Most of these effects have been traced to the inhibition
of mitochondrial respiratory chain Complex I for two main reasons: First, over the past 20
years, different laboratories have reproducibly observed that metformin inhibits mitochondrial
respiratory chain Complex I (4–20). Second, these pleiotropic effects have been reproduced by well
identified Complex I inhibitors [gluconeogenesis (21, 22), cell death (18, 23–28)]. METFORMIN PHARMACOKINETICS Metformin is a hydrophilic compound charged positively at
physiological pH. Its hydrophilicity limits its permeability
through lipid membranes. Metformin enters and leaves cells by
the presence of several transporters including Organic Cation
Transporters (OCTs) and multidrug and toxin extrusion (MATE)
transporters (29). This leads to a steady-state concentration
of metformin inside cells, depending on both the amount
and activity of such transporters as well as metformin plasma
concentration. Despite this, many authors have hypothesized that metformin
accumulates in mitochondria (5, 13, 15, 44). This scenario
may reconcile the observation that millimolar concentrations
of metformin are necessary to inhibit Complex I in isolated
mitochondria (see below) while, when used at the therapeutic
dose, the plasma metformin concentration remains in the
micromolar range in both humans and animals (31, 36, 42). The pharmacological inhibition or the genetic ablation of
OCTs reduce the distribution of metformin to the liver, small
intestine and kidney (30–32) while the overexpression of OCT1
in HEK293 and CHO cells increases metformin uptake (30, 33). The pharmacological inhibition or the genetic ablation of MATE1
cause hepatic and kidney accumulation of metformin (32, 34). In humans, the genomic variations of metformin transporters
can affect its pharmacokinetics (concentration, clearance, volume
of distribution) (35, 36) suggesting that such genomic variations
affect metformin concentration in tissues. From a theoretical point of view, this hypothesis is
plausible. Indeed, because the mitochondrial respiratory chain
transfers protons from the matrix to the intermembrane space,
mitochondria build up and maintain an electrical mitochondrial
membrane potential that drives the accumulation of positively
charged molecules into mitochondria, provided the molecule
crosses the membrane. In these conditions, Nernst equation
indicates that for a physiological mitochondrial membrane
potential of −180 mV the thermodynamic equilibrium is reached
after a 1,000-fold accumulation of a positively charged molecule
if the molecule has one charge. Since metformin is a positively
charged molecule and assuming the presence of a still unknown
carrier for metformin in the inner membrane, its mitochondrial
concentration could reach the millimolar range despite a
cytosolic concentration within the micromolar range (see
Figure 1). In addition, assuming a plasma membrane potential
of −36 mV and the absence of kinetic constraints on metformin
transporters (OCT and MATE), the cytosolic concentration of
metformin would be 4 times that of plasma. Citation: Fontaine E (2018) Metformin-Induced
Mitochondrial Complex I Inhibition:
Facts, Uncertainties, and
Consequences. Front. Endocrinol. 9:753. doi: 10.3389/fendo.2018.00753 December 2018 | Volume 9 | Article 753 Frontiers in Endocrinology | www.frontiersin.org 1 Metformin and Mitochondrial Complex I Fontaine the studies (35, 40–42). With a volume of distribution of 1.12 ±
0.08 L/kg in healthy volunteers (40), metformin is not supposed
to accumulate dramatically in tissues. The amount of metformin
in the liver ranges from 2 to 5 times that of plasma -depending on
the studies (32, 35, 42, 43)- and increases up to 10 times that of
plasma in small intestinal walls (32). However, the mechanism by which metformin affects the
activity of Complex I remains debated. In order to clarify
whether the different conclusions found in the literature may
be due to methodological differences, this review compares
results obtained in vivo or with intact cells, to results obtained
with isolated mitochondria or isolated Complex I. In this last
case, authors tend to assume that metformin accumulates in
mitochondria, here we will discuss evidence supporting or not
this assumption. Finally, since pro- and anti-apoptotic effects of
metformin are observed in intact cells, we will examine the role of
metformin concentrations as a potential cause of these conflicting
observations. Thus, the pharmacokinetic studies indicate that metformin
enters but does not accumulate in large amounts in cells. Whether
its metabolic activity depends on its diffusion inside the cells is
supported by several but not all studies. Once in the cell, as metformin inhibits Complex I it
is
tempting
to
speculate
that
metformin
penetrates
the
mitochondria. The composition of the mitochondrial matrix
(the space delimited by the inner mitochondrial membrane)
is different from that of the cytosol. In order to maintain such
a different metabolite composition, the inner membrane is
impermeable to almost all hydrophilic molecules which enter or
leave the mitochondria through specific transporters. Among the
numerous recognized mitochondrial carriers, no specific carrier
for metformin has been identified yet. Frontiers in Endocrinology | www.frontiersin.org December 2018 | Volume 9 | Article 753 METFORMIN PHARMACOKINETICS Whether the activities of the metformin transporters (i.e., the
metformin concentration in tissues) affect the metabolic effects
of metformin is not systematically reported in the literature. On
the one hand, metformin failed to reduce fasting plasma glucose
concentration in OCT1-knockout mice submitted to a high-
fat diet for 8 weeks and failed to suppress glucagon-stimulated
glucose production in OCT1−/−hepatocytes (30). On the other
hand, the effect of metformin on glucose tolerance tests was
similar in animal controls and OCT1/2-knockout animals (31). A broad variation in clinical efficacy of metformin has long
been recognized as well as a reduced function polymorphism of
OCT1 in humans. However, if some authors reported a decreased
effect of metformin in type-2 diabetes patients carrying reduced
function polymorphism of OCT1 (30, 36), others did not observe
such a correlation (37, 38). However, the hypothesis that metformin accumulates in
mitochondria contradicts several observations. First of all, the accumulation of numerous positive charges in
the matrix compensated by proton extrusion by the respiratory
chain, should lead to a collapse of mitochondrial membrane
potential associated with an increase in delta pH. However, note
that metformin did not depolarize isolated mitochondria (8). To the best of my knowledge, no study correlating metformin
concentration in tissue (or cells) and metformin-induced
Complex I inhibition was ever published. Secondly, assuming that the total mitochondrial volume
represents approximately 20% of hepatocytes, a 1,000-fold
accumulation of metformin inside mitochondria would represent
an approximately 200-fold accumulation of metformin in liver Drugs that are extensively sequestered in organelles have a
very large apparent volume of distribution and a prolonged half-
life in vivo (39). Metformin is not metabolized and is secreted by
the kidneys with a half-life of 1.74–7.3 h in humans depending on December 2018 | Volume 9 | Article 753 2 Fontaine Metformin and Mitochondrial Complex I FIGURE 1 | Mechanism of action of metformin on complex I: the direct hypothesis and the indirect hypothesis. Metformin enters cells via Organic Cation Transporters
(OCTs) and leaves cells via multidrug and toxin extrusion (MATE) transporters. Assuming a plasma membrane potential of 35 mV and a passive mechanism of OCTs
and MATE, the cytosolic metformin concentration is expected to be 4 times that of the plasma concentration. METFORMIN PHARMACOKINETICS In the direct hypothesis (left), metformin is supposed to
enter mitochondria via a hypothetical carrier reaching a matrix concentration 1,000 times that of the cytosol for mitochondrial membrane potential of 180 mV. In the
indirect hypothesis (right), metformin does not enter mitochondria but stimulates a hypothetical signaling pathway that eventually modifies Complex I conformation,
making it less active. FIGURE 1 | Mechanism of action of metformin on complex I: the direct hypothesis and the indirect hypothesis. Metformin enters cells via Organic Cation Transporters
(OCTs) and leaves cells via multidrug and toxin extrusion (MATE) transporters. Assuming a plasma membrane potential of 35 mV and a passive mechanism of OCTs
and MATE, the cytosolic metformin concentration is expected to be 4 times that of the plasma concentration. In the direct hypothesis (left), metformin is supposed to
enter mitochondria via a hypothetical carrier reaching a matrix concentration 1,000 times that of the cytosol for mitochondrial membrane potential of 180 mV. In the
indirect hypothesis (right), metformin does not enter mitochondria but stimulates a hypothetical signaling pathway that eventually modifies Complex I conformation,
making it less active. compounds in the mitochondrial matrix at a concentration in
the millimolar range. If metformin accumulated spontaneously
in the mitochondria, the addition of molecules targeting the
mitochondria would be unnecessary, which is clearly not the case. (without accounting for accumulation in the cytosol). Such an
accumulation is 2 orders of magnitude higher than that measured
by several groups (32, 35, 42, 43). Thirdly, a large mitochondrial accumulation is not compatible
with the low volume of distribution of metformin and its short
half-live (see above). Fourthly, using radioactive [14C] metformin, the radioactivity
was not found to accumulate in liver mitochondria of rats
treated orally with metformin (45) and no radioactivity was
measured inside mitochondria when Xenopus laevis oocytes were
exposed to concentrations of metformin that led to Complex
I inhibition (6). Importantly, Complex I remained inhibited
after mitochondrial isolation. Although this result does not
definitively exclude a possible accumulation of metformin in
mitochondria as a cause of Complex I inhibition (accumulated
metformin may diffuse during the isolation procedure), it rules
out the hypothesis that the inhibition of Complex I by metformin
requires metformin inside mitochondria. Frontiers in Endocrinology | www.frontiersin.org CHARACTERISTICS OF COMPLEX I
INHIBITION ACCORDING TO THE MODELS
USED Although this may seem odd, it has been reported by
several different laboratories that the concentrations required to
inhibit Complex I are lower for intact cells than for isolated
mitochondria (4–6, 13, 19). Note however that the characteristics
of Complex I inhibition reveals some differences depending on
whether metformin acts on intact cells (animal models, infused
organs, isolated cells) or directly on isolated mitochondria or
isolated Complex I (see Table 1). In summary, unlike the less hydrophilic biguanides (46),
the accumulation of metformin inside the mitochondria is not
supported by direct measurements, is not consistent with the
pharmacokinetic data, and would require a transporter that has
not yet been discovered. The incubation of isolated Complex I or submitochondrial
particles in the presence of millimolar concentrations of
metformin leads to an inhibition of Complex I that can be
complete (13) with an IC50 ranging from 19 to 79 mM depending
on laboratories (5, 9, 13). It should be noted that there is no
membrane potential in these particular conditions of incubation,
thus no possibility of metformin accumulation. In other words,
the concentrations tested are the actual concentrations to which
Complex I is exposed. Derivatives combining a molecule of metformin at different
alkyl chain lengths containing a triphenylphosphonium cation
(a liposoluble cation known to accumulate in mitochondria
according to membrane potential) have been synthesized (47,
48) in order to increase the anti-cancer effect of metformin
(see below). These different compounds accumulate in cells
(47), depolarize mitochondria (48) and inhibit Complex I with
an IC50 in the micromolar range (47, 48), which according
to Nernst equation is consistent with the accumulation of The incubation of isolated mitochondria in the presence
of millimolar concentrations of metformin leads to a rather
fast (within a few minutes) inhibition of Complex I with an
“apparent” IC50 also in the millimolar range (5). December 2018 | Volume 9 | Article 753 3 Metformin and Mitochondrial Complex I Fontaine TABLE 1 | Main differences in the characteristics of Complex I inhibition according to the model used. CHARACTERISTICS OF COMPLEX I
INHIBITION ACCORDING TO THE MODELS
USED Live animal, perfused organ, intact cells
Isolated mitochondria, isolated Complex I
References
References
Speed of inhibition
Time dependent
(4) (5)
Immediate (minutes)
(14) (13)
Type of inhibition
Partial
(4) (5) (18)
Total
(13)
Affinity
Apparent IC50 approximately 1 mM after
30 min in isolated rat hepatocytes
Apparent IC50 250 µM and 330 µM after
6 h incubation in 143B and HepG2 cells
respectively
(4)
(13)
Apparent IC50 15 mM in isolated
mitochondria
IC50 79 mM in sub-mitochondrial particles
IC50 66 mM in immunocaptured Complex I
IC50 19 mM in isolated Complex I
(5)
(5)
(9)
(13)
Inhibition in State-3? Yes
(5) (11) (12)
(14) (15)
Yes
(5) (8) (9) (10)
(12) (14) (15)
Inhibition in State-4? Yes
(4) (7) (14)
No
(4) (8) (10) (15)
Inhibition after
uncoupling
Yes
(4) (7) (11) (14)
(19)
No
(4) (15) (19)
Inhibition after the
removal of metformin
Yes
(4) (14) (20)
No for metformin concentration ≤2 mM
(13)
NADH/NAD+
Increases
(4) (5) (18)
Decreases
(19) metformin concentration (hours are required for micromolar
concentrations of metformin) (5, 50). Contrary to what is
observed in isolated Complex I, the inhibition is not total and
plateaus at approximately 40% of the Vmax (4). Consistent with
a pure effect on Complex I, the inhibition leads to an increase in
the NADH/NAD+ ratio (as assessed by the Lactate/pyruvate and
3-hydroxybutyrate/ acetoacetate ratios) (4, 5). Importantly, once
cells are permeabilized (i.e., once mitochondria can be studied as
if they were isolated) the inhibition is observed in State 3, but
also in State 4 and after uncoupling (4, 11, 19). Finally, Complex
I remains inhibited in mitochondria isolated from either rat
exposed to metformin or liver perfused with metformin, even
after uncoupling (4, 14) or when NADH:quinone oxidoreductase
activity (i.e., Complex I activity) is studied directly using broken
mitochondria (4). Note that the isolation procedure removes
most of (if not all) the free metformin, while uncoupling (either
chemical or after inner membrane rupture) would release the
putative accumulated metformin. Although these results do
not exclude a possible binding of metformin in mitochondrial
membrane, they rule out the hypothesis that the inhibition of
Complex I by metformin could depend on membrane potential. This
observation
is
not
easily
reconcilable
with
the
proposal
that
metformin
accumulates
in
mitochondria. CHARACTERISTICS OF COMPLEX I
INHIBITION ACCORDING TO THE MODELS
USED Indeed,
assuming
that
metformin
did
accumulate
in
mitochondria, Complex I inhibition would have been observed
at micromolar concentrations of metformin (corresponding
to millimolar concentrations inside mitochondria), which
has not been reported. One could argue that at millimolar
concentrations of metformin, the inhibition of Complex
I
would
depolarize
mitochondria,
preventing
metformin
accumulation. However, it has been shown that millimolar
concentrations
of
metformin
did
not
depolarize
isolated
mitochondria (8). In
these
particular
conditions
of
incubation
(isolated
mitochondria
exposed
to
millimolar
concentrations
of
metformin), it has to be noted that the inhibition of Complex I
is observed almost exclusively during ATP synthesis (also called
State 3) and disappears when mitochondria are depolarized
(uncoupled State) or at rest (also called State 4) (4, 8, 15). Such
behavior is not observed with rotenone (the reference inhibitor
of Complex I) but is typical of biguanide-induced inhibition of
Complex I in isolated mitochondria (49). It has been proposed that the reason why the inhibition is
not observed after uncoupling might be due to the fact that the
driving force for metformin accumulation within mitochondria
disappears in these particular conditions. Although, as stated
above, the accumulation of metformin in mitochondria is not
supported by any evidence, this hypothesis does not explain the
lack of inhibition in State 4, a situation in which the driving force
(the membrane potential) is higher than in State 3. Frontiers in Endocrinology | www.frontiersin.org OTHER MITOCHONDRIAL EFFECTS OF
METFORMIN In intact cells the inhibition of oxygen consumption is strictly
located on Complex I. This conclusion comes from the
observation that metformin has no effect on oxygen consumption
when electrons feed the respiratory chain downstream Complex
I (using succinate for example) regardless of the respiratory State
(3, 4 and uncoupled) (4). Curiously, it has been reported that the inhibition of oxygen
consumption in isolated mitochondria is accompanied by NADH
oxidation (19). This observation is not expected in case of a
simple Complex I inhibition, suggesting an uncoupling effect of
metformin in this particular condition. On the contrary, using isolated mitochondria and millimolar
concentrations of metformin, some authors reported inhibitory
effects on complexes III and IV (16). High concentrations of
metformin have been reported to inhibit ATP hydrolysis but not The incubation of intact cells in the presence of metformin
leads to a slower inhibition of Complex I depending on December 2018 | Volume 9 | Article 753 Frontiers in Endocrinology | www.frontiersin.org 4 Metformin and Mitochondrial Complex I Fontaine ATP synthesis (13), suggesting an unconventional effect on the
ATP synthase. ATP synthesis (13), suggesting an unconventional effect on the
ATP synthase. ATP synthesis (13), suggesting an unconventional effect on the
ATP synthase. The first one (in chronological order, but second in
popularity) proposes that in vivo and in intact cells, metformin
triggers a signaling pathway that in turn induces the inhibition
of Complex I (4). Although such a signaling pathway is
yet unknown, it has been reported that Complex I exists
in
two
different
functional
conformations
(active
and
inactive) (52), while reactive thiols of several Complex I
subunits have been identified as targets for post-translational
modifications
(53,
54). However,
whether
metformin
affects reactive thiols in Complex I has not been published
yet. Some evidence suggests that Complex I can interact with
ATP synthase (51). So we may infer that in this particular
condition of incubation (isolated mitochondria exposed to
millimolar concentrations of metformin), ATP synthesis possibly
sensitizes Complex I to metformin. Although speculative, this
personal suggestion could account for the observation that
millimolar concentrations of metformin inhibit Complex I
almost exclusively in State 3. In summary, on intact cells metformin acts slowly but the
effect is visible at micromolar concentrations. The inhibition
affects only Complex I in all the respiratory states and does
not depend on mitochondrial membrane potential. WHERE DOES METFORMIN ACT ON
COMPLEX I? Apparently contradictory effects are found in the literature
regarding the effects of metformin on cell death. Some authors
have put forward its protective effects against cell death (3) while
others have reported its induction of cell death especially in
cancer cells (2). Yet, all of them have concluded that the observed
effects are due to the inhibition of Complex I (see below). The respiratory chain is a sequence of redox reactions which
couple an electron flux with a vectorial transfer of protons. Mammalian respiratory chain complex I is a large protein
complex with at least 45 subunits. It includes a hydrophobic part
embedded in the inner membrane involved in proton transfer
and a hydrophilic part protruding into the matrix in which
electrons pass from NADH to ubiquinone via a succession of
redox reactions. Complex I inhibitors rotenone and piericidin
bind at, or close to, the ubiquinone binding site, inhibiting
both electron flux and proton extrusion. Using artificial electron
acceptors, a rotenone-insensitive NADH oxidation which is not
coupled with proton pumping (i.e., a non-physiological pathway)
can occur in Complex I. OTHER MITOCHONDRIAL EFFECTS OF
METFORMIN On isolated
mitochondria (or isolated Complex I), metformin acts rapidly
but the effect requires millimolar concentrations. The inhibition
does not only affect Complex I and Complex I inhibition is not
observed in all the respiratory states. The second hypothesis necessarily involves an accumulation
of metformin in the mitochondria that would be driven by
mitochondrial membrane potential. Although proposed by
several authors, this hypothesis is not yet supported by any
evidence (see above). METFORMIN PREVENTS CELL DEATH
WHEN IT IS DUE TO PTP OPENING The permeability transition pore (PTP) is a channel located
in the inner membrane normally closed in order to maintain
a high mitochondrial membrane potential required for ATP
synthesis. Once permanently opened, the membrane potential
collapses (55), leading to a drastic inhibition of ATP synthesis. Beyond this uncoupling effect, PTP opening has many other
effects: It allows the thermodynamic equilibrium of the
mitochondrial and cytosolic redox potentials, leading to an
increase in cytosolic NAD(P)H concentration (56). It partly
inhibits Complex I (57), reallocating the electron flux for the
production of reactive oxygen species (58). Finally, it leads to the
release of mitochondrial pro-apoptotic proteins both in isolated
mitochondria (secondary to mitochondrial swelling leading to
the rupture of the outer membrane) (59) and in intact cells (most
probably by a distinct but still unknown mechanism) (56, 60–62). Using isolated Complex I and millimolar concentrations of
metformin, it has been shown that metformin does not inhibit
NADH oxidation due to artificial electron acceptors, behaves
as a non-competitive inhibitor of the physiological electron
pathway and preferentially binds Complex I when the enzyme
is in its “deactive” conformation (13). However, the exact
localization where metformin acts in this condition of incubation
remains unknown. Moreover, the exact mechanism leading to
the inhibition of Complex I in intact cells using micromolar
concentrations of metformin and where exactly it inhibits the
electron flux in Complex I has not been reported. As there are several signaling pathways involved in cell
death, there are many factors activating these pathways. To
discriminate whether a given condition leading to cell death
involves PTP opening or not, experiments are performed in the
presence or absence of a recognized PTP inhibitor (generally
cyclosporine A, but not exclusively). Using this approach,
it has been reproducibly observed that PTP opening occurs
when cell death is triggered by calcium overload or oxidative
stress (63). HYPOTHETICAL MECHANISMS OF
ACTION To account for the fact that the concentration of metformin
required to observe the inhibition of Complex I on whole cells is
lower than the concentration required to observe the inhibition
on mitochondria, two hypotheses have been proposed in the
literature (see Figure 1). December 2018 | Volume 9 | Article 753 Frontiers in Endocrinology | www.frontiersin.org 5 Metformin and Mitochondrial Complex I Fontaine The molecular nature of the PTP has long been a subject
of dispute but recent and compelling data from different
laboratories suggest that the PTP might involve ATP synthase
(51, 64). Surprisingly, the reference Complex I inhibitor rotenone
has been shown to inhibit PTP opening in all the tested cells and
tissues (either spontaneously or in the presence of cyclosporine
A) (23, 65). Although rotenone induces an energetic stress, it
also prevents cell death in the same models as cyclosporine
A (23) and does inhibit Complex I and PTP opening with a
similar concentration dependence (65). Piericidin, another well
recognized Complex I inhibitor also inhibits PTP opening (23). Thus, the activity of Complex I can be said to be a regulator of
PTP opening. Moreover, several ubiquinone analogs (known to
bind with Complex I among others) have been proved to regulate
PTP opening and cell death (57, 66–69). totally prevented by the overexpression of a metformin-resistant
Saccharomyces cerevisiae NADH dehydrogenase NDI1 (15), very
elegantly demonstrating that the toxicity of metformin is due to
its effect on Complex I. The suggestion that metformin’s toxicity is related to an
energetic stress raises several questions: Why is metformin
less toxic in non-cancer cells that are yet more dependent on
mitochondrial ATP production? How can metformin protect
against PTP-induced cell death despite its effect on ATP
production? In other words, what triggers that a same inhibition
of Complex I either prevents or induces cell death? Again, part of the answer could be found in the comparison
of metformin concentrations. While millimolar concentrations
of metformin are generally used to induce cell death in
vitro, micromolar concentrations are sufficient to prevent PTP-
opening induced cell death. Although it has been shown that
cellular energy status is inversely correlated with metformin
concentrations (11, 79), a 24-h incubation with 100 µM
metformin did not affect the AMP/ATP ratio in primary cultured
hepatocytes (11). HYPOTHETICAL MECHANISMS OF
ACTION This suggests that the metformin concentration
used to prevent PTP opening (100 µM, overnight) was not
sufficient to induce a lethal decrease in energy status. On the
contrary, this confirms that the concentrations used to kill cells
dramatically affect the energy status. Note however that some
authors have reported an anti-apoptotic effect even at millimolar
concentrations of metformin, suggesting that some cells are able
to overcome energy stress (75, 80). Knowing that metformin partly inhibits Complex I, we tested
whether it also inhibited PTP opening and related cell death. We
found that, metformin was less potent than rotenone but also
inhibited PTP opening (50). Suggesting a common mechanism
of action with rotenone, the effect of metformin was not additive
with that of rotenone, whereas it was additive with that of
cyclosporine A (65). At present, metformin has been shown
to prevent PTP opening in endothelial cells (50), KB cells
(7), INS-1 insulinoma cells (61), HeLa cells (65), LNCaP cells
(70), A375 cells (70), primary cortical neurons (71) and kidney
mitochondria (72). Accordingly, metformin prevents cell death
induced by oxidative stress in endothelial cells (50) and KB cells
(7), etoposide in primary neurons (71), gentamicin in kidneys
(72), hyperglycemia in endothelial (50) and INS-1 cells (61),
hyperfructosemia in INS-1 cells (61) and ischemia reperfusion in
INS-1 cells (73). Many other works have found a protective effect
of metformin (particularly during oxidative stress or ischemia
reperfusion injury) without having studied the role of the PTP
(18, 74–77). However, if the mechanism by which metformin kills
isolated cells can be traced to a collapse in energy status, the
concentrations that prevent cancer growth in animal models are
in the micromolar range. The practical assumption of metformin
accumulation in mitochondria has obviously been retained, but
one can wonder: why are normal cells preserved? Alternative
or complementary explanations must exist. Among them, it has
been proposed that the effect of metformin in animal models
is indirect (for example due to a decrease in blood insulin
concentration) (2). It is also possible that the accumulation of
metformin or the sensitivity of Complex I to metformin is higher
in cancer cells than in normal tissues (personal hypothesis). As
far as I know, these assumptions have not yet been tested. ANTI-NEOPLASTIC EFFECTS OF
METFORMIN Although PTP opening irremediably leads to cell death, PTP
opening is not mandatory to kill cells as cells can die with a closed
PTP. Although Complex I inhibition prevents PTP opening-
related cell death (see above), it can also induce cell death in
several models. Indeed, it has been repetitively reported that
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metformin in neurodegenerative diseases. Front Endocrinol (Lausanne). (2018) 9:400. doi: 10.3389/fendo.2018.00400 17. Boukalova
S,
Stursa
J,
Werner
L,
Ezrova
Z,
Cerny
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Bezawork-
Geleta A, et al. Mitochondrial targeting of metformin enhances its
activity against pancreatic cancer. Mol Cancer Ther. (2016) 15:2875–86. doi: 10.1158/1535-7163.MCT-15-1021 4. El-Mir MY, Nogueira V, Fontaine E, Averet N, Rigoulet M, Leverve X. Dimethylbiguanide inhibits cell respiration via an indirect effect targeted
on the respiratory chain complex I. J Biol Chem. (2000) 275:223–8. doi: 10.1074/jbc.275.1.223 18. Ling S, Shan Q, Liu P, Feng T, Zhang X, Xiang P, et al. Metformin ameliorates
arsenic trioxide hepatotoxicity via inhibiting mitochondrial complex I. Cell
Death Dis. (2017) 8:e3159. doi: 10.1038/cddis.2017.482 5. Owen MR, Doran E, Halestrap AP. Evidence that metformin exerts its
anti-diabetic effects through inhibition of complex 1 of the mitochondrial
respiratory chain. Biochem J. (2000) 348:607–14. doi: 10.1042/bj3480607 19. Cameron AR, Logie L, Patel K, Erhardt S, Bacon S, Middleton P, et al. Metformin selectively targets redox control of complex I energy transduction. Redox Biol. (2018) 14:187–97. doi: 10.1016/j.redox.2017.08.018 6. Detaille D, Guigas B, Leverve X, Wiernsperger N, Devos P. Obligatory
role of membrane events in the regulatory effect of metformin on
the respiratory chain function. Biochem Pharmacol. (2002) 63:1259–72. doi: 10.1016/S0006-2952(02)00858-4 20. Wu Y, Gao WN, Xue YN, Zhang LC, Zhang JJ, Lu SY, et al. SIRT3 aggravates
metformin-induced energy stress and apoptosis in ovarian cancer cells. Exp. Cell Res. CONCLUSIONS AND PROPOSAL As explained several times in this manuscript, the concentration
with which experiments were conducted is the main misleading
point regarding the effect of metformin on Complex I. On the one hand, it is obvious that the assumption that
metformin accumulates in mitochondria suits many authors. This hypothesis can bridge the gap between concentrations
measured in vivo and those used in vitro. On the other
hand, two different laboratories that attempted to measure such
an accumulation put forward a total absence of metformin
accumulation in mitochondria (6, 45) in which Complex
I was nevertheless inhibited (6). Furthermore, although the
pharmacokinetic data are indirect evidence, they are not
compatible with an accumulation of metformin in mitochondria. Cancer cells are known to be generally highly glycolytic
(Warburg effect) and are thus not supposed to be very sensitive
to mitochondrial poison. But is it so simple? As soon as cells
consume oxygen at the mitochondrial level, they are supposed
to produce mitochondrial ATP. Thus, even if the proportion of
mitochondrial ATP production is reduced in cancer cells, this
mitochondrial ATP production exists and its reduction could
be toxic. Supporting this proposal, it has been reported that
metformin inhibits the proliferation of HCT116 p53−/−cancer
cells in the presence of glucose, while it induces cell death in case
of glucose deprivation (15). Moreover, the effect of metformin is December 2018 | Volume 9 | Article 753 6 Fontaine Metformin and Mitochondrial Complex I generally accepted hypothesis of metformin accumulation in
mitochondria. Facing the facts, one must admit that there is either a technical
mistake in the studies that did not find metformin accumulation
in mitochondria or there is absolutely no experiment performed
at millimolar concentrations of metformin that reflect what
occurs in vivo. This includes a lot of articles both on
its antidiabetic role and on its anticancer effect. There
is an urgent need to solve this problem for good, and
this could be performed easily by fast cell fractionation
coupled to mass spectrometry (or other technics to detect
metformin) in order to confirm if metformin is found in
large amount in mitochondria of cells exposed to metformin. Currently
the published evidence does not support the ACKNOWLEDGMENTS I thank Christophe Cottet and Emily Tubbs for the corrections of
the English version of this paper. I thank Christophe Cottet and Emily Tubbs for the corrections of
the English version of this paper. AUTHOR CONTRIBUTIONS The author confirms being the sole contributor of this work and
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S, et al. Neuroprotective role of antidiabetic drug metformin against apoptotic
cell death in primary cortical neurons. J Mol Neurosci. (2008) 34:77–87. doi: 10.1007/s12031-007-9002-1 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. 72. Morales AI, Detaille D, Prieto M, Puente A, Briones E, Arevalo M,
et al. Metformin prevents experimental gentamicin-induced nephropathy
by a mitochondria-dependent pathway. Kidney Int. (2010) 77:861–9. doi: 10.1038/ki.2010.11 Copyright © 2018 Fontaine. 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. Frontiers in Endocrinology | www.frontiersin.org December 2018 | Volume 9 | Article 753 REFERENCES No use, distribution or
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Richard MJ, et al. Respective effects of oxygen and energy substrate
deprivation on beta cell viability. Biochim Biophys Acta (2015) 1847:629–39. doi: 10.1016/j.bbabio.2015.04.002 December 2018 | Volume 9 | Article 753 Frontiers in Endocrinology | www.frontiersin.org 9
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https://www.biorxiv.org/content/biorxiv/early/2019/07/20/708719.full.pdf
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English
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Plasma-derived HIV Nef+ exosomes persist in ACTG384 study participants despite successful virological suppression
|
bioRxiv (Cold Spring Harbor Laboratory)
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cc-by
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CC-BY 4.0 International license
a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
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;
https://doi.org/10.1101/708719
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bioRxiv preprint . CC-BY 4.0 International license
a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
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doi:
bioRxiv preprint . CC-BY 4.0 International license
a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
this version posted July 20, 2019. ;
https://doi.org/10.1101/708719
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certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
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bioRxiv preprint 1
Plasma-derived HIV Nef+ exosomes persist in ACTG384 study participants despite successful 1
Plasma-derived HIV Nef+ exosomes persist in ACTG384 study participants despite successful 1
Plasma-derived HIV Nef+ exosomes persist in ACTG384 study participants despite successful 2
virological suppression. Andrea D. Raymond 1, Michelle J. Lang 1, Jane Chu 1, Tamika Campbell-Sims 1, 2
virological suppression. Andrea D. Raymond 1, Michelle J. Lang 1, Jane C 3
Mahfuz Khan1, Vincent C. Bond 1, Richard B. Pollard*, David M. Asmuth*, and Michael D. Powell 1. 4
Morehouse School of Medicine1, *University of California-Davis, and the ACTG 384 team 6
Short title: HIV Nef+ exosomes impact immune recovery 7
Keywords: Nef, exosomes, microvesicles, discordant CD count/viral load, CD4 recovery,
8
ACTG384 study 18 1 1 . CC-BY 4.0 International license
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ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
this version posted July 20, 2019. ;
https://doi.org/10.1101/708719
doi:
bioRxiv preprint . CC-BY 4.0 International license
a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
this version posted July 20, 2019. ;
https://doi.org/10.1101/708719
doi:
bioRxiv preprint . CC-BY 4.0 International license
a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
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bioRxiv preprint 19
Abstract 19
Abstract
20
Human Immunodeficiency Virus (HIV) accessory protein Negative factor (Nef) is detected in the
21
plasma of HIV+ individuals associated with exosomes. The role of Nef+ exosomes (exNef) in HIV
22
pathogenesis is unknown. We perform a retrospective longitudinal analysis to determine
23
correlative clinical associations of exNef plasma levels in ARV-treated HIV+ patients with or
24
without immune recovery. exNef concentration in a subset of AIDS Clinical Trial Group (ACTG)
25
384 participants with successful virological suppression and with either high (∆ >100 CD4 cell
26
recovery/High Immunological Responders (High-IR) or low (∆ ≤100 CD4 cell recovery/ Low
27
Immunologic Responders (Low-IR) immunologic recovery was measured and compared for
28
study weeks 48, 96, and 144. CD4 recovery showed a negative correlation with exNef at study
29
week 144 (r = -0.3573, *p=.0366). Plasma exNef concentration in high IRs negatively correlated
30
with naïve CD4 count and recovery (r = -0.3249, *p = 0. 0348 (High-IR); r =0.2981, *p= #0.0513 (Low-
31
IR)). However, recovery of CD4 memory cells positively correlated with exNef (r =.4534,
32
*p=.0358) in Low-IRs but not in High-IRs. Regimen A (Didanosine, Stavudine, Efavirenz) lowered
33
exNef levels in IRs by 2-fold compared to other regimens. Nef+ exosomes persist in ART-treated
34
HIV+ individuals despite undetectable viral loads, negatively correlates with naive and memory
35
CD4 T cell restoration and may be associated with reduced immunological recovery. Taken
36
together, these data suggest that exNef may represent a novel mechanism utilized by HIV to
37
promote immune dysregulation 20
Human Immunodeficiency Virus (HIV) accessory protein Negative factor (Nef) is detected in the 38 39 2 2 . CC-BY 4.0 International license
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ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
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bioRxiv preprint . CC-BY 4.0 International license
a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
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doi:
bioRxiv preprint 40
Introduction 41
The prognosis for patients infected with HIV has improved significantly with the advent of
42
combination antiretroviral therapy (cART) which can lead to suppression of plasma viremia
43
usually associated with marked improvements in CD4+ T cell counts [1, 2]. However, a subset of
44
patients do not experience a robust immune recovery despite viral suppression. While host
45
factors such as age and baseline CD4+ T-cells with a naïve phenotype have been observed to be
46
negatively associated with the magnitude of CD4+ T-cell count improvement [1, 3-6], virological
47
factors that contribute to these immunologic outcomes are less well defined. 48
Immune reconstitution can be defined as an increase in the number of peripheral CD4 T-
49
cells to greater than 350-500 cells/dL after 4 years of effective cART [3]. Discordant responses
50
characterized by a lack of immune recovery despite viral suppression occur in 7-39% of
51
participants receiving cART [4-7]. The reasons for this phenomenon are not understood. 52
The HIV-encoded Negative factor (Nef) has been implicated in pathogenesis based on
53
studies examining HIV-infected long term non-progressors (LTNP) and elite controllers (EC). 54
LTNPs remain asymptomatic for years with stable CD4 counts while ECs control plasma HIV
55
RNA in the absence of ART [12]. Although several hypotheses have been presented to account
56
for the protective factors leading to delayed disease progression, replication defective virions/nef-
57
defective virions have been associated with these populations in some cases [9-11]. Recent
58
reports have shown that the viruses within some LTNPs and ECs are actually replication
59
competent and control of HIV may be due to host and/or viral factors that maintain low levels of
60
viremia [11]. HIV-1 Nef may be integral to the control of viral replication and/or T-cell responses
61
in an HIV-infected individual. Early in vitro studies have shown that soluble Nef is cytotoxic to 48
Immune reconstitution can be defined as an increase in the number of peripheral CD4 T-
49
cells to greater than 350-500 cells/dL after 4 years of effective cART [3]. Discordant responses
50
characterized by a lack of immune recovery despite viral suppression occur in 7-39% of
51
participants receiving cART [4-7]. The reasons for this phenomenon are not understood. 3
52
The HIV-encoded Negative factor (Nef) has been implicated in pathogenesis based on
53
studies examining HIV-infected long term non-progressors (LTNP) and elite controllers (EC). 40
Introduction 54
LTNPs remain asymptomatic for years with stable CD4 counts while ECs control plasma HIV
55
RNA in the absence of ART [12]. Although several hypotheses have been presented to account
56
for the protective factors leading to delayed disease progression, replication defective virions/nef-
57
defective virions have been associated with these populations in some cases [9-11]. Recent
58
reports have shown that the viruses within some LTNPs and ECs are actually replication
59
competent and control of HIV may be due to host and/or viral factors that maintain low levels of
60
viremia [11]. HIV-1 Nef may be integral to the control of viral replication and/or T-cell responses
61
in an HIV-infected individual. Early in vitro studies have shown that soluble Nef is cytotoxic to 3
52
The HIV-encoded Negative factor (Nef) has been implicated in pathogenesis based on
53
studies examining HIV-infected long term non-progressors (LTNP) and elite controllers (EC). 54
LTNPs remain asymptomatic for years with stable CD4 counts while ECs control plasma HIV
55
RNA in the absence of ART [12]. Although several hypotheses have been presented to account
56
for the protective factors leading to delayed disease progression, replication defective virions/nef-
57
defective virions have been associated with these populations in some cases [9-11]. Recent
58
reports have shown that the viruses within some LTNPs and ECs are actually replication
59
competent and control of HIV may be due to host and/or viral factors that maintain low levels of
60
viremia [11]. HIV-1 Nef may be integral to the control of viral replication and/or T-cell responses
61
in an HIV-infected individual. Early in vitro studies have shown that soluble Nef is cytotoxic to 3 3 . CC-BY 4.0 International license
a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
this version posted July 20, 2019. ;
https://doi.org/10.1101/708719
doi:
bioRxiv preprint . CC-BY 4.0 International license
a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
this version posted July 20, 2019. 40
Introduction It is made available under
The copyright holder for this preprint (which was not
this version posted July 20, 2019. ;
https://doi.org/10.1101/708719
doi:
bioRxiv preprint . CC-BY 4.0 International license
a
ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
this version posted July 20, 2019. ;
https://doi.org/10.1101/708719
doi:
bioRxiv preprint e Leukemia Virus) [28-32]. Given the immunomodulatory functions of exosomes, we
to explore whether exNef may selectively impair CD4+ T-cells recovery during cART
. CC-BY 4.0 International license
a
by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
this version posted July 20, 2019. ;
https://doi.org/10.1101/708719
doi:
preprint . CC-BY 4.0 International license
a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
this version posted July 20, 2019. ;
https://doi.org/10.1101/708719
doi:
bioRxiv preprint 83
Murine Leukemia Virus) [28-32]. Given the immunomodulatory functions of exosomes, we
84
sought to explore whether exNef may selectively impair CD4+ T-cells recovery during cART. 85
For this study, Nef concentration was determined in plasma-derived exosomes isolated
86
from a subset of the ACTG-384 cohort with and without immune recovery post-cART. We show
87
that Nef+ exosomes persist and can be detected in study participants with undetectable viral
88
loads even after 144 weeks of therapy. This suggests that exNef production is independent of
89
plasma viral load. Interestingly, we demonstrate that Low-IRs have significantly higher levels of
90
exNef compared to High- IRs at 144 weeks post-treatment. Recovery of naive CD4 T-cells
91
(CD45RA+, CD62L+) and total CD4 cells negatively correlated with exNef. Overall, we report that
92
Nef+ exosomes are detected in the plasma despite viral suppression and that exNef is negatively
93
associated with changes in naïve and total CD4 count. Taken together these results suggest that
94
exNef may ultimately affect CD4 T-cell recovery and be a biomarker of immune recovery. . CC-BY 4.0 International license
a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 40
Introduction It is made available under 5
84
sought to explore whether exNef may selectively impair CD4+ T-cells recovery during cART. 85
For this study, Nef concentration was determined in plasma-derived exosomes isolated
86
from a subset of the ACTG-384 cohort with and without immune recovery post-cART. We show
87
that Nef+ exosomes persist and can be detected in study participants with undetectable viral
88
loads even after 144 weeks of therapy. This suggests that exNef production is independent of
89
plasma viral load. Interestingly, we demonstrate that Low-IRs have significantly higher levels of
90
exNef compared to High- IRs at 144 weeks post-treatment. Recovery of naive CD4 T-cells
91
(CD45RA+, CD62L+) and total CD4 cells negatively correlated with exNef. Overall, we report that
92
Nef+ exosomes are detected in the plasma despite viral suppression and that exNef is negatively
93
associated with changes in naïve and total CD4 count. Taken together these results suggest that
94
exNef may ultimately affect CD4 T-cell recovery and be a biomarker of immune recovery. 95
96
Material and Methods
97
Study Population. A subset of cryopreserved plasma samples (n = 240) taken from participants
98
in ACTG 384 were used in this study [1] (Robbins, GK 2003; Shafer 2003). As previously
99
described ACTG 384 was a factorial multi-center randomized controlled trial conducted in the
100
United States and Italy that compared sequential three-drug regimens for treatment of HIV
101
infection. Nine hundred eighty ART-naive HIV-1+ subjects were randomized and treated with
102
stavudine/didanosine or zidovudine/lamivudine with nelfinavir, efavirenz, or both nelfinavir
103
and efavirenz. If virological failure occurred, then participants were placed on another regimen 85
For this study, Nef concentration was determined in plasma-derived exosomes isolated
86
from a subset of the ACTG-384 cohort with and without immune recovery post-cART. We show
87
that Nef+ exosomes persist and can be detected in study participants with undetectable viral
88
loads even after 144 weeks of therapy. This suggests that exNef production is independent of
89
plasma viral load. Interestingly, we demonstrate that Low-IRs have significantly higher levels of
90
exNef compared to High- IRs at 144 weeks post-treatment. Recovery of naive CD4 T-cells
91
(CD45RA+, CD62L+) and total CD4 cells negatively correlated with exNef. 40
Introduction ;
https://doi.org/10.1101/708719
doi:
bioRxiv preprint 62
T-cells and is released from HIV-infected cells in plasma derived microvesicles that can be
63
detected in HIV-infected patients [13-23]. These studies suggest that Nef does not exist as a
64
soluble protein in vivo but instead non-virion associated Nef is found in microvesicles or
65
exosome-like microvesicles (exNef) [7, 8]. 66
Several in vitro studies have identified pathogenic activities of soluble Nef including its
67
capacity to reduce surface expression of CD4 and MHC class II, to increase HIV infectivity, to
68
stimulate primary macrophages to release pro-inflammatory cytokines and chemokines, and
69
induce apoptosis in CD4 T-cells [13, 18, 24-27]. We have previously shown that expression of nef
70
in HEK293 cells is sufficient to produce exNef, and the resultant exosomes can be absorbed by T-
71
cells and macrophage but only induces apoptosis in T-cells [14]. Taken together, these
72
observations suggest that Nef+ microvesicles/exosome could have an impact on CD4 T-cell
73
recovery. We hypothesize that Nef microvesicles/exosomes are released from HIV infected cells
74
despite successful cART-induced viral suppression and contribute to pathogenesis by impeding
75
CD4-T-cell recovery. 76
Exosomes range in size from 30 -100 nm and are released from hematopoetic (i.e. B-and T-
77
lymphocytes, dendritic cells, monocytes, mast cells, reticulocytes) and non-hematopoetic (i.e. 78
neurons, intestinal epithelial cells, and tumors) cells. Exosomes are detected in physiological
79
fluids such as plasma, urine, malignant effusions, and amniotic fluids. Several functions have
80
been attributed to exosomes/microvesicles including the modulation of cell signaling, cellular
81
homeostasis, intercellular communication, shuttling genetic material, establishment of tissue
82
polarity, regulation of immune responses, and enhancing the site of budding (i.e. HIV and 76
Exosomes range in size from 30 -100 nm and are released from hematopoetic (i.e. B-and T-
77
lymphocytes, dendritic cells, monocytes, mast cells, reticulocytes) and non-hematopoetic (i.e. 78
neurons, intestinal epithelial cells, and tumors) cells. Exosomes are detected in physiological
79
fluids such as plasma, urine, malignant effusions, and amniotic fluids. Several functions have
80
been attributed to exosomes/microvesicles including the modulation of cell signaling, cellular
81
homeostasis, intercellular communication, shuttling genetic material, establishment of tissue
82
polarity, regulation of immune responses, and enhancing the site of budding (i.e. HIV and 4 . CC-BY 4.0 International license
a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 40
Introduction Overall, we report that
92
Nef+ exosomes are detected in the plasma despite viral suppression and that exNef is negatively
93
associated with changes in naïve and total CD4 count. Taken together these results suggest that
94
exNef may ultimately affect CD4 T-cell recovery and be a biomarker of immune recovery. 96
Material and Methods 97
Study Population. A subset of cryopreserved plasma samples (n = 240) taken from participants
98
in ACTG 384 were used in this study [1] (Robbins, GK 2003; Shafer 2003). As previously
99
described ACTG 384 was a factorial multi-center randomized controlled trial conducted in the
100
United States and Italy that compared sequential three-drug regimens for treatment of HIV
101
infection. Nine hundred eighty ART-naive HIV-1+ subjects were randomized and treated with
102
stavudine/didanosine or zidovudine/lamivudine with nelfinavir, efavirenz, or both nelfinavir
103
and efavirenz. If virological failure occurred, then participants were placed on another regimen 5 . CC-BY 4.0 International license
a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
this version posted July 20, 2019. ;
https://doi.org/10.1101/708719
doi:
bioRxiv preprint . CC-BY 4.0 International license
a
ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
this version posted July 20, 2019. ;
https://doi.org/10.1101/708719
doi:
bioRxiv preprint . CC-BY 4.0 International license
a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
this version posted July 20, 2019. ;
https://doi.org/10.1101/708719
doi:
bioRxiv preprint 104
sequentially. In this study, samples from three distinct groups were obtained and assayed. The
105
groups included 1) Treatment failure (TF) defined as those with a virologic failure with a
106
detectable viral load ( HIV RNA > 50 RNA copies) what at any time point before 144 weeks, 2). 107
Immunologic responders defined as those with high CD4 improvement of > 100 cells/mm3
108
(achieved at any point post study initiation) and suppressed viremia (< 50 RNA copies) (High
109
IRs) and 3) Immunologic non-responders defined as those with CD4 improvement of < 100
110
cells/mm3 and suppressed viremia (Low IRs) (Figure 1). To avoid confounding effects of
111
multiple-treatment regimens, the samples utilized for this sub-cohort were derived from subjects
112
on their first treatment regimen and those with samples from three- or four time intervals (0, 48,
113
96, and/or 144 weeks). . 96
Material and Methods CC-BY 4.0 International license
a 104
sequentially. In this study, samples from three distinct groups were obtained and assayed. The
105
groups included 1) Treatment failure (TF) defined as those with a virologic failure with a
106
detectable viral load ( HIV RNA > 50 RNA copies) what at any time point before 144 weeks, 2). 107
Immunologic responders defined as those with high CD4 improvement of > 100 cells/mm3
108
(achieved at any point post study initiation) and suppressed viremia (< 50 RNA copies) (High
109
IRs) and 3) Immunologic non-responders defined as those with CD4 improvement of < 100
110
cells/mm3 and suppressed viremia (Low IRs) (Figure 1). To avoid confounding effects of
111
multiple-treatment regimens, the samples utilized for this sub-cohort were derived from subjects
112
on their first treatment regimen and those with samples from three- or four time intervals (0, 48,
113
96, and/or 144 weeks). . CC-BY 4.0 International license
a 114
Isolation of plasma-derived exosomes. Plasma exosomes were isolated as previously described
115
[14, 23, 36]. Plasma was pre-cleared by centrifugation at 10,000g for 30 minutes to remove
116
particulates. Microvesicles were pelleted from pre-cleared plasma via ultra-centrifugation for 1
117
hour at 300,000 x g and then re-suspended in 250 µl of phosphate buffered saline (PBS). Immune
118
complexes within the re-suspended exosomes were removed using acid-dissociation prior to Nef
119
measurement similar to p24 antigen measurements from plasma. Exosomes/high speed pellets
120
(100 µl) were treated with 100 µl of 0.3N hydrochloric (HCl, Sigma) and allowed to incubate for 1
121
hour at 37 C. The acid mixture was neutralized with 100 µl of 0.3N sodium hydroxide (NaCl,
122
Sigma) prior to assaying for Nef. 123
Nef Enzyme-linked Immunosorbent Assay (ELISA). Nef concentration in acid-dissociated
124
microvesicle/exosome preparations was measured using a commercially available anti-Nef 123
Nef Enzyme-linked Immunosorbent Assay (ELISA). Nef concentration in acid-dissociated 6 . CC-BY 4.0 International license
a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
this version posted July 20, 2019. ;
https://doi.org/10.1101/708719
doi:
bioRxiv preprint . CC-BY 4.0 International license
a
ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
this version posted July 20, 2019. ;
https://doi.org/10.1101/708719
doi:
bioRxiv preprint . CC-BY 4.0 International license
a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
this version posted July 20, 2019. ;
https://doi.org/10.1101/708719
doi:
bioRxiv preprint 125
sandwich ELISA kit (Immunodiagnostics, Bedford, MA) according to the manufacturer’s
126
instructions. Briefly, the neutralized microvesicle preparations were diluted 1:1 with sample
127
diluent (Component C) and added to ELISA plates coated with anti-Nef (Component A). 128
Following 1-hour incubation at room temperature the plates were washed three times with wash
129
buffer (Component B), and 100 µl of anti-Nef-HRP labeled antibody solution (component E) was
130
then added to each well. Plates were incubated for 1 hour at room temperature, washed three
131
times with component B, and developed by adding 100 µl of alkaline phosphatase substrate
132
(component F) per well. Plates were developed for approximately 10 minutes after which 100 µl
133
of stop solution (Component G) was added to each well and the absorbance at 450 nm
134
determined using a Spectramax spectrophotometer. 125
sandwich ELISA kit (Immunodiagnostics, Bedford, MA) according to the manufacturer’s
126
instructions. Briefly, the neutralized microvesicle preparations were diluted 1:1 with sample
127
diluent (Component C) and added to ELISA plates coated with anti-Nef (Component A). 128
Following 1-hour incubation at room temperature the plates were washed three times with wash
129
buffer (Component B), and 100 µl of anti-Nef-HRP labeled antibody solution (component E) was
130
then added to each well. Plates were incubated for 1 hour at room temperature, washed three
131
times with component B, and developed by adding 100 µl of alkaline phosphatase substrate
132
(component F) per well. 123
Nef Enzyme-linked Immunosorbent Assay (ELISA). Nef concentration in acid-dissociated Plates were developed for approximately 10 minutes after which 100 µl
133
of stop solution (Component G) was added to each well and the absorbance at 450 nm
134
determined using a Spectramax spectrophotometer. 125
sandwich ELISA kit (Immunodiagnostics, Bedford, MA) according to the manufacturer’s 135
Statistical Analysis. Exosomal Nef concentrations were correlated with various immunologic
136
outcomes (immune recovery, change in cell populations, etc.) in each group using the Spearman
137
rank test. The Kruskal-Wallis test was used to compare continuous outcomes between these three
138
groups. The Mann-Whitney rank-sum test or the Dunn’s multiple comparison tests were used
139
appropriately to compare Nef levels in Treatment failures and immunological responders. 140
Statistical analysis was performed using MYSTAT software version 12 (Systat software, Inc. 141
2007). 7
144
Baseline characteristics of ACTG384 sub-cohort. This sub-cohort consisted primarily of males
145
(88.4%) (Table 1). Plasma HIV RNA levels at baseline were not significantly different between 7 . CC-BY 4.0 International license
a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
this version posted July 20, 2019. ;
https://doi.org/10.1101/708719
doi:
bioRxiv preprint . CC-BY 4.0 International license
a
ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
this version posted July 20, 2019. ;
https://doi.org/10.1101/708719
doi:
bioRxiv preprint . CC-BY 4.0 International license
a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
this version posted July 20, 2019. 123
Nef Enzyme-linked Immunosorbent Assay (ELISA). Nef concentration in acid-dissociated ;
https://doi.org/10.1101/708719
doi:
bioRxiv preprint 150
151
152
153
154
155
156
157
158
159
160
161
Characteristics
Complete subcohort
Treatment
Failures
>200 ∆CD4
cell count
<200 ∆CD4
cell count
sex[n,%]
(n=47)
(n=14)
(n=15)
(n=18)
Male
41,(88.4%)
10(71%)
13(87%)
18(100%)
Female
6,(11.6%)
4(29%)
2(13%)
0
Age
(Baseline)
37
36
37
39
Viral load(log10)
Baseline
5.447
4.998
5.563
5.425
96 weeks
1.68
3.32
1.699
1.699
CD4 Count
***
***
***
*
Baseline
116
252
61
87
96 weeks
330
372
409
253.5
Activated
CD4/CD38/HLA(%)
***
***
***
***
Baseline
28
21
22
31
96 weeks
7
11
7
6.5
Activated
CD8/CD38/HLA(%)
***
*
***
***
Baseline
53
52
57
53
96 weeks
20
29
22.5
18.5
Naïve CD4
(cells/mm3)
***
ns
***
***
Baseline
6.4
42.7
9.9
5.3
96 weeks
102.3
58.5
151.8
60.9
Memory CD4
***
ns
***
***
Baseline
24.5
78.9
22.5
33.9
96 weeks
205.1
161.7
271.4
174.9 161 162 8 . CC-BY 4.0 International license
a
ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
this version posted July 20, 2019. ;
https://doi.org/10.1101/708719
doi:
bioRxiv preprint . CC-BY 4.0 International license
a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
this version posted July 20, 2019. ;
https://doi.org/10.1101/708719
doi:
bioRxiv preprint . CC-BY 4.0 International license
a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
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bioRxiv preprint 163
Table 1. 123
Nef Enzyme-linked Immunosorbent Assay (ELISA). Nef concentration in acid-dissociated Statistical analysis – Intergroup analysis (Column) compared gender and group association using chi-
164
square; ethnicity and group composition at baseline, using Chi-square analysis, #Trend p-value <0.1; Median age of
165
groups compared using ANOVA – Kruskal-Wallis, ns-not significant; Viral load at baseline between groups at
166
baseline and 96 weeks, Kruskall-Wallis, ns- not significant.; Baseline CD4 count, ns-not significantly different
167
between groups; Post-96 weeks CD4 count significantly different between Low and High Immunological responders
168
(IR); Kruskall-Wallis, Dunnett’s Multiple comparisons, ***p-value<.001; CD4 count different between baseline and 96
169
weeks for each group Mann-Whitney ***p-value <.0001; Activated CD4 and CD8 T-cell counts are ns-(not
170
significant) between groups at baseline and 96 weeks, Kruskall-Wallis, Dunnett’s Multiple; Naïve CD4 counts is
171
significantly different between high and low IRs at baseline but at 96 weeks differ significantly, Kruskall-Wallis,
172
Dunnett’s Multiple comparisons ***p-value<.001; Intra-group difference in IR memory differ significantly, Mann-
173
Whitney ***p-value <.0001 174
groups, either at baseline or post 96 weeks of treatment. (Table 1). Other cell subsets B-cells and
175
natural killer (NK) cells were not significantly different between High-IRs and Low-IRs (data not
176
shown). However, naïve and memory CD4 T-cell subsets were significantly different between
177
High-and Low_IRs (Table 1) suggesting that these cell subsets are integral to immune recovery. 178
179
CD4 recovery among immunological responders negatively correlates with plasma exNef
180
concentration. ART-induced decrease in viral load (VL) to <50 RNA copies did not necessarily
181
reduce Nef concentration in plasma or result in CD4 recovery for all HIV+ patients within the
182
sub-cohort. We examined study participants receiving anti-retrovirals with successful viral
183
suppression that had CD4 recovery above or below 300 cells/mm3 at study week 144 (Fig 1A). At
184
study week 144, 40% of the sub-cohort exhibited discordant VL and CD4 cell recovery along with
185
increased exNef level. In the absence of detectable viral replication, exNef could be detected in
186
the plasma and the levels correlated with total CD4 recovery (Fig 1B). 174
groups, either at baseline or post 96 weeks of treatment. (Table 1). Other cell subsets B-cells and
175
natural killer (NK) cells were not significantly different between High-IRs and Low-IRs (data not
176
shown). However, naïve and memory CD4 T-cell subsets were significantly different between
177
High-and Low_IRs (Table 1) suggesting that these cell subsets are integral to immune recovery. 123
Nef Enzyme-linked Immunosorbent Assay (ELISA). Nef concentration in acid-dissociated 179
CD4 recovery among immunological responders negatively correlates with plasma exNef
180
concentration. ART-induced decrease in viral load (VL) to <50 RNA copies did not necessarily
181
reduce Nef concentration in plasma or result in CD4 recovery for all HIV+ patients within the
182
sub-cohort. We examined study participants receiving anti-retrovirals with successful viral
183
suppression that had CD4 recovery above or below 300 cells/mm3 at study week 144 (Fig 1A). At
184
study week 144, 40% of the sub-cohort exhibited discordant VL and CD4 cell recovery along with
185
increased exNef level. In the absence of detectable viral replication, exNef could be detected in
186
the plasma and the levels correlated with total CD4 recovery (Fig 1B). 179
CD4 recovery among immunological responders negatively correlates with plasma exNef
180
concentration. ART-induced decrease in viral load (VL) to <50 RNA copies did not necessarily
181
reduce Nef concentration in plasma or result in CD4 recovery for all HIV+ patients within the
182
sub-cohort. We examined study participants receiving anti-retrovirals with successful viral
183
suppression that had CD4 recovery above or below 300 cells/mm3 at study week 144 (Fig 1A). At
184
study week 144, 40% of the sub-cohort exhibited discordant VL and CD4 cell recovery along with
185
increased exNef level. In the absence of detectable viral replication, exNef could be detected in
186
the plasma and the levels correlated with total CD4 recovery (Fig 1B). 9 9 . CC-BY 4.0 International license
a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
this version posted July 20, 2019. ;
https://doi.org/10.1101/708719
doi:
bioRxiv preprint . CC-BY 4.0 International license
a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
this version posted July 20, 2019. ;
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doi:
bioRxiv preprint 187
Figure 1: ARV-treated HIV+ patients exhibit differential CD4 T-cell recovery, which negatively correlates with
188
exNef level despite successful viral suppression. (A) At study weeks 48 (n=38), 96(n=32), and 144 (n=25) the
189
change in CD4 count (as measured by flow cytometry) was tracked in in the participants with VL >50 RNA copies
190
and compared to baseline CD4 count. 123
Nef Enzyme-linked Immunosorbent Assay (ELISA). Nef concentration in acid-dissociated 187
Figure 1: ARV-treated HIV+ patients exhibit differential CD4 T-cell recovery, which negatively correlates with
188
exNef level despite successful viral suppression. (A) At study weeks 48 (n=38), 96(n=32), and 144 (n=25) the
189
change in CD4 count (as measured by flow cytometry) was tracked in in the participants with VL >50 RNA copies
190
and compared to baseline CD4 count. High = change in CD4 count >100; Low= change in CD count <100 cells. 191
Statistical significance determined by Kruskal-Wallis test statistic, ***P<.001, Dunn’s Multiple comparison’s **P<.01. 192
(B) Plasma-derived exNef concentration negatively correlates with the change in CD4 count. *P<0.05, Spearman r. 193
194
Trend in exosomal Nef correlates with immunological response status. cART initiation reduces
195
viral load to undetectable levels over time but the effects of ART on exNef production is
196
unknown. Cross-sectional reports have demonstrated that Nef can be detected in the plasma in
197
the absence and presence of ART [36]. Since exNef may play a role in HIV immunopathogenesis
198
and impact CD4 recovery we examined the longitudinal changes of exNef in High IRs. 199
Interestingly, exNef was detected in the plasma of these patients despite suppression of viral
200
replication (Fig 2A). A retrospective longtitudinal analysis shows that at the initiation of therapy,
201
Nef levels were not significantly different between the Low_IRs and High_IRs (Fig 2A, upper
202
panel). However, by study week 144, the Low-IRs had significantly higher levels of plasma exNef
203
than the High IRs (Fig 2A, lower panel). The median baseline plasma Nef level in the sub- cohort
204
at 48 weeks was approximately 2.5 ng per 1 ml plasma and by 144 weeks Nef levels increased to
205
almost 5 ng per 1 ml plasma in the Low IRs. In fact Low-IRs exhibit an increasing trend in exNef
206
levels from study weeks 48 through 144 while in High-IRs exNef level decreased over the study
207
(Fig 2B). This finding suggests that plasma exNef may play a role immunological recovery. 208
Figure 2: Exosomal Nef is significantly different between low and high immunological responders and
209
trends upward in low immunological responders. (A) exNef concentration as measured by anti-Nef ELISA is
210
increased in Low responders at study weeks 48(Low-n=25, High=5 , 96(Low-n=16; High-n-14), and 144 weeks (Low- 194
Trend in exosomal Nef correlates with immunological response status. 123
Nef Enzyme-linked Immunosorbent Assay (ELISA). Nef concentration in acid-dissociated High = change in CD4 count >100; Low= change in CD count <100 cells
191
Statistical significance determined by Kruskal-Wallis test statistic, ***P<.001, Dunn’s Multiple comparison’s **P<.01
192
(B) Plasma-derived exNef concentration negatively correlates with the change in CD4 count. *P<0.05, Spearman r. 193
194
Trend in exosomal Nef correlates with immunological response status. cART initiation reduces
195
viral load to undetectable levels over time but the effects of ART on exNef production is
196
unknown. Cross-sectional reports have demonstrated that Nef can be detected in the plasma in
197
the absence and presence of ART [36]. Since exNef may play a role in HIV immunopathogenesis
198
and impact CD4 recovery we examined the longitudinal changes of exNef in High IRs
199
Interestingly, exNef was detected in the plasma of these patients despite suppression of viral
200
replication (Fig 2A). A retrospective longtitudinal analysis shows that at the initiation of therapy
201
Nef levels were not significantly different between the Low_IRs and High_IRs (Fig 2A, upper
202
panel). However, by study week 144, the Low-IRs had significantly higher levels of plasma exNef
203
than the High IRs (Fig 2A, lower panel). The median baseline plasma Nef level in the sub- cohort
204
at 48 weeks was approximately 2.5 ng per 1 ml plasma and by 144 weeks Nef levels increased to
205
almost 5 ng per 1 ml plasma in the Low IRs. In fact Low-IRs exhibit an increasing trend in exNef
206
levels from study weeks 48 through 144 while in High-IRs exNef level decreased over the study
207
(Fig 2B). This finding suggests that plasma exNef may play a role immunological recovery. . CC BY 4.0 International license
a 187
Figure 1: ARV-treated HIV+ patients exhibit differential CD4 T-cell recovery, which negatively correlates with
188
exNef level despite successful viral suppression. (A) At study weeks 48 (n=38), 96(n=32), and 144 (n=25) the
189
change in CD4 count (as measured by flow cytometry) was tracked in in the participants with VL >50 RNA copies
190
and compared to baseline CD4 count. High = change in CD4 count >100; Low= change in CD count <100 cells. 191
Statistical significance determined by Kruskal-Wallis test statistic, ***P<.001, Dunn’s Multiple comparison’s **P<.01. 192
(B) Plasma-derived exNef concentration negatively correlates with the change in CD4 count. *P<0.05, Spearman r. 123
Nef Enzyme-linked Immunosorbent Assay (ELISA). Nef concentration in acid-dissociated cART initiation reduces
195
viral load to undetectable levels over time but the effects of ART on exNef production is
196
unknown. Cross-sectional reports have demonstrated that Nef can be detected in the plasma in
197
the absence and presence of ART [36]. Since exNef may play a role in HIV immunopathogenesis
198
and impact CD4 recovery we examined the longitudinal changes of exNef in High IRs. 199
Interestingly, exNef was detected in the plasma of these patients despite suppression of viral
200
replication (Fig 2A). A retrospective longtitudinal analysis shows that at the initiation of therapy,
201
Nef levels were not significantly different between the Low_IRs and High_IRs (Fig 2A, upper
202
panel). However, by study week 144, the Low-IRs had significantly higher levels of plasma exNef
203
than the High IRs (Fig 2A, lower panel). The median baseline plasma Nef level in the sub- cohort
204
at 48 weeks was approximately 2.5 ng per 1 ml plasma and by 144 weeks Nef levels increased to
205
almost 5 ng per 1 ml plasma in the Low IRs. In fact Low-IRs exhibit an increasing trend in exNef
206
levels from study weeks 48 through 144 while in High-IRs exNef level decreased over the study
207
(Fig 2B). This finding suggests that plasma exNef may play a role immunological recovery. 208
Figure 2: Exosomal Nef is significantly different between low and high immunological responders and
209
trends upward in low immunological responders. (A) exNef concentration as measured by anti-Nef ELISA is
210
increased in Low responders at study weeks 48(Low-n=25, High=5 , 96(Low-n=16; High-n-14), and 144 weeks (Low- 194
Trend in exosomal Nef correlates with immunological response status. cART initiation reduce 10 . CC-BY 4.0 International license
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certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
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doi:
bioRxiv preprint . CC-BY 4.0 International license
a
ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
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bioRxiv preprint 211
n=10 ;High n=16). Statistical significance determined via Mann-Whitney, *p-value <0.05, Mann-Whitney. (B) High
212
and low immunological responders defined in sub-cohort defined by change in CD4 count from baseline. High –
213
change 100 (cells/mm3); Low change <100 cells/mm3) (B) Median Nef concentration in exosomes isolated from
214
plasma of subjects with Treatment Failure (n=10) or Success (with high, and low CD4 recovery; n=30) subjects. Nef
215
quantified by anti-Nef ELISA. 216
Naïve CD4 count and recovery inversely correlate with exNef. W 216
Naïve CD4 count and recovery inversely correlate with exNef. We then investigated whether
217
Nef+ exosome levels correlated with recovery of naïve CD4 cell counts specifically. 218
Immunological recovery (as defined in Methods) 48 weeks post treatment and close to 90% (18
219
out of 20) by week 96 of treatment (Fig 3A). However, none of the Low-IRs exhibited increases in
220
CD4 T-cell count close to 350 cells/mm3 by 144 weeks (Fig 3A). Although naïve T-cells do recover
221
in both High- or Low-IR the IRs have appreciably less naïve CD4 cells than the IRs 96- and 144
222
weeks post treatment initiation (Fig 3C). Notably the changes in both CD4 T-cell count and CD4
223
naïve T-cells negatively correlated with the Nef concentration in plasma-derived microvesicles
224
(Fig 3, B and D) suggesting that in vivo microvesicular Nef may be associated with immune
225
recovery/ CD4 T-cell rebound. 11
226
Figure #3: Naïve CD4 counts inversely correlate with exNef. (A) Naïve counts (as measured by flow cytometry)
227
significantly higher in the High_IR group at study week 48 and (B) study week 96. (C ) Nef-level inversely correlates
228
with both Naïve CD T-cell count and the (D)change in Naïve CD4 count post ARV-treatment. Correlation determined
229
by Spearman r, *p-value<.05, #p-value <.1 (trend). Statistical significance determined by One-Way ANOVA Kruskal-
230
Wallis test statistic and Dunn’s Multiple comparison * P-value<0.05, **p-value<.01, and ***p-value<.001. 231
232
Memory CD4 T-cells are significantly different in high low and responders. Given the role of
233
memory cells in immunological recovery, we sought to determine whether exNef also impacted
234
CD4 memory cell recovery. Interestingly at weeks 48 and 96 post-therapy High-IRs exhibited 226
Figure #3: Naïve CD4 counts inversely correlate with exNef. (A) Naïve counts (as measured by flow cytometry)
227
significantly higher in the High_IR group at study week 48 and (B) study week 96. (C ) Nef-level inversely correlates
228
with both Naïve CD T-cell count and the (D)change in Naïve CD4 count post ARV-treatment. Correlation determined
229
by Spearman r, *p-value<.05, #p-value <.1 (trend). Statistical significance determined by One-Way ANOVA Kruskal-
230
Wallis test statistic and Dunn’s Multiple comparison * P-value<0.05, **p-value<.01, and ***p-value<.001. 226
Figure #3: Naïve CD4 counts inversely correlate with exNef. 216
Naïve CD4 count and recovery inversely correlate with exNef. W (A) Naïve counts (as measured by flow cytometry)
227
significantly higher in the High_IR group at study week 48 and (B) study week 96. (C ) Nef-level inversely correlates
228
with both Naïve CD T-cell count and the (D)change in Naïve CD4 count post ARV-treatment. Correlation determined
229
by Spearman r, *p-value<.05, #p-value <.1 (trend). Statistical significance determined by One-Way ANOVA Kruskal-
230
Wallis test statistic and Dunn’s Multiple comparison * P-value<0.05, **p-value<.01, and ***p-value<.001. 238
Figure #4: Memory CD4 T-cells are significantly different in high low and responders. (A) CD4 memory cell 238
Figure #4: Memory CD4 T-cells are significantly different in high low and responders. (A) CD4 memory cell
239
counts (as determined by flow cytometry) in high-immunological responders are significantly increased compared to
240
low responders at study weeks 48 (Low-n=25; High-n=5); and 96 (High- n=14; Low-n=16). Significance from baseline
241
determined via Kruskal-Wallis, intergroup differences compared via Dunn’s Multiple comparison *p-value<0.05, **p-
242
value<.01, and ***p-value<.001. (B) The change in CD4 memory count from baseline(n=33) to study week 96 (n=30)
243
directly correlates with exNef level in low- immunological responders (n=18). Correlation determined using
244
Spearman r, *p-value<0.05, ns=not significant. 238
Figure #4: Memory CD4 T-cells are significantly different in high low and responders. (A) CD4 memory cell
239
counts (as determined by flow cytometry) in high-immunological responders are significantly increased compared to
240
low responders at study weeks 48 (Low-n=25; High-n=5); and 96 (High- n=14; Low-n=16). Significance from baseline
241
determined via Kruskal-Wallis, intergroup differences compared via Dunn’s Multiple comparison *p-value<0.05, **p-
242
value<.01, and ***p-value<.001. (B) The change in CD4 memory count from baseline(n=33) to study week 96 (n=30)
243
directly correlates with exNef level in low- immunological responders (n=18). Correlation determined using
244
Spearman r, *p-value<0.05, ns=not significant. 232
Memory CD4 T-cells are significantly different in high low and responders. Given the role of 11 . CC-BY 4.0 International license
a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
this version posted July 20, 2019. ;
https://doi.org/10.1101/708719
doi:
bioRxiv preprint . CC-BY 4.0 International license
a
ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
this version posted July 20, 2019. ;
https://doi.org/10.1101/708719
doi:
bioRxiv preprint . CC-BY 4.0 International license
a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
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bioRxiv preprint 235
appreciably more CD4 memory cells than the Low_IRs (Fig 4, panel A). The degree of CD4
236
increase directly correlated with exNef in the Low_IRs (Fig 4, panel B), suggesting that CD4
237
memory cells could be one of the sources of exNef during anti-viral suppression. 245
PI-sparing regimen significantly reduced plasma exNef. ACTG 384 was a prospective double- 245
PI-sparing regimen significantly reduced plasma exNef. ACTG 384 was a prospective double-
246
blinded study using a factorial design to compare sequential three-drug regimens. Study arms
247
are depicted in Table 2. Basically, two NRTIs zidovudine and lamuvidine or didanosine and
248
stauvidine followed by either efavirenz or nelfinavir were compared (Fig 5A, upper panel). 249
Previous results from the ACTG-384 cohort demonstrated that the combination of zidovudine,
250
lamivudine, and efavirenz lead to the shortest time to viral suppression suggesting that this
251
combination was the most efficacious combination [2]. However, we are still able to detect exNef
252
in the sub-cohort of ACTG384 participants with successful viral suppression. If exNef negatively
253
affects immunological recovery, then we must identify a treatment regimen that suppresses both
254
viral replication and exNef release. To determine how treatment regimen impacts exNef
255
production we stratified participants by treatment regimen and compared their respective exNef
256
level. By weeks 96 and 144 exNef was reduced 2-fold only in participants (with undetectable
257
viral load) that received regimen A (Didanosine, Stauvidine and Efavirenz) (Fig 5, lower panel). 12 12 . CC-BY 4.0 International license
a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
this version posted July 20, 2019. ;
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doi:
bioRxiv preprint . CC-BY 4.0 International license
a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
this version posted July 20, 2019. ;
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doi:
bioRxiv preprint his suggests that drug regimen may also dictate exNef levels and that PI-sparing regimens
duce both viral load and exNef level. . CC-BY 4.0 International license
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ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
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bioRxiv preprint 258
This suggests that drug regimen may also dictate exNef levels and that PI-sparing regimens
259
reduce both viral load and exNef level. 245
PI-sparing regimen significantly reduced plasma exNef. ACTG 384 was a prospective double- It is made available under
The copyright holder for this preprint (which was not
this version posted July 20, 2019. ;
https://doi.org/10.1101/708719
doi:
bioRxiv preprint . CC-BY 4.0 International license
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ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
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bioRxiv preprint 276
immunological recovery. We posit that exNef may represent a novel mechanism utilized by HIV
277
to promote viral replication in resting CD4 T-cells. The impact of exNef on HIV pathogenesis
278
and CD4 T-cell recovery during cART is unknown. Additionally, we demonstrate that exNef
279
level was not only elevated in cART-treated HIV+ patients with low immunological recovery but
280
also negatively correlated with CD4 count recovery in these participants. This clearly suggests a
281
role for exNef in immunological recovery. 276
immunological recovery. We posit that exNef may represent a novel mechanism utilized by HIV
277
to promote viral replication in resting CD4 T-cells. The impact of exNef on HIV pathogenesis
278
and CD4 T-cell recovery during cART is unknown. Additionally, we demonstrate that exNef
279
level was not only elevated in cART-treated HIV+ patients with low immunological recovery but
280
also negatively correlated with CD4 count recovery in these participants. This clearly suggests a
281
role for exNef in immunological recovery. 282
Plasma exNef could theoretically determine immune recovery potential. Detection of Nef
283
microvesicles is not unexpected since several studies have shown that in order to lower surface
284
expression of CD4 and MHC class II Nef interact with component of the endocytic and exocytic
285
machinery [13, 22]. Nef has no enzymatic activity and functions primarily as an adaptor within
286
an infected cell. The function of extracellular Nef is unclear. However, several in vitro studies
287
have demonstrated that extracellular soluble Nef is cytotoxic to cells, induces cytokine and
288
chemokine release from macrophage activation, increases viral infectivity and alters innate
289
immune signaling pathways [13, 18, 27, 37-40]. We know from in vitro and ex vivo studies that
290
Nef is released in exosome-like vesicles from nef-transfected and HIV-infected cells and that these
291
vesicles are detected in the plasma of HIV+ patients [14, 23]. 245
PI-sparing regimen significantly reduced plasma exNef. ACTG 384 was a prospective double- 260 Initial
Initial NNRTI
Initial PI
Initial PI
+NNRTI
Efavirenz
Nelfinavir
NFV +
EFV
NRTI Factor
(EFV)
(NFV)
DDI+D4T
A (5)
B (3)
E (11)
ZDV /3TC
C (10)
D (7)
F(14)
261 13
261
262
Table 2: Study arms of ACTG384. Drug regimen groupings as described by Smeaton et al 2001
263
followed by the number of participants (in parentheses) within regimen group. 264
Figure #5: PI-sparing regimen significantly reduced plasma exNef by study weeks 96 and 144. (Upper panel)
265
Schematic of drug regimens administered to sub-cohort in the first tier of the ACTG384 study. Participants on
266
regimens A (n=5) or C (n=10) received efavirenz with DDI/4TC or ZDV/4TC while those given regimen D(n=7)
267
received ZDV/4TC and Nelfinavir(Lower Panel). Longtitudinal comparison of plasma exNef level as measured by
268
anti-Nef ELISA in participants taking regimen A,C, and D. Statistical significance determined via Kruskal-Wallis, and
269
Dunn’s multiple comparison *p-value <0.05, **p-value<.01, ***p-value<.001, and ns=not significant. 270
271
Discussion
272
This study shows that exNef is detected in the plasma of cART-treated HIV+ patients with
273
successful viral suppression. Immunological recovery does not occur in 40% of cART-treated
274
HIV+ patients. Understanding the factors involved in poor immunological recovery could lead to
275
the development of novel therapeutics that inhibit viral replication while promoting 261 262
Table 2: Study arms of ACTG384. Drug regimen groupings as described by Smeaton et al 2001
263
followed by the number of participants (in parentheses) within regimen group. 262
Table 2: Study arms of ACTG384. Drug regimen groupings as described by Smeaton et al 2001
263
followed by the number of participants (in parentheses) within regimen group. 264
Figure #5: PI-sparing regimen significantly reduced plasma exNef by study weeks 96 and 144. (Upper panel)
265
Schematic of drug regimens administered to sub-cohort in the first tier of the ACTG384 study. Participants on
266
regimens A (n=5) or C (n=10) received efavirenz with DDI/4TC or ZDV/4TC while those given regimen D(n=7)
267
received ZDV/4TC and Nelfinavir(Lower Panel). Longtitudinal comparison of plasma exNef level as measured by
268
anti-Nef ELISA in participants taking regimen A,C, and D. Statistical significance determined via Kruskal-Wallis, and
269
Dunn’s multiple comparison *p-value <0.05, **p-value<.01, ***p-value<.001, and ns=not significant. 13 . CC-BY 4.0 International license
a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 245
PI-sparing regimen significantly reduced plasma exNef. ACTG 384 was a prospective double- Although the biological role of Nef+
292
exosomes is still unknown we expected exNef to have similar cellular and functional affects
293
ascribed to soluble Nef. 294
It has recently been reported that released Nef microvesicles/exosomes similar to soluble Nef
295
can trigger activation induced cell death (apoptosis) in peripheral blood leukocytes thus
296
promoting the depletion of CD4+ T-cells [21]. Another report indicated that Nef induces massive 294
It has recently been reported that released Nef microvesicles/exosomes similar to soluble Nef
295
can trigger activation induced cell death (apoptosis) in peripheral blood leukocytes thus
296
promoting the depletion of CD4+ T-cells [21]. Another report indicated that Nef induces massive 14 14 14 . CC-BY 4.0 International license
a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
this version posted July 20, 2019. ;
https://doi.org/10.1101/708719
doi:
bioRxiv preprint . CC-BY 4.0 International license
a
ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
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bioRxiv preprint 297
secretion of microvesicle clusters in HIV-infected T-cells and that extracellular Nef is then passed
298
to uninfected bystander by cell-to-cell contact via an ERK-1/2 dependent mechanism [41]. Other
299
studies have shown that extracellular Nef vesicles are taken-up/absorbed by T-cells and that
300
these released Nef microvesicles cause activation-induced cell death in primary peripheral blood
301
leukocytes [14, 21]. Primary leukocytes exposed to exNef as observed with soluble Nef release
302
chemokines MIP-1a and MIP-1b (unpublished result). 297
secretion of microvesicle clusters in HIV-infected T-cells and that extracellular Nef is then passed
298
to uninfected bystander by cell-to-cell contact via an ERK-1/2 dependent mechanism [41]. Other
299
studies have shown that extracellular Nef vesicles are taken-up/absorbed by T-cells and that
300
these released Nef microvesicles cause activation-induced cell death in primary peripheral blood
301
leukocytes [14, 21]. Primary leukocytes exposed to exNef as observed with soluble Nef release
302
chemokines MIP-1a and MIP-1b (unpublished result). 245
PI-sparing regimen significantly reduced plasma exNef. ACTG 384 was a prospective double- 303
Here we show that plasma-derived microvesicles/exosomes from participants within the
304
ACTG384 cohort contain Nef even in the presence of successful HAART therapy and are elevated
305
in low immunological responders. This suggests that exNef may play a role in preventing
306
sufficient CD4 T-cell recovery thereby promoting immunodeficiency despite successful HAART
307
outcomes. Taken together, these studies show that extracellular Nef exosomes function similar to
308
soluble Nef in that exNef is absorbed by T-cells, induce apoptosis and may play a role in
309
discordant successful viral suppression and CD4 T-cell recovery. 310
Most interestingly Nef+ microvesicles/exosomes persist and are detectable over a 144-week
311
period suggesting that exNef may simply be a product of chronic HIV infection. We observed
312
that significant reduction of viral load correlated with an increase in exNef level suggesting that
313
exNef may regulate viral replication and/or cell function. These findings are novel in HIV-
314
pathobiology but may not be a unique to HIV. Indeed, recent reports demonstrated that EBV-
315
infected nasopharyngeal cells release inhibitory exosomes containing the EBV-encoded protein
316
latent membrane protein-1 (LMP-1) [42]. These LMP-1+ exosomes inhibited T-cell activation and 15 . CC-BY 4.0 International license
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certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
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a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
this version posted July 20, 2019. ;
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bioRxiv preprint anti-EBV immune responses [42]
So exNef released from HIV-infected cells may have a similar
. CC-BY 4.0 International license
a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
this version posted July 20, 2019. ;
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doi:
bioRxiv preprint nti-EBV immune responses [42]. So exNef released from HIV-infected cells may have a similar
fect on anti-HIV immune responses. . 245
PI-sparing regimen significantly reduced plasma exNef. ACTG 384 was a prospective double- CC-BY 4.0 International license
a
ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
this version posted July 20, 2019. ;
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doi:
bioRxiv preprint . CC-BY 4.0 International license
a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
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bioRxiv preprint 317
anti-EBV immune responses [42]. So exNef released from HIV-infected cells may have a similar
318
effect on anti-HIV immune responses. 317
anti-EBV immune responses [42]. So exNef released from HIV-infected cells may have a similar 319
Aside from the ACTG384, several studies have shown HIV+ patients with successful virological
320
responses to HAART yet incomplete CD4 recovery have increased mortality [43-45]. Age and
321
prolonged periods of immunodeficiency prior to successful HAART are risk factors for
322
incomplete/insufficient CD4 T-cell recovery [46]. We provide evidence in this study that plasma
323
derived Nef+ microvesicles may be associated with immune recovery. We detected increased in
324
exNef only in Low_IRs. In these Low-IRs exNef concentration correlated negatively and positively
325
with the recovery of CD4 Naïve and memory T-cell counts, respectively. This result suggests that
326
naïve CD4 cells are a potential exNef targets while the CD4 memory cells could be a source of the
327
Nef+ exosomes. 328
Treatment regimens not only directly impact viral replication but also appear to affect the
329
generation of Nef+ exosomes. PI-sparing HAART regimens (e.g. Regimen A: Didanosine,
330
Stauvidine, and Efavirenz) reduced both viral load and exNef level. This result suggests that
331
treatment regimen may dictate exNef level and exNef in turn may be developed as a prognostic
332
indicator of CD4 immune recovery during HAART. 333
We posit that the increased level of Nef+ exosomes early in High_IRs (by week 48) are
334
indicative of reduced virus production and suggests that in the absence of productive viral
335
replication HIV-infected cells release more Nef+ exosomes. If these exosomes affect T-cell
336
activation or viability, then this may impact immune recovery. By 144 weeks however, exNef is
337
significantly reduced in High-IRs participants relative to Low-IRs participants. Taken together 16 16 . 245
PI-sparing regimen significantly reduced plasma exNef. ACTG 384 was a prospective double- CC-BY 4.0 International license
a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
this version posted July 20, 2019. ;
https://doi.org/10.1101/708719
doi:
bioRxiv preprint . CC-BY 4.0 International license
a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
this version posted July 20, 2019. ;
https://doi.org/10.1101/708719
doi:
bioRxiv preprint ur study suggests that extended use of combination therapy lacking a HIV protease inhibitor
mpairs the release of Nef+ exosomes while NRTI/NNRTIs have no long-term effects on exNef. . CC-BY 4.0 International license
a
ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
this version posted July 20, 2019. ;
https://doi.org/10.1101/708719
doi:
bioRxiv preprint 338
our study suggests that extended use of combination therapy lacking a HIV protease inhibitor
339
impairs the release of Nef+ exosomes while NRTI/NNRTIs have no long-term effects on exNef. 340
Overall, these data also suggest that increased Nef levels maybe a double-edged sword – in
341
terms of viral suppression- high exNef is associated with decreased viral load but in regard to
342
immune recovery- elevated exNef is associated with reduced CD4 T-cell rebound/immune
343
recovery. Recently, regions within Nef important for secretion have been identified. Disruption
344
of the secretion modification region (SMR) within the Nef gene appears to abolish its release in
345
microvesicles/exosomes [61]. Peptide-targeted disruption of the SMR also inhibited Nef release
346
suggesting that SMR peptides could be developed as a therapeutic agent. . CC-BY 4.0 International license
a 338
our study suggests that extended use of combination therapy lacking a HIV protease inhibitor
339
impairs the release of Nef+ exosomes while NRTI/NNRTIs have no long-term effects on exNef. 338
our study suggests that extended use of combination therapy lacking a HIV protease inhibitor
339
impairs the release of Nef+ exosomes while NRTI/NNRTIs have no long-term effects on exNef. 245
PI-sparing regimen significantly reduced plasma exNef. ACTG 384 was a prospective double- 340
Overall, these data also suggest that increased Nef levels maybe a double-edged sword – in
341
terms of viral suppression- high exNef is associated with decreased viral load but in regard to
342
immune recovery- elevated exNef is associated with reduced CD4 T-cell rebound/immune
343
recovery. Recently, regions within Nef important for secretion have been identified. Disruption
344
of the secretion modification region (SMR) within the Nef gene appears to abolish its release in
345
microvesicles/exosomes [61]. Peptide-targeted disruption of the SMR also inhibited Nef release
346
suggesting that SMR peptides could be developed as a therapeutic agent. 347
Ultimately, we provide evidence of exNef as a novel virological factor contributing to the
348
dissociation of viral load and immunological recovery. Since, drug regimens can alter exNef
349
levels, therapies need to be developed that both successfully lower viral load and Nef+ exosomes
350
levels. Our findings also suggest that clinicians could monitor exNef level along with CD4 T-cell
351
count in patients undergoing ARV-treatment to assess the effectiveness of therapeutic regimens. 17 . CC-BY 4.0 International license
a
ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
this version posted July 20, 2019. ;
https://doi.org/10.1101/708719
doi:
bioRxiv preprint . CC-BY 4.0 International license
a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
this version posted July 20, 2019. ;
https://doi.org/10.1101/708719
doi:
bioRxiv preprint . CC-BY 4.0 International license
a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
this version posted July 20, 2019. ;
https://doi.org/10.1101/708719
doi:
bioRxiv preprint 364
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434. 375
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virus coinfection: the Swiss HIV Cohort Study. Lancet 2000; 356(9244):1800-1805. 378
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virological and immunological responses to HAART. Biomed Pharmacother 2005; 59(8):446-451
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invariably occur following long-term HIV-1 suppression during antiretroviral therapy. 382
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208 365
1. Smeaton LM, DeGruttola V, Robbins GK, Shafer RW. ACTG (AIDS Clinical Trials Group) 384: a
366
strategy trial comparing consecutive treatments for HIV-1. Control Clin Trials 2001; 22(2):142-
367
159. 68
2. Robbins GK, De G, V, Shafer RW, Smeaton LM, Snyder SW, Pettinelli C, et al. Comparison of
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sequential three-drug regimens as initial therapy for HIV-1 infection. N Engl J Med 2003;
70
349(24):2293-2303. 372
treatment-related factors on immunologic recovery after initiation of antiretroviral therapy in
373
HIV-1-positive subjects: results from ACTG 384. J Acquir Immune Defic Syndr 2006; 42(4):426-
374
434. 375
4. Greub G, Ledergerber B, Battegay M, Grob P, Perrin L, Furrer H, et al. Clinical progression, survival,
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virus coinfection: the Swiss HIV Cohort Study. Lancet 2000; 356(9244):1800-1805. 378
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379
virological and immunological responses to HAART. Biomed Pharmacother 2005; 59(8):446-451. 380
6. Pakker NG, Kroon ED, Roos MT, Otto SA, Hall D, Wit FW, et al. Immune restoration does not
381
invariably occur following long-term HIV-1 suppression during antiretroviral therapy. 382
INCAS Study Group. AIDS 1999; 13(2):203-212. 375
4. Greub G, Ledergerber B, Battegay M, Grob P, Perrin L, Furrer H, et al. Clinical progression, survival,
376
and immune recovery during antiretroviral therapy in patients with HIV-1 and hepatitis C
377
virus coinfection: the Swiss HIV Cohort Study. Lancet 2000; 356(9244):1800-1805. 378
5. Jevtovic D, Salemovic D, Ranin J, Pesic I, Zerjav S, Djurkovic-Djakovic O. The dissociation between
379
virological and immunological responses to HAART. Biomed Pharmacother 2005; 59(8):446-451. 380
6. Pakker NG, Kroon ED, Roos MT, Otto SA, Hall D, Wit FW, et al. Immune restoration does not
381
invariably occur following long-term HIV-1 suppression during antiretroviral therapy. 382
INCAS Study Group. AIDS 1999; 13(2):203-212. 383
7. Piketty C, Castiel P, Belec L, Batisse D, Si MA, Gilquin J, et al. Discrepant responses to triple
384
combination antiretroviral therapy in advanced HIV disease. AIDS 1998; 12(7):745-750. 385
8. 353
Acknowledgements 354
We would like to acknowledge the assistance of Dr. Gale Newman (Morehouse School of
355
Medicine). We also acknowledge the support of RCMI core facilities at Morehouse School of
356
Medicine (U54MD007602), the Emory Center for AIDS Research (P30AI050409), and the NIH
357
AIDS Reagent program. We are grateful for funding support from grants S06-GM08428, R21
358
A1060370, and R21 NS105577). Research reported in this publication was supported by the
359
National Institute of Allergy and Infectious Diseases of the National Institutes of Health under
360
Award Number UM1AI068634, UM1 AI068636 and UM1 AI106701. The content is solely the
361
responsibility of the authors and does not necessarily represent the official views of the National
362
Institutes of Health”. 18 . CC-BY 4.0 International license
a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
this version posted July 20, 2019. ;
https://doi.org/10.1101/708719
doi:
bioRxiv preprint . CC-BY 4.0 International license
a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
this version posted July 20, 2019. ;
https://doi.org/10.1101/708719
doi:
bioRxiv preprint . CC-BY 4.0 International license
a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
this version posted July 20, 2019. ;
https://doi.org/10.1101/708719
doi:
bioRxiv preprint 364
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9. Alexander L, Weiskopf E, Greenough TC, Gaddis NC, Auerbach MR, Malim MH, et al. Unusual
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survivors of human immunodeficiency virus type 1 infection. N Engl J Med 1995; 332(4):201-
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cytoplasmic tail disrupts MHC-I trafficking. J Virol 2002; 76(23):12173-12184. 419
23. Raymond AD, Campbell-Simms TC, Lang MJ, Khan M, Powell MD. HIV-1 Nef is secreted in CD
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survival of myeloid cells by a Stat3-dependent pathway. J Biol Chem 2001; 276(27):25605-
423
25611. 424
25. Fujii Y, Otake K, Tashiro M, Adachi A. In vitro cytocidal effects of human immunodeficiency vir
425
type 1 Nef on unprimed human CD4+ T cells without MHC restriction. J Gen Virol 1996;
426
Pt 12):2943-2951. 427
26. Le GS, Erdtmann L, Benichou S, Berlioz-Torrent C, Liu L, Benarous R, et al. Nef interacts with th
428
subunit of clathrin adaptor complexes and reveals a cryptic sorting signal in MHC I
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molecules. Immunity 1998; 8(4):483-495. 430
27. Olivetta E, Percario Z, Fiorucci G, Mattia G, Schiavoni I, Dennis C, et al. HIV-1 Nef induces the
431
release of inflammatory factors from human monocyte/macrophages: involvement of Ne
432
endocytotic signals and NF-kappa B activation. J Immunol 2003; 170(4):1716-1727. 433
28. Pelchen-Matthews A, Kramer B, Marsh M. Infectious HIV-1 assembles in late endosomes in prim
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macrophages. In: 2003. pp. 443-455. 411
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20. Kirchhoff F, Schindler M, Specht A, Arhel N, Munch J. Role of Nef in primate lentiviral
413
immunopathogenesis. Cell Mol Life Sci 2008; 65(17):2621-2636. 414
21. Lenassi M, Cagney G, Liao M, Vaupotic T, Bartholomeeusen K, Cheng Y, et al. HIV Nef is secreted in
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microvesicles and triggers apoptosis in bystander CD4+ T cells. Traffic 2010; 11(1):110-122. 415
microvesicles and triggers apoptosis in bystander CD4+ T cells. Traffic 2010; 11(1):110-122
416
22. Williams M, Roeth JF, Kasper MR, Fleis RI, Przybycin CG, Collins KL. Direct binding of human
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immunodeficiency virus type 1 Nef to the major histocompatibility complex class I (MHC
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422
survival of myeloid cells by a Stat3-dependent pathway. J Biol Chem 2001; 276(27):25605-
423
25611. 424
25. Fujii Y, Otake K, Tashiro M, Adachi A. In vitro cytocidal effects of human immunodeficiency virus
425
type 1 Nef on unprimed human CD4+ T cells without MHC restriction. J Gen Virol 1996; 77 (
426
Pt 12):2943-2951. 427
26. Le GS, Erdtmann L, Benichou S, Berlioz-Torrent C, Liu L, Benarous R, et al. Nef interacts with the mu
428
subunit of clathrin adaptor complexes and reveals a cryptic sorting signal in MHC I
429
molecules. Immunity 1998; 8(4):483-495. 430
27. Olivetta E, Percario Z, Fiorucci G, Mattia G, Schiavoni I, Dennis C, et al. HIV-1 Nef induces the
431
release of inflammatory factors from human monocyte/macrophages: involvement of Nef
432
endocytotic signals and NF-kappa B activation. J Immunol 2003; 170(4):1716-1727. 433
28. Pelchen-Matthews A, Kramer B, Marsh M. Infectious HIV-1 assembles in late endosomes in primary
434
macrophages. In: 2003. pp. 443-455. 20 . CC-BY 4.0 International license
a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not
this version posted July 20, 2019. ;
https://doi.org/10.1101/708719
doi:
bioRxiv preprint 435
29. Stoorvogel W, Kleijmeer MJ, Geuze HJ, Raposo G. The biogenesis and functions of microvesicle
436
3. Traffic 2002; 3(5):321-330. 437
30. Van NG, Raposo G, Candalh C, Boussac M, Hershberg R, Cerf-Bensussan N, et al. Intestinal
438
epithelial cells secrete exosome-like vesicles
439
4. Gastroenterology 2001; 121(2):337-349. 440
31. Van NG, Porto-Carreiro I, Simoes S, Raposo G. Microvesicles: a common pathway for a specializ
441
function
442
3. J Biochem 2006; 140(1):13-21. 443
32. Wiley RD, Gummuluru S. Immature dendritic cell-derived microvesicles can mediate HIV-1 tra
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k
l b
h
A
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h h
d f 471
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https://doi.org/10.1101/708719
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bioRxiv preprint . CC-BY 4.0 International license
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certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
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this version posted July 20, 2019. ;
https://doi.org/10.1101/708719
doi:
bioRxiv preprint . CC-BY 4.0 International license
a
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
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this version posted July 20, 2019. ;
https://doi.org/10.1101/708719
doi:
bioRxiv preprint 509
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https://www.cdc.gov/pcd/issues/2015/pdf/15_0290.pdf
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Consumption of Alcoholic Beverages and Liquor Consumption by Michigan High School Students, 2011
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Preventing chronic disease
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public-domain
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Katherine R. Gonzales, MPH; Thomas W. Largo, MPH; Corinne Miller, PhD, DDS;
Dafna Kanny, PhD; Robert D. Brewer, MD, MSPH Katherine R. Gonzales, MPH; Thomas W. Largo, MPH; Corinne Miller, PhD, DDS;
Dafna Kanny, PhD; Robert D. Brewer, MD, MSPH Katherine R. Gonzales, MPH; Thomas W. Largo, MPH; Corinne Miller, PhD, DDS;
Dafna Kanny, PhD; Robert D. Brewer, MD, MSPH antly more likely than nonbinge drinkers to consume vodka and to
mix alcohol with energy drinks (49.0% vs 18.2%, respectively). Suggested citation for this article: Gonzales KR, Largo TW,
Miller C, Kanny D, Brewer RD. Consumption of Alcoholic
Beverages and Liquor Consumption by Michigan High School
Students, 2011. Prev Chronic Dis 2015;12:150290. DOI: http://
dx.doi.org/10.5888/pcd12.150290. Introduction Excessive alcohol consumption was responsible for approxim-
ately 4,300 annual deaths in the United States among people
younger than 21 from 2006 through 2010. Underage drinking cost
the United States $24.6 billion in 2006. Previous studies have
shown that liquor is the most common type of alcohol consumed
by high school students. However, little is known about the types
of liquor consumed by youth or about the mixing of alcohol with
energy drinks. Annually from 2006 through 2010, excessive alcohol consump-
tion was responsible for approximately 4,300 deaths and 260,000
years of potential life lost in the United States among people
younger than 21 years (1). In 2006, underage drinking cost the
United States approximately $24.6 billion (2). More than 90% of
the alcohol consumed by underage drinkers occurs during binge
drinking episodes (3). Underage drinking is also associated with
many health and social consequences, such as poor academic per-
formance, interpersonal violence, injuries, risky sexual behavior,
and unplanned pregnancies (4–6). Alcohol consumption by youth
is also strongly correlated with alcohol consumption by adults at
the state level (7), and youth often obtain the alcohol they con-
sume from adults (8). Conclusions Liquor is the most common type of alcoholic beverage consumed
by Michigan high school students; vodka is the most common type
of liquor consumed. Mixing alcohol and energy drinks is common,
particularly among binge drinkers. Community Guide strategies
for reducing excessive drinking (eg, increasing alcohol taxes) can
reduce underage drinking. Methods The 2011 Michigan Youth Tobacco Survey was used to assess
usual alcohol beverage consumption and liquor consumption and
the mixing of alcohol with energy drinks by Michigan high school
students. Beverage preferences were analyzed by demographic
characteristics and drinking patterns. Recent studies identified liquor (eg, vodka, rum, scotch, bourbon,
whiskey) as the usual type of alcohol consumed by high school
students (9–13). Students who reported usually consuming liquor
were also more likely to report frequent alcohol consumption and
binge drinking than students who reported consuming other types
of alcoholic beverages (11). However, limited information is avail-
able on the type of liquor that is consumed by underage youth and
whether specific types of alcohol are more likely to be mixed with
other types of beverages, such as energy drinks (eg, Monster, Red
Bull). Understanding the type of alcohol usually consumed by Results Overall, 34.2% of Michigan high school students consumed alco-
hol in the past month, and 20.8% reported binge drinking. Among
current drinkers, liquor was the most common type of alcohol con-
sumed (51.2%), and vodka was the most prevalent type of liquor
consumed by those who drank liquor (53.0%). The prevalence of
liquor consumption was similar among binge drinkers and non-
binge drinkers, but binge drinkers who drank liquor were signific- www.cdc.gov/pcd/issues/2015/15_0290.htm • Centers for Disease Control and Prevention 1 VOLUME 12, E194
NOVEMBER 2015 vey of beverage-specific alcohol consumption (12). Students were
asked about their 30-day consumption of alcohol; binge drinking;
the largest number of drinks consumed on any occasion; the usual
type of alcohol consumed (ie, students could select one alcoholic
beverage type that they usually consumed in the past 30 days); the
usual type of liquor consumed; the usual brand of liquor, beer, or
flavored alcoholic beverage consumed; how frequently they mixed
alcohol with energy drinks; their usual drinking location; and vari-
ous direct consequences that could have resulted from alcohol
consumption (eg, getting into a fight, riding with a driver who had
been drinking). youth is important for developing and targeting interventions to
prevent underage and binge drinking, because alcohol control
policies (eg, alcohol taxes) and alcohol advertising vary by bever-
age type (9,10). In addition, previous studies suggested that youth
who mix alcohol with energy drinks are more likely to binge
drink, consume more total drinks, have higher levels of alcohol in-
toxication, and experience alcohol-attributable harms (eg, sexual
assault) than those who do not mix energy drinks with alcohol
(14,15). As part of its efforts to reduce underage drinking, the Michigan
Department of Health and Human Services added supplemental
questions on alcohol to its 2011 Youth Tobacco Survey. The pur-
pose of this study was to use these survey findings to assess the
type of alcoholic beverage (eg, beer, wine, liquor) usually con-
sumed by Michigan high school students, the type of liquor con-
sumed by students who drank liquor, the prevalence of mixing al-
cohol with energy drinks, and how these drinking behaviors var-
ied by the demographic characteristics and drinking patterns (eg,
binge drinking) of these students. Current drinkers were defined as students who reported consum-
ing at least 1 alcoholic drink in the 30 days before survey adminis-
tration. Binge drinkers were defined as current drinkers who re-
ported consuming 5 or more alcoholic drinks in a row (ie, within a
couple of hours) during the 30 days before survey administration. Nonbinge drinkers were students who identified themselves as
current drinkers but did not report consuming 5 or more alcoholic
drinks in a row during the past 30 days. Methods Analyses were conducted using SAS, version 9.2 (SAS Institute,
Inc) to account for the complex survey design and to produce es-
timates that were representative of Michigan’s high school stu-
dent population. Prevalence estimates with 95% confidence inter-
vals were calculated. Pearson’s χ2was used to test for differences
in the prevalence of beverage-specific alcohol consumption
between groups defined by various demographic factors (ie, sex,
grade, and race/ethnicity) and by drinking patterns (eg, drinking
frequency, drinking intensity, usual place of consumption, usual
alcohol type consumed, usual liquor type consumed, mixing alco-
hol and energy drinks). The Michigan Youth Tobacco Survey (Michigan YTS) is a
school-based survey of a random sample of public-school stu-
dents in grades 9 through 12. Students surveyed complete an an-
onymous, self-administered questionnaire that consists of ques-
tions on tobacco use and on various environmental factors related
to tobacco use, such as access to tobacco products in retail set-
tings. In 2011, 4,142 Michigan high school students from 42
schools completed the Michigan YTS. The school response rate
was 68%, the student response rate was 93%, and the overall re-
sponse rate was 63%. Data were weighted by school, student non-
response, and selected demographic characteristics (ie, the sex,
race/ethnicity, and grade of students). Because the Michigan YTS
is an established public health surveillance system, it does not re-
quire review of the Michigan Department of Health and Human
Services Institutional Review Board. PREVENTING CHRONIC DISEASE
PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY VOLUME 12, E194
NOVEMBER 2015 Centers for Disease Control and Prevention • www.cdc.gov/pcd/issues/2015/15_0290.htm The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services,
the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions.
2
Centers for Disease Control and Prevention • www cdc gov/pcd/issues/2015/15 0290 htm The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services,
the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. VOLUME 12, E194
NOVEMBER 2015 had more than twice the prevalence of mixing energy drinks with
alcohol compared with nonbinge drinkers (49.0% vs 18.2%; P <
.001). ergy drinks by high school students in a state. We found that more
than half of the Michigan high school students who drank alcohol
consumed liquor and that approximately half of the students who
drank liquor usually consumed vodka. Liquor consumption was
also associated with other dangerous drinking behaviors, includ-
ing binge drinking and the mixing of alcohol and energy drinks. This finding is concerning because binge drinking increases the
risk of alcohol-attributable harms, including sexual assault, and
mixing of alcohol and energy drinks can increase binge drinking
intensity, further increasing the risk of alcohol-attributable injur-
ies and alcohol poisoning (15). Most Michigan high school students who drank alcohol reported
usually consuming liquor (51.2%) (Table 3). The prevalence of li-
quor consumption was slightly higher for binge drinkers (54.2%)
than for nonbinge drinkers (46.9%). Liquor was the most fre-
quently reported usual type of alcohol consumed by current drink-
ers across all demographic groups and drinking patterns and was
reported by most students who reported mixing alcohol and en-
ergy drinks (52.7%). Girls reported a significantly higher preval-
ence of drinking flavored alcoholic beverages than boys (21.0% vs
8.1%; P < .001), whereas boys reported a significantly higher pre-
valence of drinking beer than girls (24.3% vs 11.1%; P < .001). Other types of alcohol (wine and cordials) accounted for a small
proportion (11.2%) of the usual type of alcohol consumed by
Michigan high school students. Only 5.2% of current drinkers re-
ported not having a usual beverage type. The finding that liquor is the usual type of alcohol consumed by
Michigan high school students who drink is consistent with the
findings of other studies (10–13). This is likely due to the fact that
liquor has a higher alcohol concentration, is more portable and
easily concealed than other alcoholic beverages, and can easily be
mixed with other beverages, thereby making liquor more palat-
able to youth (16). Youth exposure to alcohol advertising in the
United States increased by 71% from 2001 through 2009, largely
because of an increase in liquor advertising on television (17), and
youth exposure to alcohol advertising is associated with both the
initiation of alcohol consumption by youth and the amount con-
sumed per drinking occasion (18). VOLUME 12, E194
NOVEMBER 2015 The high prevalence of vodka
consumption among high school students who drank liquor is also
consistent with the reported increase in vodka sales in the United
States and in Michigan (19,20), which has likely made vodka
more accessible to youth, as well, particularly given that most high
school students obtain the alcohol they consume from someone
else (10). Vodka consumption was reported by 37.3% of current drinkers,
making it by far the most common type of liquor consumed by
Michigan high school students (Table 4). Vodka consumption was
more common among binge drinkers (42.1%) than among non-
binge drinkers (30.7%) and was reported by almost half of the stu-
dents who reported mixing alcohol and energy drinks. Vodka was
the most common type of liquor consumed by current drinkers
across all demographic groups, but it was somewhat more com-
mon among girls (42.8%) than boys (31.7%). Students who drank
in someone else’s home also had a significantly higher prevalence
of vodka consumption (49.7%) than those who usually drank in
their own home (30.9%) or at some public place (30.9%). However, the prevalence of vodka consumption by current drink-
ers did not vary significantly by drinking frequency (the number
of drinking days) or intensity (the largest number of drinks con-
sumed on 1 occasion). Rum consumption was somewhat more
common among boys than girls (15.4% vs 7.8%), as was the con-
sumption of whiskey (13.0% vs 4.4%,). However, the prevalence
of tequila consumption did not vary significantly by sex. The high prevalence of mixing liquor, such as vodka, with energy
drinks, particularly among binge drinkers, is especially concern-
ing because caffeine can mask some of the sensory cues that alert
drinkers to their level of intoxication. This may lead youth to
binge drink at higher intensity levels, increasing the risk of alco-
hol-attributable harms, including alcohol poisoning, motor vehicle
crashes, and sexual assault (21). In fact, one study involving col-
lege students found that those who mixed energy drinks and alco-
hol were twice as likely to report being hurt or injured, riding in a
car with an intoxicated driver, or requiring medical treatment than
those who did not mix energy drinks and alcohol, even after ad-
justing for the amount of alcohol consumed (15). Vodka was the most common liquor type among those who repor-
ted usually consuming liquor in the past 30 days (53.0%). Results Overall, 34.2% of Michigan high school students reported current
alcohol use; 20.8% of students were binge drinkers (Table 1). Binge drinking prevalence was higher in each successive grade,
ranging from 14.3% among 9th-grade students to 35.9% among
12th-grade students. However, there were no significant differ-
ences in binge drinking prevalence by sex or by race/ethnicity. To assess beverage-specific alcohol consumption by youth, 11
state-added questions on alcohol consumption were included in
the 2011 Michigan YTS. These questions were developed in col-
laboration with the Excessive Alcohol Use Prevention Program of
the National Center for Chronic Disease Prevention and Health
Promotion at the Centers for Disease Control and Prevention, the
Center on Alcohol Marketing and Youth at the Johns Hopkins
Bloomberg School of Public Health, the Michigan Liquor Control
Commission, and other experts in alcohol and public health. The
questions added were based on questions that were used in other
surveys, such as the Youth Risk Behavior Survey and a panel sur- Among students who reported drinking in the past 30 days, binge
drinkers had a significantly higher prevalence of drinking in
someone else’s home than did nonbinge drinkers (61.6% vs
44.0%; P < .001) and had more than 5 times the prevalence of
drinking on 10 or more days in the past 30 days than nonbinge
drinkers (24.4% vs 4.5%; P < .001) (Table 2). Binge drinkers also The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services,
the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. or Disease Control and Prevention • www.cdc.gov/pcd/issues/2015/15_0290.htm VOLUME 12, E194
NOVEMBER 2015 Girls
who reported usually consuming liquor had a higher prevalence of
vodka consumption (60.3%) than boys (44.8%), but the difference
between the 2 was not significant (data not shown). Although liquor consumption was common among all Michigan
high school students who were current drinkers, girls were some-
what more likely than boys to report consuming liquor. This is
particularly concerning because women tend to achieve higher
blood alcohol concentrations than men at the same consumption
level, even taking into account differences in body size, food con- PREVENTING CHRONIC DISEASE
PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY VOLUME 12, E194
NOVEMBER 2015 VOLUME 12, E194
NOVEMBER 2015 VOLUME 12, E194
NOVEMBER 2015 shown that reducing the threshold for defining binge drinking
from 5 drinks to 4 drinks increases the relative prevalence of binge
drinking by more than one-third (27). sumption, and other factors, thus increasing the risk of their exper-
iencing alcohol-attributable harms, including unintended and alco-
hol-exposed pregnancies and adverse reproductive outcomes (22). These data highlight the need for additional and targeted efforts to
prevent binge drinking among girls, including increased efforts to
monitor and reduce youth exposure to alcohol marketing, particu-
larly given that underage girls are overexposed to alcohol market-
ing relative to women to an even greater extent than underage
boys are overexposed to alcohol marketing relative to men (23). This study shows that current drinking, particularly binge drink-
ing, by high school students in Michigan remains a serious public
health problem and that high school students who drink are most
likely to drink liquor, particularly vodka. Surveillance on bever-
age-specific alcohol consumption among youth can be useful for
planning prevention strategies that target specific beverage types
(eg, retail access to liquor) (9–11). Furthermore, because alcohol
taxes are beverage-specific, knowing the type of alcoholic bever-
age usually consumed by youth can help inform alcohol tax
policies in states and communities to prevent underage drinking
(9–11). The Task Force on Community Preventive Services re-
commends several population-level, evidence-based strategies for
preventing excessive alcohol use, including underage drinking,
that can help reduce beverage-specific alcohol consumption by
youth. These include increasing alcohol excise taxes, regulating al-
cohol outlet density, establishing commercial host liability, en-
hanced enforcement of the age 21 minimum legal drinking-age
(eg, through compliance checks in which minors or youthful-look-
ing adults attempt to purchase alcohol from retail establishments),
and avoiding privatization of alcohol sales (28). Previous research
has demonstrated that teenagers and young adults are especially
responsive to increases in the price of alcohol (29). Improved
compliance with the voluntary industry threshold for the place-
ment of alcohol advertising in television programs and in other
media venues (ie, 28.4% youth audience composition), particu-
larly advertising that is placed on cable nonsports television pro-
grams, could also help reduce the risk of underage drinking (30). Finally, states can routinely assess beverage-specific alcohol con-
sumption using the YTS and use these data to help guide efforts to
reduce youth exposure to alcohol marketing. VOLUME 12, E194
NOVEMBER 2015 For example, the
Michigan Department of Health and Human Services has worked
closely with state and local coalitions to collect and report inform-
ation on excessive alcohol use and related harms and to develop a
statewide plan to reduce underage drinking. This study also demonstrated the potential usefulness of the YTS
for assessing alcohol consumption among high school students. Although primarily focused on tobacco consumption among
youth, the YTS has the flexibility to accommodate state-added
questions on alcohol consumption, including questions on bever-
age-specific alcohol consumption, and is relatively inexpensive to
administer. Furthermore, the collection of alcohol information in
the YTS facilitates public health surveillance by providing a more
specific assessment of the relationship between alcohol consump-
tion and smoking by youth, including the potential impact of to-
bacco control measures and alcohol interventions on the drinking
and smoking behaviors of high school students. The results are subject to at least 7 limitations. First, this study as-
sessed only the alcohol consumption patterns of Michigan stu-
dents in 9th through 12th grade; therefore, these findings may not
be generalizable to high school students in other states. Second,
the YTS sample population was not large enough to assess signi-
ficant differences between the demographic characteristics of li-
quor drinkers by type of liquor consumed (eg, vodka, rum, whis-
key). Third, the YTS sample includes only students in public high
schools, and although more than 90% of Michigan youth attend
public schools, the results may not be representative of those who
attend private or other nonpublic schools or students who do not
attend school; previous research has demonstrated that students
who attend alternative schools may have even higher rates of alco-
hol use than public school students (24). Fourth, all prevalence es-
timates are based on self-report, which are likely to underestimate
alcohol consumption because of social desirability and recall bi-
ases (25). Fifth, about one-third of Michigan public high schools
declined to participate in the YTS. The exclusion of the students in
these schools from the survey may have affected the representat-
iveness of the survey findings. Sixth, the data in this study were
collected in 2011, and usual beverage consumption may have
changed since then. However, liquor has consistently been repor-
ted to be the usual type of alcohol consumed by high school stu-
dents in other studies (10–13), and it therefore seems unlikely this
has changed since 2011. Discussion To our knowledge, this is the first study to use the YTS to assess
the types of alcohol consumed and the mixing of alcohol with en- www.cdc.gov/pcd/issues/2015/15_0290.htm • Centers for Disease Control and Prevention 3 VOLUME 12, E194
NOVEMBER 2015 Finally, the definition of binge drinking
that was used in the YTS (ie, 5 or more drinks within a couple of
hours) was not sex-specific (26), and studies among women have The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services,
the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions.
4 Centers for Disease Control and Prevention • www.cdc.gov/pcd/issues/2015/15_0290.htm Centers for Disease Control and Prevention • www.cdc.gov/pcd/issues/2015/15_0290.htm The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services,
the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions.
4
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NOVEMBER 2015 VOLUME 12, E194
NOVEMBER 2015 References Thombs DL, O’Mara RJ, Tsukamoto M, Rossheim ME, Weiler
RM, Merves ML, et al. Event-level analyses of energy drink
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use among high school students — Georgia, 2007. MMWR
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Drinking. Washington (DC): US Department of Health and
Human Services, Office of the Surgeon General; 2007. 4. Bonnie RJ, O’Connell ME, editors. Reducing underage
drinking: a collective responsibility. Washington (DC): The
National Academies Press; 2004. http://www.nap.edu/
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students. Pediatrics 2007;119(1):76–85. 6. Distilled Spirits Council of the United States. Vodka: the spirit
of the industry. http://www.discus.org/assets/1/7/
Vodka2010.pdf. Accessed August 28, 2015. 19. Nelson DE, Naimi TS, Brewer RD, Nelson HA. State alcohol-
use estimates among youth and adults, 1993–2005. Am J Prev
Med 2009;36(3):218–24. 7. Michigan Liquor Control Commission. Annual financial
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statbook2011_new_397864_7.pdf. Acknowledgments We thank David Jernigan, PhD, Department of Health, Behavior,
and Society, Johns Hopkins Bloomberg School of Public Health,
Baltimore, Maryland, for his assistance in developing the ques-
tions and providing comments on this manuscript. None of the au-
thors have any conflicts of interest or financial disclosures. The
findings and conclusions in this article are those of the authors and
do not necessarily represent the official position of the Centers for
Disease Control and Prevention. or Disease Control and Prevention • www.cdc.gov/pcd/issues/2015/15_0290.htm Author Information Corresponding Author: Dafna Kanny, PhD, Division of Population
Health, National Center for Chronic Disease Prevention and
Health Promotion, Centers for Disease Control and Prevention,
4770 Buford Hwy NE, MS F-78, Atlanta, GA 30341-3717. Telephone: 770-488-5411. E-mail: dkanny@cdc.gov. Cremeens JL, Miller JW, Nelson DE, Brewer RD. Assessment
of source and type of alcohol consumed by high school
students: analyses from four States. J Addict Med 2009;
3(4):204–10. 10. Siegel MB, Naimi TS, Cremeens JL, Nelson DE. Alcoholic
beverage preferences and associated drinking patterns and risk
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40(4):419–26. 11. Author Affiliations: Katherine R. Gonzales, Thomas W. Largo,
Corinne Miller, Bureau of Disease Control, Prevention and
Epidemiology, Michigan Department of Health and Human
Services, Lansing, Michigan; Robert D. Brewer, Alcohol Program,
Epidemiology and Surveillance Branch, Division of Population
Health, National Center for Chronic Disease Prevention and
Health Promotion, Centers for Disease Control and Prevention,
Atlanta, Georgia. Siegel M, DeJong W, Naimi TS, Heeren T, Rosenbloom DL,
Ross C, et al. Alcohol brand preferences of underage youth:
results from a pilot survey among a national sample. Subst
Abus 2011;32(4):191–201. 12. Tanski SE, McClure AC, Jernigan DH, Sargent JD. Alcohol
brand preference and binge drinking among adolescents. Arch
Pediatr Adolesc Med 2011;165(7):675–6. 13. VOLUME 12, E194
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beverages usually consumed by students in 9th–12th grades —
four states, 2005. MMWR Morb Mortal Wkly Rep 2007;
56(29):737–40. 9. PREVENTING CHRONIC DISEASE
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M, Jones-Webb R. Sources of alcohol for underage drinkers. J
Stud Alcohol 1996;57(3):325–33. 8. Ferreira SE, de Mello MT, Pompéia S, de Souza-Formigoni
ML. Effects of energy drink ingestion on alcohol intoxication. Alcohol Clin Exp Res 2006;30(4):598–605. 21. g to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services,
e, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services,
the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. www.cdc.gov/pcd/issues/2015/15_0290.htm • Centers for Disease Control and Prevention 5 5 Centers for Disease Control and Prevention • www.cdc.gov/pcd/issues/2015/15_0290.htm The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services,
the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. PREVENTING CHRONIC DISEASE
PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY VOLUME 12, E194
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NOVEMBER 2015 Naimi TS, Lipscomb LE, Brewer RD, Gilbert BC. Binge
drinking in the preconception period and the risk of unintended
pregnancy: implications for women and their children. Pediatrics 2003;111(5 Pt 2):1136–41. 22. Jernigan DH, Ostroff J, Ross C, O’Hara JA 3d. Sex differences
in adolescent exposure to alcohol advertising in magazines. Arch Pediatr Adolesc Med 2004;158(7):629–34. 23. Grunbaum JA, Lowry R, Kann L. Prevalence of health-related
behaviors among alternative high school students as compared
with students attending regular high schools. J Adolesc Health
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Davis CG, Thake J, Vilhena N. Social desirability biases in
self-reported alcohol consumption and harms. Addict Behav
2010;35(4):302–11. 25. National Institute on Alcohol Abuse and Alcoholism. NIAAA
Council approves binge drinking defınition. NIAAA
Newsletter 2004;(3):3. Alcohol Abuse and Alcoholism. http://
pubs.niaaa.nih.gov/publications/Newsletter/winter2004/
Newsletter_Number3.pdf. Accessed August 28, 2015. 26. Chavez PR, Nelson DE, Naimi TS, Brewer RD. Impact of a
new gender-specific definition for binge drinking on
prevalence estimates for women. Am J Prev Med 2011;
40(4):468–71. 27. Task Force on Community Preventive Services. Preventing
excessive alcohol consumption. In: The guide to community
preventive services. New York (NY): Oxford University Press;
2005. http://www.thecommunityguide.org/alcohol/index.html. Accessed on April 28, 2014. 28. Xu X, Chaloupka FJ. The effects of prices on alcohol use and
its consequences. Alcohol Res Health 2011;34(2):236–45. 29. Centers for Disease Control and Prevention. Youth exposure to
alcohol advertising on television — 25 markets, United States,
2010. MMWR Morb Mortal Wkly Rep 2013;62(44):877–80. 30. Abbreviation: CI, confidence interval.
a Binge drinkers were defined as students who had 5 or more drinks of alcohol in a row within a couple of hours on at least 1 day during the 30 days before the
survey.
b Nonbinge drinkers were defined as students who had at least 1 drink of alcohol on at least 1 day during the 30 days before the survey who did not report binge
drinking.
c Nondrinkers were defined as students who reported consuming no alcoholic drinks in the 30 days before the survey. Tables Table 1. Prevalence of Binge, Nonbinge, or Nondrinking Among Michigan 9th Through 12th Grade Students, by Demographic Char-
acteristics, Michigan Youth Tobacco Survey, 2011
Demographic Characteristic
Binge Drinkersa, % (95% CI)
Nonbinge Drinkersb, % (95%
CI)
Nondrinkersc, % (95% CI)
Overall (n = 3,985)
20.8 (17.5–24.1)
13.4 (11.9–15.0)
65.8 (62.1–69.4)
Sex
Female (n = 2,009)
19.4 (16.2–22.7)
15.0 (13.2–16.9)
65.5 (61.8–69.3)
Male (n = 1,947)
22.2 (17.9–26.5)
11.8 (9.6–14.0)
66.0 (61.1–70.9)
Grade
9th (n = 1,612)
14.3 (10.9–7.7)
11.9 (9.7–14.2)
73.8 (69.5–78.1)
10th (n = 1,308)
16.2 (12.8–19.6)
12.9 (10.8–14.9)
71.0 (66.7–75.2)
11th (n = 663)
18.7 (14.3–23.2)
14.4 (10.8–18.0)
66.9 (61.5–72.2)
12th (n = 394)
35.9 (27.7–44.0)
14.8 (11.2–18.4)
49.3 (39.7–58.9)
Race/ethnicity
White (n = 2,988)
21.3 (17.2–25.4)
13.4 (11.8–15.1)
65.2 (60.5–70.0)
Black (n = 478)
18.4 (11.4–25.4)
15.5 (11.0–20.0)
66.1 (58.7–73.5)
Hispanic (n = 182)
24.2 (15.6–32.8)
14.6 (9.0–20.3)
61.1 (52.9–69.4)
Other (n = 285)
18.4 (10.5–26.2)
6.2 (4.2–8.3)
75.4 (67.3–83.5)
Abbreviation: CI, confidence interval. a Binge drinkers were defined as students who had 5 or more drinks of alcohol in a row within a couple of hours on at least 1 day during the 30 days before the
survey. b Nonbinge drinkers were defined as students who had at least 1 drink of alcohol on at least 1 day during the 30 days before the survey who did not report binge
drinking. c Nondrinkers were defined as students who reported consuming no alcoholic drinks in the 30 days before the survey. e, or Nondrinking Among Michigan 9th Through 12th Grade Students, by Demographic Char-
urvey, 2011 The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services,
the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. g to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services,
ce, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. www.cdc.gov/pcd/issues/2015/15_0290.htm • Centers for Disease Control and Prevention 7 www.cdc.gov/pcd/issues/2015/15_0290.htm • Centers for Disease Control and Preventio Centers for Disease Control and Prevention • www.cdc.gov/pcd/issues/2015/15_0290.htm The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services,
the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. PREVENTING CHRONIC DISEASE
PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY VOLUME 12, E194
NOVEMBER 2015 PREVENTING CHRONIC DISEASE
PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY VOLUME 12, E194
NOVEMBER 2015 Table 3. PREVENTING CHRONIC DISEASE
PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY Distribution of Usual Types of Alcohol Consumeda Among Current (Binge and Nonbinge) Drinkers, by Sociodemographic
Characteristics and Consumption Patterns, Michigan Youth Tobacco Survey, 2011
Characteristic
Liquor, % (95% CI)
Beer, % (95% CI)
Flavored Alcoholic
Beverages, % (95% CI)
No Usual Type,
% (95% CI)
Other Typeb, %
(95% CI)
Overall (n = 963)
51.2 (47.5–54.8) 17.9 (14.4–21.5)
14.5 (11.0–18.1)
5.2 (3.2–7.3)
11.2 (7.0–15.3)
Sex
Female (n = 485)
53.6 (46.2–61.0)
11.1 (7.2–15.1)
21.0 (15.3–26.6)
5.8 (2.6–8.9)
8.5 (3.9–13.1)
Male (n = 473)
49.0 (45.4–52.6) 24.3 (18.6–29.9)
8.1 (4.8–11.5)
4.7 (2.3–7.1)
13.9 (8.7–19.0)
Grade
9th (n = 324)
51.4 (44.6–58.1) 16.2 (10.7–21.8)
15.1 (11.0–19.2)
6.0 (3.3–8.6)
11.4 (7.1–15.6)
10th (n = 294)
53.7 (47.6–59.7)
13.5 (9.6–17.5)
16.5 (10.9–22.2)
7.1 (4.0–10.1)
9.2 (5.5–13.0)
11th (n = 174)
49.9 (42.5–57.3)
13.4 (7.3–19.5)
18.6 (11.0–26.3)
2.4 (0.0–4.7)
15.7 (5.5–25.9)
12th (n = 170)
50.5 (41.6–59.4) 24.6 (18.6–30.6)
10.3 (4.5–16.1)
5.6 (0.8–10.4)
9.0 (4.1–13.9)
Race/ethnicity
White (n = 746)
52.1 (48.5–55.7) 19.6 (15.3–23.9)
12.8 (9.0–16.7)
5.8 (3.7–7.9)
9.7 (5.5–13.8)
Black (n = 110)
48.9 (35.7–62.0)
10.1 (1.9–18.3)
23.4 (10.9–35.9)
3.1 (0.0–8.2)
14.5 (6.1–22.9)
Hispanic (n = 50)
55.1 (36.8–73.4)
16.0 (1.4–30.7)
11.7 (2.4–21.0)
4.0 (0.0–9.1)
13.1 (0.1–26.2)
Drinking status
Nonbinge drinkersc (n = 406)
46.9 (39.7–54.1) 17.1 (13.2–20.9)
17.6 (12.4–22.9)
5.5 (1.5–9.6)
12.9 (5.3–20.5)
Binge drinkersd (n = 551)
54.2 (49.1–59.3) 18.6 (13.5–23.7)
12.1 (8.2–16.1)
5.1 (2.7–7.4)
10.0 (6.4–13.7)
Usual place of consumption
My home (n = 340)
37.9 (30.5–45.3) 21.2 (15.5–26.9)
18.3 (12.6–24.0)
6.8 (3.9–9.8)
15.7 (9.2–22.2)
Someone else’s home (n = 546)
60.2 (54.7–65.7)
15.4 (9.8–21.1)
13.4 (8.0–18.7)
5.0 (3.3–6.6)
6.0 (2.8–9.2)
Other placee (n = 163)
41.7 (31.6–51.7) 20.3 (12.7–27.9)
13.1 (6.0–20.2)
6.5 (0.3–12.7) 18.4 (11.9–24.9)
Number of drinking days
1 or 2 (n = 463)
47.8 (41.5–54.1) 17.8 (13.6–22.0)
18.1 (12.5–23.7)
5.0 (1.6–8.4)
11.4 (5.1–17.7)
3–9 (n = 355)
57.6 (51.8–63.5) 16.2 (10.7–21.6)
11.7 (7.8–15.5)
4.9 (2.8–6.9)
9.7 (5.4–13.9)
10–30 (n = 145)
45.9 (36.6–55.2) 22.6 (14.9–30.4)
10.7 (2.4–19.0)
6.7 (0.5–13.0)
14.0 (6.8–21.3)
Largest number of drinks on any occasion
1–3 (n = 474)
46.2 (41.0–51.3) 16.7 (11.9–21.5)
18.7 (14.2–23.1)
4.8 (1.3–8.3)
13.7 (7.7–19.7)
4 or 5 (n = 176)
56.9 (48.1–65.7)
12.3 (5.5–19.1)
18.3 (11.3–25.4)
5.2 (2.4–8.0)
7.3 (2.4–12.2)
≥6 (n = 328)
55.1 (49.9–60.3) 23.4 (17.0–29.8)
7.1 (4.0–10.3)
5.2 (1.3–9.1)
9.1 (5.5–12.8)
Mixed energy drinks and alcohol (n = 432)
52.7 (45.1–60.3) 18.0 (12.1–23.9)
12.2 (7.0–17.4)
5.2 (2.8–7.6)
12.0 (6.7–17.2)
Abbreviation: CI, confidence interval. VOLUME 12, E194
NOVEMBER 2015 Table 2. Distribution of Alcohol Consumption Characteristics Among Students Who Were Binge or Nonbinge Drinkers, Michigan
Youth Tobacco Survey, 2011
Characteristic
Binge Drinkersa, n = 698, % (95% CI)
Nonbinge Drinkersb, n = 504, % (95%
CI)
Usual place of consumption
My home (n = 307)
22.7 (18.0–27.5)
37.7 (32.2–43.2)
Someone else’s home (n = 529)
61.6 (55.4–67.7)
44.0 (37.6–50.4)
Restaurant/bar (n = 30)
3.6 (1.9–5.3)
2.7 (0.6–4.9)
Other placec (n = 123)
12.1 (8.5–15.7)
15.6 (10.3–20.8)
Number of drinking days
1 or 2 (n = 577)
27.3 (24.0–30.6)
75.3 (71.2–79.3)
3–9 (n = 434)
48.3 (44.2–52.4)
20.3 (16.4–24.1)
10–30 (n = 191)
24.4 (21.1–27.7)
4.5 (1.9–7.0)
Mixed energy drinks and alcohol (n = 431)
49.0 (43.7–54.2)
18.2 (13.1–23.3)
Abbreviation: CI, confidence interval. a Binge drinkers were defined as students who had 5 or more drinks of alcohol in a row within a couple of hours on at least 1 day during the 30 days before the
survey. b Nonbinge drinkers were defined as students who had at least 1 drink of alcohol on at least 1 day during the 30 days before the survey who did not report binge
drinking. c Other place was defined as riding or driving in a car; at a beach, park, concert, or sporting event; or on school property. Abbreviation: CI, confidence interval. a Binge drinkers were defined as students who had 5 or more drinks of alcohol in a row within a couple of hours on at least 1 day during the 30 days before the
survey. b Nonbinge drinkers were defined as students who had at least 1 drink of alcohol on at least 1 day during the 30 days before the survey who did not report binge
drinking. c Other place was defined as riding or driving in a car; at a beach, park, concert, or sporting event; or on school property. 8 www.cdc.gov/pcd/issues/2015/15_0290.htm • Centers for Disease Control and Prevention 9 PREVENTING CHRONIC DISEASE
PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY a Survey question to assess usual types of alcohol was, “During the past 30 days, what type of alcohol did you usually drink? (Select only one response)”. b Other type of alcohol includes wine and cordials. c Nonbinge drinkers were defined as students who had at least 1 drink of alcohol on at least 1 day during the 30 days before the survey, but did not report binge
drinking. d Binge drinkers were defined as students who had 5 or more drinks of alcohol in a row within a couple of hours on at least 1 day during the 30 days before the
survey. e Other place was defined as a bar or restaurant; riding or driving in a car; at a park, beach, concert, or sporting event; or on school property. The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services,
the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. Abbreviation: CI, confidence interval. www.cdc.gov/pcd/issues/2015/15_0290.htm • Centers for Disease Control and Prevention 9 9 0 Centers for Disease Control and Prevention • www.cdc.gov/pcd/issues/2015/15_0290.htm pes of alcohol was, “During the past 30 days, what type of liquor did you usually drink? (Select only one response)”. Survey question to assess usual types of alcohol was, During the past 30 days, what type of liquor did you usually drink? (Select only one response) .
b Nonbinge drinkers were defined as students who had at least 1 drink of alcohol on at least 1 day during the 30 days before the survey, but did not report binge
drinking.
c Binge drinkers were defined as students who had 5 or more drinks of alcohol in a row within a couple of hours on at least 1 day during the 30 days before the sur-
vey.
d Other place was defined as bar or restaurant; while riding or driving in a car; at a park, beach, concert, or sporting event; or on school property.
(continued on next page)
The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services,
the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. PREVENTING CHRONIC DISEASE
PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY (Select only one response)”. b Abbreviation: CI, confidence interval. a Survey question to assess usual types of alcohol was, “During the past 30 days, what type of liquor did you usually drink? (Select only one response)”. b N
bi g d i k
d fi
d
t d
t
h h d t l
t 1 d i k f l
h l
t l
t 1 d
d
i g th 30 d
b f
th
b t did
t
t bi g a Survey question to assess usual types of alcohol was, “During the past 30 days, what type of liquor did you usually drink? (Select only one response)”. b Nonbinge drinkers were defined as students who had at least 1 drink of alcohol on at least 1 day during the 30 days before the survey, but did not report binge
drinking. c Binge drinkers were defined as students who had 5 or more drinks of alcohol in a row within a couple of hours on at least 1 day during the 30 days before the sur-
vey. d Other place was defined as bar or restaurant; while riding or driving in a car; at a park, beach, concert, or sporting event; or on school property. (continued on next page)
The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services,
the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. 10
Centers for Disease Control and Prevention
www cdc gov/pcd/issues/2015/15 0290 htm a Survey question to assess usual types of alcohol was, “During the past 30 days, what type of liquor did you usually drink? (Select only one response)”. b Nonbinge drinkers were defined as students who had at least 1 drink of alcohol on at least 1 day during the 30 days before the survey, but did not report binge
drinking Survey question to assess usual types of alcohol was, During the past 30 days, what type of liquor did you usually drink? (Select only one response) . b Nonbinge drinkers were defined as students who had at least 1 drink of alcohol on at least 1 day during the 30 days before the survey, but did not report binge
drinking. PREVENTING CHRONIC DISEASE
PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY VOLUME 12, E194
NOVEMBER 2015 Table 4. Distribution of Usual Type of Liquor Consumeda Among Current (Binge and Nonbinge) Drinkers, by Demographic Charac-
teristics and Consumption Patterns, Michigan Youth Tobacco Survey, 2011
Characteristic
Didn’t Drink
Liquor,
% (95% CI)
Vodka,
% (95% CI)
Rum,
% (95% CI)
Whiskey,
% (95% CI)
Tequila,
% (95% CI)
No Usual Type,
% (95% CI)
Other Type,
% (95% CI)
Overall (n =
1,146)
19.1
(15.6–22.5)
37.3
(33.2–41.1)
11.6 (8.9–14.2)
8.7 (5.6–11.7)
4.6 (3.0–6.3)
10.1 (7.8–12.5)
8.6 (6.0–11.2)
Sex
Female (n =
577)
16.7
(11.4–22.0)
42.8
(36.4–49.1)
7.8 (5.5–10.1)
4.4 (1.8–7.0)
5.8 (3.6–8.0)
13.0 (8.9–17.0)
9.6 (5.4–13.8)
Male (n = 561)
21.4
(18.6–24.3)
31.7
(28.2–35.2)
15.4
(11.5–19.4)
13.0 (9.3–16.8)
3.2 (1.2–5.2)
7.4 (4.1–10.6)
7.8 (4.8–10.8)
Grade
9th (n = 384)
21.1
(13.9–28.3)
37.0
(30.9–43.2)
9.7 (5.8–13.6)
9.4 (5.8–13.1)
4.4 (2.4–6.4)
8.0 (4.2–11.9)
10.4 (6.5–14.2)
10th (n = 359)
20.6
(13.4–27.8)
33.5
(26.5–40.4)
9.1 (5.6–12.6)
8.2 (4.3–12.1)
4.1 (2.0–6.3)
11.6 (7.7–15.5)
12.9 (8.0–17.8)
11th (n = 212)
23.6
(16.4–30.8)
36.8
(27.6–45.9)
10.9 (6.5–15.4)
5.7 (3.1–8.4)
3.6 (1.3–5.9)
10.0 (4.7–15.3)
9.4 (3.9–15.0)
12th (n = 190)
13.7 (7.2–20.2)
40.4
(31.1–49.6)
14.8
(10.5–19.1)
10.6 (3.9–17.3)
5.7 (1.8–9.6)
10.6 (5.8–15.5)
4.2 (1.3–7.1)
Race/ethnicity
White (n = 871)
19.9
(15.6–24.1)
39.4
(34.2–44.5)
12.3 (9.7–15.0)
8.9 (5.5–12.4)
3.7 (1.8–5.6)
9.7 (7.1–12.4)
6.0 (4.4–7.6)
Black (n = 141)
16.5 (9.2–23.9)
32.9
(21.4–44.5)
7.3 (1.2–13.3)
5.1 (1.3–8.9)
5.8 (0.5–11.1)
12.6 (3.6–21.5)
19.8
(11.0–28.6)
Hispanic (n =
63)
15.2 (7.4–23.0)
29.8
(16.5–43.1)
12.0 (2.5–21.4)
10.5 (1.4–19.7)
18.5 (2.2–34.9)
7.9 (2.0–13.9)
6.1 (0.1–12.0)
Drinking behavior
Nonbinge
drinkersb (n =
491)
26.2
(22.3–30.0)
30.7
(23.5–38.0)
10.0 (5.9–14.2)
6.1 (3.3–8.9)
5.0 (2.3–7.7)
11.5 (7.7–15.3)
10.5 (6.4–14.6)
Binge drinkersc
(n = 647)
14.1
(10.0–18.2)
42.1
(38.2–46.1)
12.6 (9.3–15.9)
10.5 (6.8–14.3)
4.4 (2.0–6.8)
9.3 (6.5–12.1)
6.9 (4.3–9.5)
Usual place of consumption
My home (n =
304)
14.2 (8.4–20.0)
30.9
(22.6–39.2)
10.5 (6.8–14.2)
10.9 (5.4–16.4)
7.6 (4.2–11.0)
14.3 (8.4–20.3)
11.6 (5.7–17.5)
Someone else’s
home (n = 513)
8.3 (5.2–11.5)
49.7
(43.8–55.7)
12.7 (8.8–16.5)
7.9 (4.8–11.0)
3.0 (1.1–4.8)
10.1 (7.0–13.3)
8.2 (4.9–11.6)
Other placed (n =
144)
13.2 (8.3–18.0)
30.9
(23.2–38.7)
20.0
(11.6–28.4)
9.5 (1.5–17.5)
8.1 (4.6–11.6)
10.0 (1.9–18.1)
8.3 (4.5–12.2)
Abbreviation: CI, confidence interval. iquor Consumeda Among Current (Binge and Nonbinge) Drinkers, by Demographic Charac-
chigan Youth Tobacco Survey, 2011 Abbreviation: CI, confidence interval. a Survey question to assess usual types of alcohol was, “During the past 30 days, what type of liquor did you usually drink? Abbreviation: CI, confidence interval.
a Survey question to assess usual types of alcohol was, “During the past 30 days, what type of liquor did you usually drink? (Select only one response)”.
b Nonbinge drinkers were defined as students who had at least 1 drink of alcohol on at least 1 day during the 30 days before the survey, but did not report binge
drinking.
c Binge drinkers were defined as students who had 5 or more drinks of alcohol in a row within a couple of hours on at least 1 day during the 30 days before the sur-
vey.
d Other place was defined as bar or restaurant; while riding or driving in a car; at a park, beach, concert, or sporting event; or on school property. PREVENTING CHRONIC DISEASE
PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY c Binge drinkers were defined as students who had 5 or more drinks of alcohol in a row within a couple of hours on at least 1 day during the 30 days before the sur-
vey. d Other place was defined as bar or restaurant; while riding or driving in a car; at a park, beach, concert, or sporting event; or on school property. (continued on next page)
The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services,
the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. 10 Centers for Disease Control and Prevention • www.cdc.gov/pcd/issues/2015/15_0290.htm 10 Centers for Disease Control and Prevention • www.cdc.gov/pcd/issues/2015/15_0290.htm 10 PREVENTING CHRONIC DISEASE
PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY pes of alcohol was, “During the past 30 days, what type of liquor did you usually drink? (Select only one response)”. Abbreviation: CI, confidence interval.
a PREVENTING CHRONIC DISEASE
PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY VOLUME 12, E194
NOVEMBER 2015 Abbreviation: CI, confidence interval.
a Survey question to assess usual types of alcohol was, “During the past 30 days, what type of liquor did you usually drink? (Select only one response)”.
b Binge drinkers were defined as students who had 5 or more drinks of alcohol in a row within a couple of hours on at least 1 day during the 30 days before the sur
vey.
d Other place was defined as bar or restaurant; while riding or driving in a car; at a park, beach, concert, or sporting event; or on school property. Abbreviation: CI, confidence interval. (continued) (continued)
Table 4. Distribution of Usual Type of Liquor Consumeda Among Current (Binge and Nonbinge) Drinkers, by Demographic Charac-
teristics and Consumption Patterns, Michigan Youth Tobacco Survey, 2011
Characteristic
Didn’t Drink
Liquor,
% (95% CI)
Vodka,
% (95% CI)
Rum,
% (95% CI)
Whiskey,
% (95% CI)
Tequila,
% (95% CI)
No Usual Type,
% (95% CI)
Other Type,
% (95% CI)
Number of drinking days
1 or 2 (n = 556)
23.7
(19.2–28.2)
34.9
(28.4–41.4)
10.2 (7.0–13.4)
5.0 (1.6–8.4)
4.2 (2.1–6.4)
10.9 (7.5–14.2)
11.1 (7.2–15.0)
3–9 (n = 412)
15.4
(10.2–20.6)
42.2
(36.9–47.6)
13.4 (9.4–17.4)
11.2 (6.4–15.9)
4.4 (1.9–6.8)
8.8 (5.7–12.0)
4.6 (2.1–7.1)
10–30 (n = 178)
14.2 (7.7–20.6)
32.7
(24.5–40.9)
11.3 (5.9–16.8)
13.5 (5.2–21.9)
6.5 (1.2–11.8)
11.1 (4.3–17.9)
10.7 (5.5–15.9)
Largest number of drinks on any occasion
1–3 (n = 510)
21.7
(17.4–26.1)
36.6
(30.6–42.6)
9.5 (6.1–13.0)
6.3 (3.1–9.5)
4.3 (1.8–6.7)
10.2 (6.1–14.3)
11.3 (7.2–15.5)
4 or 5 (n = 188)
14.7 (8.0–21.4)
41.9
(33.8–50.0)
10.5 (4.9–16.0)
9.1 (5.2–13.0)
5.9 (2.2–9.6)
14.2 (6.9–21.4)
3.9 (0.7–7.0)
≥6 (n = 366)
12.3 (7.9–16.7)
41.0
(34.6–47.4)
13.9 (8.2–19.6)
10.9 (5.8–16.1)
5.1 (1.8–8.4)
8.9 (4.3–13.4)
7.8 (4.2–11.5)
Mixed energy
drinks and
alcohol (n =
404)
5.9 (2.1–9.7)
47.5
(42.2–52.7)
12.6 (8.2–17.1)
10.8 (6.4–15.1)
6.3 (3.3–9.3)
9.4 (5.4–13.4)
7.5 (4.5–10.5) Table 4. Distribution of Usual Type of Liquor Consumeda Among Current (Binge and Nonbing
teristics and Consumption Patterns, Michigan Youth Tobacco Survey, 2011 The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services,
the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. g to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services,
ce, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services,
the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. www cdc gov/pcd/issues/2015/15 0290 htm • Centers for Disease Control and Prevention
11 www.cdc.gov/pcd/issues/2015/15_0290.htm • Centers for Disease Control and Prevention 11 www.cdc.gov/pcd/issues/2015/15_0290.htm • Centers for Disease Control and Preven
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https://openalex.org/W3091487379
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https://bmcpublichealth.biomedcentral.com/counter/pdf/10.1186/s12889-020-09558-9
|
English
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Propensity for COVID-19 severe epidemic among the populations of the neighborhoods of Fortaleza, Brazil, in 2020
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BMC public health
| 2,020
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cc-by
| 5,729
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Open Access Propensity for COVID-19 severe epidemic
among the populations of the
neighborhoods of Fortaleza, Brazil, in 2020 Jose Ueleres Braga1,2*
, Alberto Novaes Ramos Jr3,4, Anderson Fuentes Ferreira4, Victor Macêdo Lacerda5,
Renan Monteiro Carioca Freire5 and Bruno Vieira Bertoncini6,7 Abstract Background: The state of Ceará (Northeast Brazil) has shown a high incidence of coronavirus disease (COVID-19),
and most of the cases that were diagnosed during the epidemic originated from the capital Fortaleza. Monitoring
the dynamics of the COVID-19 epidemic is of strategic importance and requires the use of sensitive tools for
epidemiological surveillance, including consistent analyses that allow the recognition of areas with a greater
propensity for increased severity throughout the cycle of the epidemic. This study aims to classify neighborhoods in
the city of Fortaleza according to their propensity for a severe epidemic of COVID-19 in 2020. Methods: We conducted an ecological study within the geographical area of the 119 neighborhoods located in
the city of Fortaleza. To define the main transmission networks (infection chains), we assumed that the spatial
diffusion of the COVID-19 epidemic was influenced by population mobility. To measure the propensity for a severe
epidemic, we calculated the infectivity burden (ItyB), infection burden (IonB), and population epidemic vulnerability
index (PEVI). The propensity score for a severe epidemic in the neighborhoods of the city of Fortaleza was
estimated by combining the IonB and PEVI. Results: The neighborhoods with the highest propensity for a severe COVID-19 epidemic were Aldeota, Cais do
Porto, Centro, Edson Queiroz, Vicente Pinzon, Jose de Alencar, Presidente Kennedy, Papicu, Vila Velha, Antonio
Bezerra, and Cambeba. Importantly, we found that the propensity for a COVID-19 epidemic was high in areas with
differing socioeconomic profiles. These areas include a very poor neighborhood situated on the western border of
the city (Vila Velha), neighborhoods characterized by a large number of subnormal agglomerates in the Cais do
Porto region (Vicente Pinzon), and those located in the oldest central area of the city, where despite the wealth,
low-income groups have remained (Aldeota and the adjacent Edson Queiroz). * Correspondence: ueleres@gmail.com * Correspondence: ueleres@gmail.com p
g
1Department of Epidemiology, Institute of Social Medicine, State University of
Rio de Janeiro, Rua São Francisco Xavier 524 / 7017D, 7° floor, Rio de Janeiro,
RJ CEP 20550-013, Brazil 1Department of Epidemiology, Institute of Social Medicine, State University of
Rio de Janeiro, Rua São Francisco Xavier 524 / 7017D, 7° floor, Rio de Janeiro,
RJ CEP 20550-013, Brazil 2Department of Epidemiology and Quantitative Methods, Sergio Arouca
National School of Public Health (ENSP), Oswaldo Cruz Foundation
(FIOCRUZ), Rio de Janeiro, RJ, Brazil
Full list of author information is available at the end of the article Braga et al. BMC Public Health (2020) 20:1486
https://doi.org/10.1186/s12889-020-09558-9 Braga et al. BMC Public Health (2020) 20:1486
https://doi.org/10.1186/s12889-020-09558-9 (Continued from previous page) (Continued from previous page)
Conclusion: Although measures against COVID-19 should be applied to the entire municipality of Fortaleza, the
classification of neighborhoods generated through this study can help improve the specificity and efficiency of
these measures. Keywords: COVID-19, Public health surveillance, Disease outbreaks Background overall epidemic burden and requires the use of sensitive
tools for epidemiological surveillance, including analyses
that allow the recognition of areas with a greater pro-
pensity for increased severity throughout the epidemic
cycle. In addition to their technical-scientific character,
these analyses must be strongly integrated with operational
aspects of surveillance and control, with cooperation of
public managers of the municipality in the health, trans-
port, and education sectors, to achieve timely and effective
integrated responses like allocation of resources and med-
ical personnel depending on determined propensity of
severe epidemic. The first case of coronavirus disease (COVID-19) was
recorded on December 8, 2019, in Wuhan, Hubei prov-
ince, China. Approximately 3 months later, on March
11, 2020, this disease was declared as a pandemic by the
World Health Organization (WHO) [1]. On February 3,
2020, the Brazilian Ministry of Health declared COVID-19
as a Public Health Emergency of National Importance
(ESPIN), through Ordinance MS n. 188 of 2020, in line
with Decree n. 7616, of November 17, 2011. The first confirmed COVID-19 case in Latin America
was reported on February 26, 2020, in the city of São
Paulo, the most populous city in the Southern Hemisphere
(approximately 12 million inhabitants), and involved a
person who had travelled to northern Italy (Lombardia
region) [2, 3]. Approximately a month later, on March
20, Ordinance MS n. 454 was published in the Official
Gazette (Edition: 55-F / Section 1 - Extra), declaring
the existence of community transmission of COVID-19
throughout the national territory of Brazil. Since then,
Brazil has registered the largest number of confirmed
cases in Latin America (n = 20,727, 18:00, April 11,
2020) [4]. The study proposes to increase the understanding of
the COVID-19 epidemic in Fortaleza, enabling the rec-
ognition of urban spaces with a greater propensity for a
severe epidemic based on population mobility in order
to promote evidence-based actions by the municipality
public authorities. This study aims to classify neighbor-
hoods in the city of Fortaleza by their propensity for a
severe COVID-19 epidemic in 2020. © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,
which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give
appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if
changes were made. The images or other third party material in this article are included in the article's Creative Commons
licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons
licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain
permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the
data made available in this article, unless otherwise stated in a credit line to the data. Page 2 of 11 Braga et al. BMC Public Health (2020) 20:1486 Study design Ceará has become one of the states with the highest
number of cases of the disease (1374), with only São
Paulo and Rio de Janeiro reporting higher numbers
(6708 and 1938, respectively). While the average inci-
dence rate in Brazil is 7.5 cases per 100,000 inhabitants,
the rate in Ceará is double at 14.1 per 100,000 inhabi-
tants [5]. The highest number of cases and deaths is
concentrated in Fortaleza (1291 confirmed cases and 53
deaths), making it the Brazilian state capital with the
highest incidence rate (35/100,000), and a mortality rate
of 3.6% among confirmed cases [4]. This is an ecologic study based on the geographical unit
of the 119 neighborhoods in the municipality of Forta-
leza (Fig. 1). In order to define the main transmission
networks (and chains), we hypothesized that the spatial
diffusion of the COVID-19 epidemic is influenced by
population mobility. We also postulated that the spatial
spread of the COVID-19 epidemic follows the hierarch-
ical model based on networks and population mobility
that plays a key role in the constitution of the viral
transmission chains. The rationale of the study assumed that based on the
incidence rate of the initial phase of the epidemic (in-
fectivity load), the flow of passengers moving between
neighborhoods can influence the infection load in each
neighborhood of the municipality. If one considers the
epidemic vulnerability attributed to populations living in
these neighborhoods, combined with the mentioned
infection burden, one can estimate the propensity for a
serious COVID-19 epidemic. Numerous factors have been considered important to
explain the magnitude, intensity, and early viral circula-
tion of the epidemic in Fortaleza. Of these, the following
two aspects appear to be the most crucial: firstly, the
Pinto Martins International Airport in Fortaleza, which
became a “flight hub” in 2018, facilitated the influx of a
large number of national and international flights and
tourists; and secondly, the measures adopted by the state
epidemiological surveillance services included testing a
relatively large number of suspected cases. To measure the propensity of a severe epidemic, the
following events were initially calculated: (i) infectivity
burden - ItyB; (ii) infection burden - IonB; and (iii) popu-
lation epidemic vulnerability index (PEVI). Then, the (iv) Monitoring the dynamics of the COVID-19 epidemic
in Fortaleza is of strategic importance for reducing Braga et al. BMC Public Health (2020) 20:1486 Page 3 of 11 Fig. Infectivity burden Infectivity burden was calculated using the epidemio-
logical surveillance records of COVID-19 in Fortaleza,
so the number of confirmed cases were denoted as in-
fectivity burden. These data were formally obtained from
the Municipal Health Department on April 7, 2020. The
suspected cases of COVID-19 had been investigated
according to the recommendations of the Ministry of
Health. All confirmed COVID-19 cases with SARS-CoV-2
PCR positive status reported to the Municipal Health De-
partment of Fortaleza until March 12, 2020, residing in the
municipality, were included. This period corresponds to the
initial phase of the epidemic, when predominantly imported
cases (travelers) initiated the transmission; indigenous cases Study design 1 Map of the 119 neighborhoods in the municipality of Fortaleza Fig. 1 Map of the 119 neighborhoods in the municipality of Fortaleza Propensity score for a severe epidemic in the neighbor-
hoods of the city of Fortaleza was estimated. would be detected later after being in contact with the
initial cases through community transmission [6]. In
this phase, the Fortaleza City Council adopted more
general measures for surveillance, prevention, and con-
trol of COVID-19 [7]. Infection burden The Infection burden was measured by combining the
infectivity burden and population mobility between the
neighborhoods of Fortaleza. The mobility burden - MtyB,
was evaluated through the daily travel flows, by looking
at public transport use with work motivation between
the two neighborhoods. Notably, the measurement of
the flow of people between the neighborhoods of Forta-
leza (excluding displacements within the same neighbor-
hood) only became possible after a strategic study was
conducted by the city of Fortaleza and other institutions Braga et al. BMC Public Health (2020) 20:1486 Page 4 of 11 Page 4 of 11 Page 4 of 11 on this theme in 2019 called Home Origin-Destination
Survey (OD survey). on this theme in 2019 called Home Origin-Destination
Survey (OD survey). HDUs were designed to generate more homogeneous areas,
based on socioeconomic conditions, than the weighted
areas of the IBGE. To calculate the proportion of subnor-
mal agglomerations, data from the IBGE were used, consid-
ering the population living under these conditions by the
total population of the neighborhood, thereby obtaining the
percentage of people living in subnormal agglomerations
per neighborhood. HDUs were designed to generate more homogeneous areas,
based on socioeconomic conditions, than the weighted
areas of the IBGE. To calculate the proportion of subnor-
mal agglomerations, data from the IBGE were used, consid-
ering the population living under these conditions by the
total population of the neighborhood, thereby obtaining the
percentage of people living in subnormal agglomerations
per neighborhood. The OD survey comprised a sample survey performed
through interviews in households, providing the values
of the variables in this analysis with a detailed matrix of
the trip patterns and travel choices. Data were collected
in a database, which describes the various attributes of
the activities and trips of the city’s inhabitants, as well as
the respective socioeconomic status and characteristics
of individuals and their families, in order to describe a
pattern of displacement of people and the chain of their
activities throughout a typical business day. The infor-
mation from the OD Matrix is of immense importance
in the analysis of transport systems, comprising funda-
mental elements for planning and decision making, and
has therefore been integrated in this study. PEVI The PEVI was constructed according to the Urban
Health Index approach recommended by WHO [8], to
demonstrate the population attributes that best repre-
sent, from a collective point of view, the vulnerability of
the population to COVID-19. This index comprises
seven (7) sociodemographic indicators, based on the
2010 Brazilian census of the Brazilian Institute of Geog-
raphy and Statistics (IBGE). The indicators that make-up
the PEVI are: (i) proportion of households with more
than two residents per bedroom, (ii) illiteracy proportion,
(iii) proportion of the population in extreme poverty, (iv)
proportion of households without running water and sani-
tation, (v) proportion of unemployment, (vi) Gini of family
income, and (vii) proportion of people living in subnormal
agglomerations. Since the Ip values are obtained for all indicators and
units, the next step was to integrate Ip into a single com-
posite index, here called VEPI. VEPI is calculated for
each unit, using the geometric mean of the Ip values for
each unit. Considering that there are j indicators, the
formula used for this calculation was: VEPI ¼
Y
j
i¼1
Ip
i
!1
j Where, Ip
i is the standardized value of the seven indi-
cators for a given neighborhood, and j corresponds to all
other neighborhoods. In addition to the point estimate, VEPI variance and
standard error were also calculated, with respective
confidence interval estimates based on these measures. Considering that the purpose of this index is to identify
the “geographical” disparities of the studied phenomenon,
a diagnosis of its distribution, evaluating the differences
between the highest and lowest values as well as its visual
comparison with the homogeneous distribution of these
values, was carried out through the “qqnorm” graph
(Fig. 2). IBGE classifies subnormal agglomerates groups as
consisting of 51 or more housing units, characterized by
the absence of ownership titles, and at least one of the
following
characteristics:
irregularity
of
circulation
routes, size and shape of the lots, and lack of essential
public services (such as garbage collection, sewage,
water, electricity, and public lighting). The data for each of the indicators above were previously
obtained and translated into the Human Development
Units (HDUs) by the Institute for Applied Economic
Research (IPEA), with the exception of the proportion of
subnormal agglomerations. The HDUs represent units of
analysis
with
relatively
homogeneous
socioeconomic
characteristics, and the original data were used to produce
the Metropolitan Region Human Development Atlas. The Infection burden The infection
burden indicator was calculated using the following
formula: After the values of the indicators were stored in a
database, the accuracy, completeness, and consistency of
the data were verified for calculation of the summary
measure. There are two main steps in calculating the
index: (1) standardization of indicators, and (2) amal-
gamation of standardized indicators. Each of these steps
can be performed in a mathematically straightforward
manner. The standardization of the values of each indicator is
performed using the following formula: Ip ¼
I −min Ið Þ
max Ið Þ −min Ið Þ IonBi ¼
X
118
j¼1
ItyBMtyB Ip ¼
I −min Ið Þ
max Ið Þ −min Ið Þ where Ip is the standardized value of I, max (I) is the
highest value of I among all observations, and min (I) is
the lowest value of I among all observations. Severe epidemic population propensity The main study outcome is the Severe Epidemic Popula-
tion Propensity. The estimated score of the propensity
for a severe epidemic amongst the population of the
Fortaleza neighborhoods was calculated by combining Braga et al. BMC Public Health (2020) 20:1486 Page 5 of 11 Fig. 2 Flow Diagram for constructing the population epidemic vulnerability index Fig. 2 Flow Diagram for constructing the population epidemic vulnerability index the infection burden with PEVI. The calculation was
carried out using the multiplicative approach, involving
the multiplication of these two indexes. distribution is, therefore, similar to the distribution of
incidence, and compatible with the surveillance strategy
adopted by the state and municipal governments for this
first stage of the epidemic curve caused by COVID-19. Results The distribution of the infection burden (Fig. 4) was
quite heterogeneous, with some areas showing higher
levels than those of the central and eastern areas, as this
measure reflects the combination of the infectivity bur-
den and population mobility between the neighborhoods
of Fortaleza. During the initial phase of the epidemic described in this
study, from February 27 to March 12, 2020, 54 cases of
COVID-19 were detected and reported, and this was the
scenario used to study the propensity for a severe
epidemic of populations living in the neighborhoods of
the municipality of Fortaleza. The VEPI index had a very heterogeneous spatial dis-
tribution, similar to the distribution of the seven indica-
tors that were combined for its construction. The
distribution shown in Fig. 5 indicates low vulnerability
in the central and central-eastern regions of the city and
increased levels of vulnerability as the neighborhoods
move away from this region. However, it can be
observed that some areas of the southern periphery, the
western zone, and the eastern coastal zone have the
highest levels of vulnerability. The spatial distribution of COVID-19 incidence in this
period indicates that the epidemic was restricted to spe-
cific areas of the city. This distribution reflects important
heterogeneity, with a concentration of cases in the
central area and in neighborhoods that represent better
socioeconomic conditions, and also in areas with more
diverse socioeconomic conditions, such as Meireles,
Aldeota, Papicu, and Cocó (Fig. 3). During the beginning of the epidemic, the spatial
distribution of the infectivity burden by neighborhood
reflects heterogeneity, concentrated mainly in central
areas and in neighborhoods with the highest economic
conditions. This pattern indicates, at this stage, a low
burden of COVID-19 in the outskirts of the city. This When looking at the distribution of vulnerability to
the COVID-19 epidemic, the combination of the various
attributes related to the seven indicators appears to re-
veal something different than the individual indicators. An example is the vulnerability of the neighborhoods in Braga et al. BMC Public Health (2020) 20:1486 Page 6 of 11 Fig. 3 Map of the distribution of confirmed cases of COVID-19 in the neighborhoods of the Municipality of Fortaleza, from February 27 to March 12, 2020 Discussion the Cais do Porto region, the eastern coastal zone, and
the eastern part of the city, which seemed to have been
mainly
influenced
by
the
existence
of
subnormal
agglomerations. The neighborhoods with the highest propensity for a
severe COVID-19 epidemic were Aldeota, Cais do
Porto, Centro, Edson Queiroz, Vicente Pinzon, Jose
de Alencar, Presidente Kennedy, Papicu, Vila Velha,
Antonio Bezerra, and Cambeba. The main finding of
this study indicates higher levels of propensity to the
COVID-19 epidemic in areas with a wide spectrum of
socioeconomic profiles, including a group of very
poor neighborhoods on the western border of the city
(Vila Velha), a set of neighborhoods characterized by
a large number of subnormal agglomerates in the
Cais do Porto region (Vicente Pizon), and neighbor-
hoods in the oldest central area of the city, where
low-income areas exist despite the overall wealth in
the area (Aldeota and the adjacent Edson Queiroz). The distribution of the propensity shown in Fig. 6
indicates that, besides being heterogeneous, the de-
tected weaknesses (epidemic vulnerability) combined
with the initial situation of the COVID-19 epidemic
(period until March 12, 2020) and the population mo-
bility lead to the prediction that neighborhoods such
as Centro, Aldeota, and Papicu are among those in
the top 10% with the highest propensity score. Other
neighborhoods are also classified in the same group
(western zone, bordered by Caucaia). These results
are not as evident when the various aspects that in-
fluence the epidemic are evaluated separately. Braga et al. BMC Public Health (2020) 20:1486 Page 7 of 11 Fig. 4 Map of the distribution of the COVID-19 infection burden in the neighborhoods of the Municipality of Fortaleza Fig. 4 Map of the distribution of the COVID-19 infection burden in the neighborhoods of the Municipality of Fortaleza Indeed, the propensity for a severe COVID-19 epi-
demic in the neighborhoods of Fortaleza is very hetero-
geneous and reflects not only the population’s mobility
in the urban space, but also the dynamics of transmis-
sion of a disease that is influenced by the living situation
of a population in a city. Recently, an article indicating
that the heterogeneity of the distribution of the inci-
dence of COVID-19 is determined by socio-economic
factors was published on the ABC American communi-
cation network. Discussion This article states that in New York
City, a ‘stark contrast’ in COVID-19 infection rates can
be observed, based on education and ethnicity [9]. in neighborhoods with better economic conditions, such
as those in the eastern and coastal zones. Almost in a
complementary way, the distribution of the proportion of
unemployment showed higher rates in neighborhoods
located in the outskirts of the municipality. A similar situation was observed for indicators of
household agglomeration (more than two people per
bedroom) and households without access to water or
sanitation. The inequality of these distributions indicates
that these phenomena are correlated and probably
express the evolution of the urban space occupation
process in Fortaleza. Very few studies have assessed the spread of the
COVID-19 epidemic, and so far, no articles have been
published that appreciat the influence of specific popula-
tion factors linked to people’s mobility and to predict
the occurrence of severe outbreaks in areas within cities. The seven sociodemographic indicators assessed sep-
arately had spatial distributions with relevant heterogen-
eity. Inequality expressed by income (data not shown)
had a distribution with an expression of less inequality
in the periphery of the municipality and greater inequality Braga et al. BMC Public Health (2020) 20:1486 Page 8 of 11 Fig. 5 Map of the distribution of the population epidemic vulnerability to COVID-19 according to neighborhoods in the Municipality of Fortaleza Spatial analysis was used by Kang et al. [10] to understand
the epidemic spread of COVID-19. While the authors
described the spatiotemporal pattern and evaluated the
spatial association of the early stages of the COVID-19
epidemic in mainland China from January 16 to February
6, 2020, they sought only to identify the occurrence of
spatial autocorrelation measured by Moran’s I for the vari-
ous periods studied. between a given location and the surrounding spatial
units by local Moran’s I (LISA). Giuliani et al. [12] studied the spatiotemporal spread
of COVID-19 in Italy. They sought to model and predict
the number of COVID-19 infections, drawing out the
effects of its spatial diffusion. They argue that “forecasts
about where and when the disease will occur may be of
great usefulness for public decision-makers, as they give
the time to intervene on the local public health systems”. However, the authors did not consider the population
heterogeneities and their influence in predicting the epi-
demic in the studied regions. Fan et al. Discussion [11] studied the epidemiology of the Novel
COVID-19 in Gansu Province, China. They concluded
that different from findings from Wuhan Province, the
spatial distribution pattern analysis indicated hot spots
and spatial outliers in Gansu Province. To detect the
spatial distribution pattern of COVID-19 cases at county
levels during the study periods, they used local indica-
tors of spatial association to evaluate the relationship The study that used the methodological approach
most similar to our study was conducted by Pluchino
et al. [13]. They proposed a data-driven framework for
assessing the epidemic risk of a geographical area (in a Braga et al. BMC Public Health (2020) 20:1486 Page 9 of 11 Fig. 6 Map of COVID-19 severe epidemic propensity according to neighborhoods in the city of Fortaleza Fi
6 M
f COVID 9
id Fig. 6 Map of COVID-19 severe epidemic propensity according to neighborhoods in the city of Fortaleza Our study aims to contribute to mathematical model-
ling studies in a complementary manner, in order to
predict the dynamics of the COVID-19 epidemic in
Brazil. Complementary methodological approaches are
required to broaden the understanding of the epidemic
and its possible determinants. Indeed, many mathemat-
ical models were used to estimate the epidemic curve of
the COVID-19 outbreak in Brazilian cities. Rocha-Filho
et al. [16] used a variant of the SEIR (Susceptible, Exposed,
Infectious, Recovered) classical model, including hospital-
ized variables (SEIHR model) and an age-stratified structure
to analyze the expected time evolution during the onset of
the epidemic in the metropolitan area of São Paulo. predictive way), and to identify high-risk areas within a
country. They constructed a risk index combining three
different features: (1) the disease hazard, (2) the infection
exposure of the area, and (3) its vulnerability. However,
vulnerability was considered based on the local data
regarding air pollution, mobility, winter temperature,
housing concentration, health care density, population
size, and age. g
Public transport is presented as a definite spatial
trend, with trips mainly concentrated in the central and
western regions of the city of Fortaleza, which are
directly related to the provision of public transport and
radio-concentric bus lines. However, as a part of the
assumptions in this approved study, these dimensions
should be considered while studying the transmission
of SARS-CoV-2, in particular using samples of other
respiratory-based infectious diseases [14, 15]. Ethics approval and consent to participate IRB approval is considered unnecessary in accordance with Resolution No. 510, of April 7, 2016 of the National Health Council of Brazil in its first
paragraph and according to the Letter of Consent issued by the Municipal
Health Secretariat of Fortaleza. Discussion One of the main limitations of this study is that the
prediction inherent to the methodological approach does
not specify the time at which the severe epidemic will
most intensely occur in the neighborhoods of Fortaleza. Page 10 of 11 Page 10 of 11 Braga et al. BMC Public Health (2020) 20:1486 Braga et al. BMC Public Health (2020) 20:1486 The simplicity of the approach used in our study, which
does not exhaust all potential factors that influence the
epidemic, is also an advantage over other methods. long as the precepts contained in the data transfer rules of Brazilian law are
met. long as the precepts contained in the data transfer rules of Brazilian law are
met. Author details
1 1Department of Epidemiology, Institute of Social Medicine, State University of
Rio de Janeiro, Rua São Francisco Xavier 524 / 7017D, 7° floor, Rio de Janeiro,
RJ CEP 20550-013, Brazil. 2Department of Epidemiology and Quantitative
Methods, Sergio Arouca National School of Public Health (ENSP), Oswaldo
Cruz Foundation (FIOCRUZ), Rio de Janeiro, RJ, Brazil. 3Department of
Community Health, School of Medicine, Federal University of Ceará (UFC),
Fortaleza, Brazil. 4Postgraduate Program in Public Health, School of Medicine,
Federal University of Ceará (UFC), Fortaleza, Brazil. 5Municipal Secretariat of
Conservation and Public Services (SCSP), Fortaleza, Brazil. 6Department of
Transport Engineering, Technology Centre, Federal University of Ceará (UFC),
Fortaleza, Brazil. 7Postgraduate Program in Transport Engineering,
h
l
d
l
f
á (
)
l
l No funding was obtained for this study. No funding was obtained for this study. References
1
WHO C ESPIN: Public Health Emergency of National Importance; OD: Origin-
Destination; HDU: Human Development Units; IPEA: Institute for Applied
Economic Research; IBGE: Brazilian Institute of Geography and Statistics;
LISA: Local Moran’s I; PEVI: Population epidemic vulnerability index;
WHO: World Health Organization 1. WHO. Coronavirus disease 2019 Situation Report – 51. 2020. https://www. who.int/emergencies/diseases/novel-coronavirus-2019. 2. Faria NR. First cases of coronavirus disease (COVID-19) in Brazil, South
America (2 genomes, 3rd march 2020) - novel 2019 coronavirus / genome
reports - Virological. 2020. http://virological.org/t/first-cases-of-coronavirus-
disease-covid-19-in-brazil-south-america-2-genomes-3rd-march-2020/409. Accessed 13 Apr 2020. 2. Faria NR. First cases of coronavirus disease (COVID-19) in Brazil, South
America (2 genomes, 3rd march 2020) - novel 2019 coronavirus / genome
reports - Virological. 2020. http://virological.org/t/first-cases-of-coronavirus-
disease-covid-19-in-brazil-south-america-2-genomes-3rd-march-2020/409. Accessed 13 Apr 2020. Competing interests The author declares that he has no competing interests. None of the authors
of this paper have a financial or personal relationship with other people or
organizations that could inappropriately influence or bias the content of the
paper. Supplementary information y
Supplementary information accompanies this paper at https://doi.org/10. 1186/s12889-020-09558-9. y
Supplementary information accompanies this paper at https://doi.org/10. 1186/s12889-020-09558-9. Supplementary information accompanie
1186/s12889-020-09558-9 Additional file 1. Letter of consent from the Municipal Health
Secretariat of Fortaleza. Document referring to the consent of the
Municipal Health Secretariat of Fortaleza secretariat giving the notification
data aggregated by neighborhood of residence, which were published in
the epidemiological bulletins and on the Fortaleza city hall website. In
this document there is also reference about the need not submit the
referred project to the ethics committee of that institution, nor of the
other institutions. Received: 27 April 2020 Accepted: 17 September 2020 Received: 27 April 2020 Accepted: 17 September 2020 Acknowledgements The authors would like to thank Edenilo Baltazar Barreira Filho, Francisco
Moraes de Oliveira Neto, Geziel dos Santos de Sousa, Jader de Oliveira
Santos, Manoel Mendonca de Castro Neto, Verônica Teixeira Franco Castelo
Branco, for the valuable suggestions on the analysis and discussion
processes. We would like to thank Joana Angélica Paiva Maciel, Fortaleza’s
municipal health secretary for the transfer of COVID-19 notification data. We
also thank Jorge Ubirajara Pedreira Junior (Polytechnic School of the Federal
University of Bahia - EPUFBA) and Márcio Santos da Natividade (Institute of
Collective Health - ISC / UFBA) for the initial conception of the study. 3. Rodriguez-Morales AJ, Gallego V, Escalera-Antezana JP, Méndez CA,
Zambrano LI, Franco-Paredes C, et al. COVID-19 in Latin America: The
implications of the first confirmed case in Brazil. Travel Med Infect Dis. 2020:
101613. https://doi.org/10.1016/j.tmaid.2020.101613. 3. Rodriguez-Morales AJ, Gallego V, Escalera-Antezana JP, Méndez CA,
Zambrano LI, Franco-Paredes C, et al. COVID-19 in Latin America: The
implications of the first confirmed case in Brazil. Travel Med Infect Dis. 2020:
101613. https://doi.org/10.1016/j.tmaid.2020.101613. 4. Brasil. COVID-19 - Painel Coronavírus. 2020. https://covid.saude.gov.br/. Accessed 13 Apr 2020. 5. Brasil. Boletim Epidemiológico 8 - COE Coronavírus. 2020. https://www. saude.gov.br/images/pdf/2020/April/09/be-covid-08-final-2.pdf. 5. Brasil. Boletim Epidemiológico 8 - COE Coronavírus. 2020. https://www. saude.gov.br/images/pdf/2020/April/09/be-covid-08-final-2.pdf. 6. WHO. Vigilância mundial da COVID-19 causada por infecção humana pelo
vírus COVID-19; 2020. p. 1–4. https://apps.who.int/iris/bitstream/handle/1
0665/331506/WHO-2019-nCoV-SurveillanceGuidance-2020.6-por. pdf?sequence=14&isAllowed=y. 6. WHO. Vigilância mundial da COVID-19 causada por infecção humana pelo
vírus COVID-19; 2020. p. 1–4. https://apps.who.int/iris/bitstream/handle/1
0665/331506/WHO-2019-nCoV-SurveillanceGuidance-2020.6-por. pdf?sequence=14&isAllowed=y. Authors’ contributions JUB was responsible for statistical modelling, data analysis, result
presentation, discussion and writing of the manuscript. ANRJ and BVB were
responsible for data analysis, result presentation, discussion, and writing of
the manuscript. AFF, VML, and RMCF were responsible for data analysis,
result presentation, and discussion. All authors reviewed, revised and
approved the manuscript. 7. Fortaleza. Novo Coronavírus - Informações da Prefeitura de Fortaleza. Prefeitura Municipal de Fortaleza. 2020. https://coronavirus.fortaleza.ce.gov. br/acoes.html. Accessed 17 Apr 2020. 8. WHO. The urban health index: a handbook for its calculation and use. Geneva PP - Geneva: World Health Organization. https://apps.who.int/iris/
handle/10665/136839. Conclusion In conclusion, although universal actions should be ap-
plied to the entire municipality of Fortaleza, the classifi-
cation of neighborhoods generated in this study can help
improve specific measures in order to be more efficient. A set of recommendations were made to the municipal
government of Fortaleza. The neighborhoods that were
identified with a greater propensity for severe epidemics
should receive special attention in the adoption of mea-
sures to control the epidemic. Funding 9. Schumaker E, In NYC. “stark contrast” in COVID-19 infection rates based on
education and race - ABC News. ABC News; 2020. p. 1. https://abcnews.go. com/Health/nyc-stark-contrast-covid-19-infection-rates-based/story?id=6992
0706. Accessed 16 Apr 2020. 9. Schumaker E, In NYC. “stark contrast” in COVID-19 infection rates based on
education and race - ABC News. ABC News; 2020. p. 1. https://abcnews.go. com/Health/nyc-stark-contrast-covid-19-infection-rates-based/story?id=6992
0706. Accessed 16 Apr 2020. Availability of data and materials Giving TB wheels: public
transportation as a risk factor for tuberculosis transmission. Tuberculosis. 2011;91(SUPPL. 1):S16–23. https://doi.org/10.1016/j.tube.2011.10.005. 15. Feske ML, Teeter LD, Musser JM, Graviss EA. Giving TB wheels: public
transportation as a risk factor for tuberculosis transmission. Tuberculosis. 2011;91(SUPPL. 1):S16–23. https://doi.org/10.1016/j.tube.2011.10.005. 16. Filho TMR, Santos FSG d, Gomes VB, TAH R, JHR C, Ramalho WM, et al. Expected impact of COVID-19 outbreak in a major metropolitan area in
Brazil. medRxiv. 2020;2020:03.14.20035873. https://doi.org/10.1101/2020.03. 14.20035873. Availability of data and materials 10. Kang D, Choi H, Kim J-H, Choi J. Spatial epidemic dynamics of the COVID-19
outbreak in China. Int J Infect Dis. 2020:1–21. https://doi.org/10.1016/j.ijid. 2020.03.076. I confirm that the map in Figs. 1, 3, 4, 5, and 6. is my own. It was done with
GIS software. The notification data for COVID-19 was obtained by request
from the municipal health department of Fortaleza and the authors made a
commitment to confidential use and confidentiality was guaranteed. Any
researcher can request this data to the municipal health department of
Fortaleza (https://www.fortaleza.ce.gov.br/institucional/a-secretaria-327) as 10. Kang D, Choi H, Kim J-H, Choi J. Spatial epidemic dynamics of the COVID-19
outbreak in China. Int J Infect Dis. 2020:1–21. https://doi.org/10.1016/j.ijid. 2020.03.076. 11. Fan J, Liu X, Pan W, Douglas MW, Bao S. Epidemiology of 2019 novel
coronavirus Disease-19 in Gansu Province, China, 2020. Emerg Infect Dis. 2020;26:1–8. https://doi.org/10.3201/eid2606.200251. 11. Fan J, Liu X, Pan W, Douglas MW, Bao S. Epidemiology of 2019 novel
coronavirus Disease-19 in Gansu Province, China, 2020. Emerg Infect Dis. 2020;26:1–8. https://doi.org/10.3201/eid2606.200251. Page 11 of 11 Braga et al. BMC Public Health (2020) 20:1486 Braga et al. BMC Public Health (2020) 20:1486 12. Giuliani D, Dickson MM, Espa G, Santi F. Modelling and Predicting the
Spatio-Temporal Spread of Coronavirus Disease 2019 (COVID-19) in Italy. SSRN Electron J. 2020. https://doi.org/10.2139/ssrn.3559569. 12. Giuliani D, Dickson MM, Espa G, Santi F. Modelling and Predicting the
Spatio-Temporal Spread of Coronavirus Disease 2019 (COVID-19) in Italy. SSRN Electron J. 2020. https://doi.org/10.2139/ssrn.3559569. 13. Pluchino A, Inturri G, Rapisarda A, Biondo AE, Moli RL, Zappala’ C, et al. A
Novel Methodology for Epidemic Risk Assessment: the case of COVID-19
outbreak in Italy. arXiv Prepr arXiv200402739. 2020; http://arxiv.org/abs/2004
02739. Accessed 16 Apr 2020. 13. Pluchino A, Inturri G, Rapisarda A, Biondo AE, Moli RL, Zappala’ C, et al. A
Novel Methodology for Epidemic Risk Assessment: the case of COVID-19
outbreak in Italy. arXiv Prepr arXiv200402739. 2020; http://arxiv.org/abs/2004. 02739. Accessed 16 Apr 2020. 14. Troko J, Myles P, Gibson J, Hashim A, Enstone J, Kingdon S, et al. Is public
transport a risk factor for acute respiratory infection? BMC Infect Dis. 2011;
11:16. https://doi.org/10.1186/1471-2334-11-16. 14. Troko J, Myles P, Gibson J, Hashim A, Enstone J, Kingdon S, et al. Is public
transport a risk factor for acute respiratory infection? BMC Infect Dis. 2011;
11:16. https://doi.org/10.1186/1471-2334-11-16. 15. Feske ML, Teeter LD, Musser JM, Graviss EA. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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https://jhiphalexu.journals.ekb.eg/article_19913_8e65a695adb6e8ffcd23e74a55fd75ad.pdf
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English
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Evaluation of Leading Safety Performance of Primary School Buildings in Alexandria, Egypt: Cross-Sectional Study
|
Journal of High Institute of Public Health
| 2,018
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cc-by-sa
| 5,869
|
Evaluation of Leading Safety Performance of Primary
School Buildings in Alexandria, Egypt: Cross-
Sectional Study Gehan R. Zaki1 ¥, Kholoud Y. Tayel2, Mayada M. Reda3, 4, Aleya H. Mahmoud3, Engy I. Labib
1 Department of Occupational Health and Air Pollution, High Institute of Public Health, Alexandria University, Egypt
2 Department of Family Health, High Institute of Public Health, Alexandria University, Egypt
3 Department of Environmental Health, High Institute of Public Health, Alexandria University, Egypt
4 Hamdan Bin Mohammed Smart University, Dubai, UAE ublic,
by
(SP)
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1.8%)
eas."
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safety;
Available on line at:
www.jhiph.alexu.edu.eg
¥Correspondence
Email: gehanra17@gmail.com
Suggested Citations: Zaki GR,
Tayel KY, Reda MM, Mahmoud
AH,
Labib
EI. Evaluation
of
Leading Safety Performance of
Primary
School
Buildings
in
Alexandria, Egypt: Cross-Sectional
Study. JHIPH. 2018;48(2):77-84. Journal of High Institute of Public Health 2018;48(2):77-84. Original Article Background: Unsafe school buildings may adversely affect students, teachers, administrative
workers, cleaning workers, and visitors. Objective(s): The aim of the present study was to evaluate the leading safety performance of public,
private, and experimental primary school buildings in Alexandria, Egypt. Objective(s): The aim of the present study was to evaluate the leading safety performance of public,
private, and experimental primary school buildings in Alexandria, Egypt. Methods: A cross-sectional study was conducted in 30 primary schools in Alexandria selected by
stratified cluster sampling. Two observational checklists were designed, validated, and used;
including school building safety inspection checklist (SBSIC), and classroom safety inspection
checklist (CSIC). The completed checklists were reviewed and coded. The safety performances (SP)
for each category and for the overall checklists were then calculated. Results: The SP in private primary schools and classrooms [(66.1±13.0%), (68.7±12.5)] were higher
than that of experimental [(59.9±14.0%), (65.1±14.2%)] and public ones [(39.3±7.8%), (46.0±11.8%)
respectively]. The most common causes of reduced school SP were "the absence of protective
measures against vectors & insects," "the non-daily refuse disposal," "the non-inspected play areas."
Other causes included "the irregular fire drills," "the absence of the alarm system," "the non-earthed
electrical equipment," "the absence of emergency plan," and "the unmarked tripping/slipping
locations." The most frequent unsafe classroom conditions were "the absence of classroom alarm
point," "the on-board glare," and "the non-compliant windows to class area ratios."
Conclusion: Many safety violations were found to occur in Alexandria primary schools. This would
d
ti
f th
f t
f
d
tl
l
k f
f t
t S f t than that of experimental [(59.9±14.0%), (65.1±14.2%)] and public ones [(39.3±7.8%), (46.0±11.8%)
respectively]. The most common causes of reduced school SP were "the absence of protective
measures against vectors & insects," "the non-daily refuse disposal," "the non-inspected play areas."
Other causes included "the irregular fire drills," "the absence of the alarm system," "the non-earthed
electrical equipment," "the absence of emergency plan," and "the unmarked tripping/slipping
locations." The most frequent unsafe classroom conditions were "the absence of classroom alarm
point," "the on-board glare," and "the non-compliant windows to class area ratios." Conclusion: Many safety violations were found to occur in Alexandria primary schools. This would
cause a reduction of the safety performance and consequently a lack of safety management. Safety
performance in private schools was better than that in experimental and public ones. Keywords: Classroom safety; Electrical safety; Emergency preparedness; Fire safety; Ground safety;
Housekeeping; Leading safety performance; School safety Abstract Background: Unsafe school buildings may adversely affect students, teachers, administrative
workers, cleaning workers, and visitors. INTRODUCTION factors that might lead to future disease or disability.”
School safety management is a vital element of the
healthy school environment, which is an essential part
of the teaching and learning activities. It focuses on
controlling the accident promoting factors within the
school.(2) According to the proverb "you cannot
manage what you cannot measure," it is essential to
assess safety performance in order to manage safety. Previously, safety performance was measured by
calculation
of
accident
parameters,
including
frequency rate, severity rate, fatality rate, and
frequency
severity
index
(lagging
indicators). Nowadays, it is periodically assessed using both factors that might lead to future disease or disability.”
School safety management is a vital element of the
healthy school environment, which is an essential part
of the teaching and learning activities. It focuses on
controlling the accident promoting factors within the
school.(2) According to the proverb "you cannot
manage what you cannot measure," it is essential to
assess safety performance in order to manage safety. Previously, safety performance was measured by
calculation
of
accident
parameters,
including
frequency rate, severity rate, fatality rate, and
frequency
severity
index
(lagging
indicators). Nowadays, it is periodically assessed using both P rimary education stage usually occurs in
primary schools, which are occupied by
students, teachers, administrative employees,
and cleaning workers. The World Health Organization
(WHO) defines a health-promoting school as “one that
constantly strengthens its capacity as a healthy setting
for living, learning and working”.(1) The American
Academy of Pediatrics defines a “healthful school
environment” as “one that protects students and staff
against immediate injury or disease and promotes
prevention activities and attitudes against known risk
P 77 Journal of High Institute of Public Health 2018;48(2):77-84. 78 classroom safety inspection checklists (SBSIC and
CSIC). The two checklists were designed with Yes,
No or Not Applicable responses. The Yes response
was scored as "one" and the No as "zero." (5-8) The first
checklist (SBSIC) consisted of six categories,
including housekeeping, maintenance, fire safety,
electrical safety, emergency preparedness, in addition
to ground safety, which had eight, four, eleven, ten,
nine, and ten statements, with score ranges of (0-8),
(0-4), (0-11), (0-10), (0-9), and (0-10) respectively
added to the school data. INTRODUCTION The second checklist (CSIC)
had three main categories, including classroom
conditions, safety and emergency preparedness, as
well as indoor air quality, which had eight, twenty
eight, and six statements in the checklist, with score
ranges of (0-8), (0-28), and (0-6) respectively in
addition to the classroom data. lagging and leading indicators. Leading safety
performance is regularly used by employing the
traditional safety checklist with yes, no, or not
applicable responses. It aims at finding out the
accident promoting factors and immediately executing
the suitable corrective actions to prevent it.(3) In Egypt, leading school safety performance is not
periodically assessed. Hence, school safety cannot be
managed. To bridge this gap it is important to assess
the current situation of school safety. So, the aim of
the present study was to evaluate the leading safety
performance of public, private, and experimental
primary school buildings in Alexandria, Egypt. METHODS A cross-sectional study design was conducted in
primary schools in Alexandria, Egypt. Multistage
stratified cluster sampling from Alexandria public,
private, and experimental primary schools was
conducted to cover all educational zones, including
Eastern, Western, Central, El-Gomrok, El-Montazah,
El-Amreya and Borg el Arab. According to the WHO
practical manual 1991(4), the minimum sample size
was 26 clusters at 95% confidence level and 5%
expected error. It was raised to 30 schools. Two
classrooms were randomly selected from each school. The sample was selected using a multistage stratified
cluster sampling technique. The strata were classified
into public, private and experimental primary schools
in Alexandria. The seven educational zones in
Alexandria Governorate (Eastern, Western, Middle,
El-Gomrok, El-Montazah, El-Amreya and Borg El-
Arab) were included in the present study. An occupational safety professor in High Institute
of Public Health, Alexandria University was consulted
to review the two checklists, which were later tested
for their validation. This was conducted through
completing the checklists by three researchers for the
same five schools. Those schools were not included
within the study sample. Pearson's correlation
coefficients were calculated for the results of the first
and second; first and third; as well as for second and
third researchers respectively.(9) The data were collected using the validated
checklists. The completed checklists were reviewed
and coded. The safety performance (percent safety
score) for each category and for the overall checklist
were then calculated according to equations (1) and
(2). It was classified into "poor" (<60%), "moderate"
(60-70%), "satisfactory" (70-80%), and "Good"
(≥80%).(9) Two observational checklists were designed after
extensive literature review, school building and The Number of Yes responses of the school = Number of Yes responses in SBSIC+ Number of Yes responses of
CSIC of Class-1+Number of Yes responses of CSIC of Class-2
(1) The Number of Yes responses of the school = Number of Yes responses in SBSIC+ Number of Yes responses of
CSIC of Class-1+Number of Yes responses of CSIC of Class-2
(1) (1) Where SP of SBSIC is the safety performance of school building inspection checklist, SP of CSIC of Class-1 is the
safety performance of first classroom inspection checklist, and SP of CSIC of Class-2 is the safety performance of
second classroom inspection checklist. No. of YES Responses × 100
No. of YES Responses + No. of NO Responses
(2) Safety Performance % =
No. of YES Responses × 100
No. of YES Responses + No. RESULTS Regarding validation of the checklists, the correlation
coefficients of the overall SP in schools and
classrooms between the first and second researchers
were 0.81, 0.87; the second and third were 0.84, 0.90; Table 1: Safety performance (percent safety scores) of primary schools and classrooms at different school
types in Alexandria, Egypt 2014
Variable
n
%
Mean %
Safety
Score
SD
Minimum
Maximum
p-
value*
School Type
Public
16
53.3
39.3
7.8
29.7
52.5
≤0.05
Private
8
26.7
66.1
13.0
50.0
91.4
Experimental
6
20.0
59.9
14.0
59.8
81.5
Classrooms
Public
32
53.3
46.0
11.8
30.9
64.3
≤0.05
Private
16
26.7
68.7
12.5
50.0
91.2
Experimental
12
20.0
65.1
14.2
53.7
92.9
*One-Way ANOVA Test; it is significant at P-value ≤ 0.0 e (percent safety scores) of primary schools and classrooms at different school mance (percent safety scores) of primary schools and classrooms at different school
gypt 2014 performance (percent safety scores) of primary schools and classrooms at different school
dria, Egypt 2014 Table 1: Safety performance (percent safety scores) of primary schools and classrooms
types in Alexandria, Egypt 2014 *One-Way ANOVA Test; it is significant at P-value ≤ 0.0 The housekeeping SP in private schools was good
(84.0%), while that of experimental and public schools
were
satisfactory
(70.0%)
and
poor
(41.0%)
respectively (Figure 1). The SPs of the housekeeping
category were significantly varied (p≤0.05, at
C.I.=95%) among different school types. Further
analysis disclosed the significant differences between
public and private as well as between public and
experimental schools (p≤0.05, at C.I.=95%). The
reduction of the housekeeping SP below the 100% was
attributed to the safety standards' violations. The major
unsafe housekeeping conditions were "the absence of
protective measures against vectors & insects"
(100.0%, 50.0%, 66.7%), and "the non-daily disposal
of Refuse" (100.0%, 12.5%, and 50.0%) in public,
private, and experimental schools respectively as
illustrated in (Table 2). The poor SPs of the
maintenance (<60%) in each of public, private, and
experimental schools (Figure 1) were obvious in the
non-conformity with the safety benchmarks. One-Way ANOVA Test revealed significant variation in
maintenance SP among the three school types (p≤0.05,
at C.I.=95%). Meanwhile, significant differences in
maintenance SP were noticed between public and
private schools as well as between public and
experimental ones (p≤0.05, at C.I.=95%). The most
frequent unsafe maintenance condition was "the non-
inspected play areas" (100.0%, 87.5%, and 100.0%)
for
public,
private,
and
experimental
schools
respectively (Table 2). Ethical Considerations The study was approved by the Institutional Review
Board and the Ethics Committee of High Institute of
Public Health. The study conformed to the principles
of Helsinki declaration (2013) and the international
ethics
guidelines. Confidentiality
of
collected
information was ensured. METHODS of NO Responses
(2) No. of YES Responses × 100 Safety Performance % = Safety Performance % = (2) Post-Hoc Multiple Comparison Least Significant
Difference (LSD) Tests were used to check the
significance of variation for variables of more than
two classes. The significance of the results was The data were entered and statistically analyzed using
IBM SPSS 21 software package (IBM SPSS Statistics,
Somers, NY, USA). The safety performance (% safety
score) was checked for normality using One-Sample
Kolmogorov-Smirov Test. One-Way ANOVA and the 79 Zaki et al. estimated at 95% confidence interval (C.I). The results
were considered significant at P-value ≤ 0.05.(10, 11) and between the first and third were 0.83, 0.89
respectively. The
SP
showed
non-significant
Kologorov Smirov Test (p>0.05, at 95% C.I.). Table
(1) indicated that the SP of private primary schools
and classrooms [(66.1±13.0%), and (68.7±12.5)] were
higher than that of experimental [(59.9±14.0%) and
(65.1±14.2%)] and public ones [(39.3±7.8%) and
(46.0±11.8%) respectively]. Significant differences
(p≤0.05, at C.I.=95%) of schools and classrooms' SP
were observed among the three school types (public,
private, and experimental). Further analysis showed
highly significant variation in the schools and
classrooms' SP between public and private as well as
between public and experimental schools (P≤0.05, at
C.I.=95%). Safety performance of schools and
classrooms
showed
non-significant
One-Way
ANOVA Test among different districts (p>0.05, at
C.I.=95%). RESULTS The fire-safety SP in private schools was
moderate (65.0%), while that of experimental (38.0%)
and public (8.0%) were poor (Figure 1). The fire SP
showed highly significant variation (p≤0.05, at
C.I.=95%) among different school types (One-Way
ANOVA Test). Further analysis showed significant
differences between public and private as well as
between public and experimental schools (p≤0.05, at
C.I.=95%). The most frequent unsafe condition was Journal of High Institute of Public Health 2018;48(2):77-84. 80 "the irregular fire drills" that present 100.0%, 75.0%,
and 83.3% of public, private, and experimental schools
respectively. "The absence of the alarm system" was
found in 100.0% of public, 62.5% of private, and
83.3% of the experimental schools (Table 3). g
p
and 83.3% of public, private, and experimental schools
respectively. "The absence of the alarm system" was
found in 100.0% of public, 62.5% of private, and
83.3% of the experimental schools (Table 3). Figure 1: Primary Schools’ Safety Performance (percent safety Scores) of different checklist categories at
different school types in Alexandria, Egypt 2014. 41
20
8
28
6
48
46
84
54
65
48
31
70
69
70
50
38
30
30
73
65
0
10
20
30
40
50
60
70
80
90
100
Housekeeping
Maintenance
Fire safety
Electrical safety
Emergency
Preparedness
Ground safety
Classroom
safety
% Safety Score of the School (Safety Performance)
Public n=16
Private n=8
Experimental n=6
≤0.05
≤0.05
≤0.05
>0.05
>0.05
≤0.05
≤0.05 41
20
8
28
6
48
46
84
54
65
48
31
70
69
70
50
38
30
30
73
65
0
10
20
30
40
50
60
70
80
90
100
Housekeeping
Maintenance
Fire safety
Electrical safety
Emergency
Preparedness
Ground safety
Classroom
safety
% Safety Score of the School (Safety Performance)
Public n=16
Private n=8
Experimental n=6
≤0.05
≤0.05
≤0.05
>0.05
>0.05
≤0.05
≤0.05 ary Schools’ Safety Performance (percent safety Scores) of different checklist categories at
types in Alexandria, Egypt 2014. Figure 1: Primary Schools’ Safety Performance (percent safety Scores) of different checklist categories at
different school types in Alexandria, Egypt 2014. Table 2: Distribution of the violated safety standards of housekeeping and maintenance at primary schools in
Alexandria, Egypt 2014
Variables
Public
n=16
Private
n=8
Experimental
n=6
No. %
No. %
No. RESULTS %
Fire Safety
Extinguishers are absent
13
81.3
1
12.5
3
50.0
Extinguishers are not properly mounted
2
12.5
1
12.5
0
0.0
Extinguishers are not accessible
2
12.5
0
0.0
0
0.0
There is not alarm system
16
100.0
5
62.5
5
83.3
Alarm system is not working
--
--
5
62.5
0
0.0
Fire lane is absent
15
93.8
6
75.0
5
83.3
Lane is not clear
1
6.3
6
75.0
0
0.0
Fire drills are not undertaken regularly
16
100.0
6
75.0
5
83.3
Electrical Safety
Electrical cords near the foot traffic area
4
25.0
4
50.0
2
33.3
All electrical cords near the foot traffic area are not tied up
0
0.0
0
0.0
0
0.0
Power Strips are not used instead of extension cords
13
81.3
4
50.0
4
66.7
Electrical Panels are not properly labeled
15
93.8
5
62.5
5
83.3
Electrical Panels have open slots
15
93.8
6
75.0
6
100.0
Electrical panels' rooms are not locked
7
43.8
3
37.5
5
83.3
Electrical switch/junction boxes are not covered
16
100.0
6
75.0
4
66.7
There are charred outlet faces
11
68.8
2
25.0
3
50.0
Flammable materials are stored in heater rooms
9
56.3
2
25.0
4
66.7
All electrical equipment are not earthed
16
100.0
8
100.0
6
100.0 on of the studied primary schools according to violated safety standards of Fire and
exandria, Egypt 2014 Concerning the SP of the electrical safety, it was poor
at the three school types (28.0%, 48.0%, and 30.0%
respectively), although the difference was not
significant (Figure 1). The main unsafe electrical
safety condition was "the non-earthed electrical
equipment" at all of the studied schools (Table 3). The
safety performance of the emergency preparedness Table 4: Distribution of the studied primary schools according to the violated safety standards of Emergency,
and of Ground safety, Alexandria, Egypt 2014
Variable
Public
n=16
Private
n=8
Experimental
n=6
No. %
No. %
No. RESULTS %
Emergency
preparedness
Emergency exits are absent
12
75.0
5
62.5
4
66.7
Emergency Exits are not clear
4
25.0
1
12.5
1
16.7
Emergency evacuation maps are absent
16
100
7
87.5
4
66.7
Emergency Planning Manual is absent
16
100
7
87.5
5
83.4
MSDSs are absent in the school
15
93.8
5
62.5
4
66.7
Ground Safety
There are slippery and broken stairs
12
75.0
2
25.0
2
33.3
Yard ground texture is slippery
2
12.5
1
12.5
0
0.0
Ground is not clean and not free from obstacles
2
12.5
0
0.0
0
0.0
Aisles, stairs, and walking surfaces are not clear of
obstacles
5
31.3
0
0.0
1
16.7
Floors are not in good conditions with missing tiles
15
93.8
1
12.5
1
16.7
Floors are slippery
0
0.0
1
12.5
0
0.0
Areas with high fall potential (around drinking
fountains, entryways, etc.) have not been marked
15
93.8
5
62.5
4
66.7
High-visibility tape or paint are not used to point
out hard to see steps, cracks, trip hazard
15
93.8
8
100.0
5
83.3 Table 3: Distribution of the studied primary schools according to violated safety standards of Fire and
electrical safety, Alexandria, Egypt 2014
Variables
Public
n=16
Private
n=8
Experimental
n=6
No. %
No. %
No. RESULTS %
Housekeeping
Presence of hazards in corridors
7
43.8
0
0
1
16.7
Unclean of workers' uniform of kitchen or
canteen
5
31.3
3
33.3
4
66.7
Absence of protective measures against
vectors & insects
16
100.0
4
50.0
4
66.7
Absence of refuse container in each room
1
6.3
0
0
1
16.7
Refuse disposal does not occur daily
16
100.0
1
12.5
3
50.0
Water basins (sinks) are not clean
11
68.8
1
12.5
0
0.0
The closets are not clean
13
81.3
1
12.5
1
16.7
Maintenance
Play areas are not inspected for accidents
hazards
16
100.0
7
87.5
6
100.0
Drinking tabs are not maintained
10
62.5
1
12.5
0
0.0
Water basins are not regularly maintained
10
62.5
2
25
2
33.3
Closets are not maintained
15
93.8
4
50
4
66.7 on of the violated safety standards of housekeeping and maintenance at primary schools in
2014 Zaki et al. 81 81 Table 3: Distribution of the studied primary schools according to violated safety standards of Fire and
electrical safety, Alexandria, Egypt 2014
Variables
Public
n=16
Private
n=8
Experimental
n=6
No. %
No. %
No. RESULTS %
Fire Safety
Extinguishers are absent
13
81.3
1
12.5
3
50.0
Extinguishers are not properly mounted
2
12.5
1
12.5
0
0.0
Extinguishers are not accessible
2
12.5
0
0.0
0
0.0
There is not alarm system
16
100.0
5
62.5
5
83.3
Alarm system is not working
--
--
5
62.5
0
0.0
Fire lane is absent
15
93.8
6
75.0
5
83.3
Lane is not clear
1
6.3
6
75.0
0
0.0
Fire drills are not undertaken regularly
16
100.0
6
75.0
5
83.3
Electrical Safety
Electrical cords near the foot traffic area
4
25.0
4
50.0
2
33.3
All electrical cords near the foot traffic area are not tied up
0
0.0
0
0.0
0
0.0
Power Strips are not used instead of extension cords
13
81.3
4
50.0
4
66.7
Electrical Panels are not properly labeled
15
93.8
5
62.5
5
83.3
Electrical Panels have open slots
15
93.8
6
75.0
6
100.0
Electrical panels' rooms are not locked
7
43.8
3
37.5
5
83.3
Electrical switch/junction boxes are not covered
16
100.0
6
75.0
4
66.7
There are charred outlet faces
11
68.8
2
25.0
3
50.0
Flammable materials are stored in heater rooms
9
56.3
2
25.0
4
66.7
All electrical equipment are not earthed
16
100.0
8
100.0
6
100.0
Concerning the SP of the electrical safety, it was poor
at the three school types (28.0%, 48.0%, and 30.0%
respectively), although the difference was not
significant (Figure 1). The main unsafe electrical
safety condition was "the non-earthed electrical
equipment" at all of the studied schools (Table 3). The
safety performance of the emergency preparedness
was poor in the public, private, and experimental
schools (6.0%, 31.0%, and 30.0% respectively), with
no significant difference was between the three school
types (p>0.05, at C.I.=95%) (Figure 1). The most
frequent unsafe emergency condition was "the absence
of emergency planning manual" (100.0%, 87.5%, and
83.4% respectively) (Table 4). Table 4: Distribution of the studied primary schools according to the violated safety standards of Emergency,
and of Ground safety, Alexandria, Egypt 2014
Variable
Public
n=16
Private
n=8
Experimental
n=6
No. %
No. %
No. RESULTS The most recurrent unsafe
classroom situations were "the absence of classroom
alarm point" (100.0%, 87.5%, and 83.3%), "the on-
board glare" (75.0%, 50.0%, and 75.0%), and "the
non-compliant windows to class area ratios" (71.9%,
75.0%,
and
58.3%)
in
public,
private,
and
experimental schools respectively. Table 5: Distribution of the studied primary schools' classrooms according to violated safety standards,
Al
d i
E
t 2014 of the studied primary schools' classrooms according to violated safety standards, Table 5: Distribution of the studied primary schools' classrooms according to violated safety standards,
Alexandria, Egypt 2014
Safety Standards
Public
n=32
Private
n=16
Experimental
n=12
No. %
No. %
No. RESULTS %
Emergency
preparedness
Emergency exits are absent
12
75.0
5
62.5
4
66.7
Emergency Exits are not clear
4
25.0
1
12.5
1
16.7
Emergency evacuation maps are absent
16
100
7
87.5
4
66.7
Emergency Planning Manual is absent
16
100
7
87.5
5
83.4
MSDSs are absent in the school
15
93.8
5
62.5
4
66.7
Ground Safety
There are slippery and broken stairs
12
75.0
2
25.0
2
33.3
Yard ground texture is slippery
2
12.5
1
12.5
0
0.0
Ground is not clean and not free from obstacles
2
12.5
0
0.0
0
0.0
Aisles, stairs, and walking surfaces are not clear of
obstacles
5
31.3
0
0.0
1
16.7
Floors are not in good conditions with missing tiles
15
93.8
1
12.5
1
16.7
Floors are slippery
0
0.0
1
12.5
0
0.0
Areas with high fall potential (around drinking
fountains, entryways, etc.) have not been marked
15
93.8
5
62.5
4
66.7
High-visibility tape or paint are not used to point
out hard to see steps, cracks, trip hazard
15
93.8
8
100.0
5
83.3 ution of the studied primary schools according to the violated safety standards of Emergency
afety, Alexandria, Egypt 2014 Journal of High Institute of Public Health 2018;48(2):77-84. 82 Considering the SP of the ground safety, it was the
highest for experimental schools (73% Satisfactory),
followed by private (70% Satisfactory) and public
(48% poor) ones (Figure 1). One-Way ANOVA Test
indicated the highly significant variation of SP
between the three school types (p≤0.05, at C.I.=95%). Further analysis disclosed statistically significant
difference between public and private as well as
between public and experimental schools (p≤0.05, at
C.I.=95%). As obvious in table (4), the unsafe
condition of the highest frequency was "the unmarked
tripping/slipping locations" (93.8%, 100.0%, and
83.3%) in public, private, and experimental schools
respectively. According to the classrooms' SP, it was the highest at private schools (69% moderate),
followed by experimental (65% moderate) and public
(46% poor) ones (Figure 1), and it was statistically
significant (p≤0.05, at C.I.=95%) at different school
types. Additional
statistical
analysis
declared
significant differences between public and private as
well as between public and experimental schools
(p≤0.05, at C.I.=95%). RESULTS %
Area available for each student is non-compliant
19
59.4
7
43.8
6
50.0
Classroom
Conditions
Board is not maintained regularly
14
59.4
1
6.3
1
8.3
Classroom is crowded
19
59.4
7
43.8
6
50.0
Illumination source is not clean
17
53.1
0
0.0
0
0.0
There is glare on board
24
75.0
8
50.0
9
75.0
Windows are not clean
24
75.0
0
0.0
8
66.7
Windows are not maintained periodically
12
37.5
1
6.3
4
33.3
There is broken glass windows
18
56.3
4
25.0
0
0.0
There is odor in the classroom
17
53.1
3
18.8
4
33.3
Classroom Safety and emergency
preparedness
Absence of emergency exits
32
100.0
10
62.5
10
83.3
Absence of emergency routes
32
100.0
10
62.5
10
83.3
Absence of fire extinguishers in the class
32
100.0
11
68.8
10
83.3
Absence of alarm point within the class
32
100.0
14
87.5
10
83.3
There are Obstacles in the classroom
1
3.1
2
12.5
2
16.7
There are extra cables in the classroom
4
12.5
4
25.0
2
16.7
There are storage area in the classroom
0
0.0
3
18.8
2
16.7
All classroom equipment is not checked on a regular
basis
21
65.6
6
37.5
2
16.7
First aid measures is absent
28
87.5
10
62.5
2
16.7
Classroom is not vacuumed regularly
19
59.4
4
25.0
2
16.7
Blackboards/whiteboards are not cleaned properly
8
25.0
0
0.0
2
16.7
Trash is not removed daily
21
67.7
0
0.0
3
9.0
Food is kept in the classroom overnight
21
65.6
10
62.5
4
33.3
Desks and lockers are not cleaned regularly
27
84.4
4
25.0
8
66.7
Indoor
air
quality
On whiteboards, markers that release high levels of
volatile organic compounds (VOCs) are used
1
4.8
0
0.0
1
8.3
Windows to class area ratio is not compliant
23
71.9
12
75.0
7
58.3
Windows are not facing each other's
7
21.9
5
31.3
0
0.0
DISCUSSION
safety performance (SP) is essential for safety safety performance (SP) is essential for safety
management,(3) which is necessary for improving
SP.(12) The SP in public schools, and classrooms were
poor. This can be explained based on two factors,
including safety expenditure, and lack of safety rules'
enforcement. The first is too high as compared with DISCUSSION This study assessed the presence and application of the
minimal safety standards in school buildings of
different types. Ensuring school safety is a very
important public health issue worldwide. Evaluation of Zaki et al. 83 the economic conditions in the Egypt. So, the private
schools had the highest SP followed by experimental
and public and ones. Safety rules in Egypt are
enforced
by
the
Ministry
of
Manpower
and
Immigration, which grants permits and approvals for
any foundation after ensuring compliance with the
minimal safety rules. Previously, it granted these
permissions routinely for governmental foundations,
but now it becomes stricter with both governmental
and private institutions. earthquakes respectively, while the unusual crises
were deaths of staff and students at school, and
terrorism.(22) Unsafe grounds may lead to falling, tripping, or
slipping. The ground safety category revealed
satisfactory SP for experimental and private schools,
and poor for public ones. The most common unsafe
situation
was
"the
unmarked
tripping/slipping
locations." In accordance with these results, the
findings revealed from a project conducted in
Kenyatta University to assess school safety reported
that about 17% of school accidents occur due to
ground problems, and more than one-quarter of the
students' injuries are on the ground.(23) There
is
strong
relationship
between
housekeeping and safety performance as stated in two
Finnish studies 1989, and 2014.(13,
14) The
housekeeping showed good SP in private, satisfactory
in experimental, and poor in public schools. The major
frequent causes of low SP in the present study were
"the absence of protective measures against vectors
and insects," and "non-daily refuse disposal." A Saudi
study 1998 conducted safety inspection in large and
small construction projects. It concluded that the
housekeeping safety scores of large projects were
good, while that of small projects were poor.(15) The classrooms' SP was moderate for private and
experimental schools and poor for public ones. The
most recurrent unsafe classroom situations at the
classroom
conditions,
safety
and
emergency
preparedness, and indoor air quality categories were
"the on-board glare," "the absence of classroom alarm
point," and "the non-compliant windows to class area
ratios" respectively. Two studies conducted in
Swedish and Arizona revealed the positive impact of
classroom safety on the students' achievements and
their ability to learn.(24, 25) Designers and managers must consider the fire-
safety standards to ensure sustainability.(16) In this
study; the fire SP was moderate for private schools,
and poor for experimental and public ones. DISCUSSION The most
common fire hazards were "the irregular fire drills" as
well as "the absence of the alarm system." In
compliance with the present study, a Chinese study
(1999) developed a fire-safety assessment system for
existing buildings, and applied it on residence in Hong
Kong. It found that the low safety score was observed
for the alarm system.(17) In contrary to the present
study, A US study 2009 checked 1052 public schools
in Texas and revealed that the most frequent cause of
reduced fire safety was the absence of fire sprinkler,
and the absence of the alarm system was common in
just 5.4% of the public schools.(18) CONCLUSIONS AND RECOMMENDATIONS The findings of the present study can lead us to
conclude that there are many safety violations that
occur in Alexandria primary schools. This causes
reduction of the safety performance and lack of safety
management. Safety performances of private schools
are better than that in experimental and public ones. Emergency preparedness category is of the least SP,
while housekeeping is of the highest in the three
school types. It is recommended to activate the licensing and
inspection roles of the Ministry of Manpower and
Immigration
to
enable
enforcement
of
safety
standards. In addition, the decision makers in the
Ministry of Education must train and motivate the
health and safety committee for each school to
implement the school safety standards. Moreover, they
must provide the suitable financial allocations
necessary for safety equipment. Also, they had to
consider
categories,
including
housekeeping,
maintenance, fire safety, electrical safety, emergency
preparedness, ground safety, and classroom safety. The electrical safety performances were poor in
the three school types. The unsafe electric situation of
the highest popularity was "the non-earthed electrical
equipment," which is an actual problem of non-
industrial buildings in Egypt. In agreement with the
present work, the results of a study conducted in
Tampere University of Technology 2010 revealed that
the most common cause of electrical accidents was the
failure to earth the electrical equipment.(19) The SP of the emergency preparedness was poor
at the three school types. The most frequent violations
were "the absence of emergency planning manual,"
and "the lack of evacuation maps." Similarly, the main
emergency problems in Saudi and Turkish schools
were the absence of emergency plan, long response
time to incidents, and lack of emergency training.(20, 21)
The New Zealand study (2017) revealed that the most
common emergencies were due to weather, fires, and REFERENCES 13. Saari J, Näsänen M. The effect of positive feedback on industrial
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8. التعليمية اال. معايير واشتراطات صالحية المواقع والمباني المدرسية بمدارس
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Russia’s Sovereignty and Emergence of Pragmatic Polycentrism
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Upravlenie i politika
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2022, Vol. 1, No. 1
Received: February 15, 2022 / Accepted: March 10, 2022
Political Science / Country in Focus 2022, Vol. 1, No. 1
Received: February 15, 2022 / Accepted: March 10, 2022
Political Science / Country in Focus 2022, Vol. 1, No. 1
Received: February 15, 2022 / Accepted: March 10, 2022
Political Science / Country in Focus Governance and Politics UDC: 323, 327, 369.032 1 Ladislav Zemánek – PhDr., Analyst and Researcher, Faculty of Arts, Institute of World History, Charles University, Czech
Republic
E-mail: zemanek.ml@gmail.com
The author declares the absence of conflict of interest Russia’s Sovereignty and
Emergence of Pragmatic
Polycentrism LADISLAV ZEMÁNEK1
Charles University (Czech Republic) LADISLAV ZEMÁNEK1 Abstract The article researches Russia’s concept of comprehensive sovereignty that is a cornerstone of
the official state paradigm. Sovereignty is analyzed in its internal as well as external dimen-
sion, both synchronically and diachronically. The concept is interconnected with national secu-
rity expanding into different realms. Sovereignty-based securitization tendencies are examined
through methods of discursive analysis based upon constructivist assumptions. The present
study puts emphasis on conceptual constructions and discursive practices significantly affecting
individual perceptions, interpretation and Weltanschauung of political leadership as a whole,
thereby shaping behavior, strategies and policies of individual actors concerned. The analy-
sis reveals an affinity between concepts and discursive practices of the actors included in the
research – Russia, the United States, China, and the European Union. It manifests itself in the
concepts of comprehensive sovereignty, America First, dual circulation, and strategic autonomy
respectively. These strategies are examined in relation to the transformation of the global order
from US-led hegemonism towards polycentrism. The author draws attention to the interactions
within the “quadrilateral” consisting of Russia, China, the EU and the US in order to demonstrate
the emergence and dynamics of autonomization or regionalization, which is to be regarded as
a dialectical moment in the globalization process towards glocalization and pragmatic polycen-
trism. The author concludes that the EU’s strategic autonomy is a positive feature, but requires
the abandonment of Euro-Atlanticism, which thwarts restructuring the Eurasian macroregion in
general, and settling Russia-Europe relations in particular. Key words
Russia, United States, China, European Union, sovereignty, national security, polycentrism, Eu-
ro-Atlanticism, strategic autonomy, hegemonism For citation
Ladislav Zemánek (2022). Russia’s Sovereignty and Emergence of Pragmatic Polycentrism. Gov-
ernance and Politics, 1(1), P. 63–99. Key words
Russia, United States, China, European Union, sovereignty, national security, polycentrism, Eu-
ro-Atlanticism, strategic autonomy, hegemonism 2 Presidential Address to Federal Assembly. Kremlin, 2019, February 20. URL: http://en.kremlin.ru/events/president/
news/59863 (accessed 20.02.2022) For citation For citation
Ladislav Zemánek (2022). Russia’s Sovereignty and Emergence of Pragmatic Polycentrism. Gov-
ernance and Politics, 1(1), P. 63–99. For citation
Ladislav Zemánek (2022). Russia’s Sovereignty and Emergence of Pragmatic Polycentrism. Gov-
ernance and Politics, 1(1), P. 63–99. 63 Ladislav Zemánek “Without sovereignty, Russia cannot be a state. Some countries can do this,
but not Russia.” These words were spoken by President Vladimir Putin during
his address to the Federal Assembly in 2019.2 Sovereignty is a crucial concept
of Russia’s hegemonic paradigm. It is of a comprehensive nature, not limited to
the realm of security or international policy. This concept is an analogous phe-
nomenon to EU’s strategic autonomy, China’s dual circulation or Donald Trump’s
America First policy. All these strategies are symptoms of the transformation of
the global order towards a polycentric model, thus embodying complex dialec-
tics of globalization and deglobalization, integration and regionalization. Global
restructuring is of utmost relevance and significance in terms of contemporary
socio-economic as well as political processes, also affecting the very concept of
governance. The present article examines Russia’s concept of sovereignty from both in-
ternal and external perspectives. A detailed discursive analysis is made focusing
on basic normative documents (Military Doctrine, National Security Strategy),
bilateral diplomatic agreements and declarations (Joint Statement on the Twen-
tieth Anniversary of the Treaty of Good Neighborliness and Friendly Cooperation
between the Russian Federation and the People’s Republic of China; Joint Statement
on the International Relations Entering a New Era and the Global Sustainable
Development), as well as articles and statements of the highest-ranking repre-
sentatives of the relevant sides (Vladimir Putin, Sergey Lavrov, Dmitry Med-
vedev, Donald Trump, Mike Pompeo, Joe Biden and Josep Borrell). The chosen
method is based upon a constructivist assumption of the importance of ideas
and ideology in social life including politics (Peter Berger and Thomas Luck-
man, Alexander Wendt). The discursivity-oriented research can be effectively
combined with related analyses of the interstate interactions, international re-
lations and practical behavior of individual actors concerned. Both diachron-
ic and synchronic methods are employed in order to shed light on the ongo-
ing processes under investigation. For citation In the article, a multidisciplinary attitude is
adopted, making use of findings from political science and philosophy (Glenn
Diesen, Alexander Lukin, Carl Schmitt, Dmitry Trenin), international relations Governance and Politics Governance and Politics 64 UDC: 323, 327, 369.032 Political Science / Country in Focus (Richard Sakwa, Alexander Sergunin, Dmitry Suslov, Ivan Timofeev), history
(Richard Pipes, Andrei Tsygankov), and last but not least social anthropology
(Chris Hann). The analysis begins with the internal model of Russia’s comprehensive
sovereignty and its constitutive elements in different fields, proceeding to the
external model. The inquiry is therefore put into the context of relations with
the United States, China, and the European Union on the one hand, and of the
increasingly strong move towards polycentrism on the other. The article thus
presents an in-depth insight into the status quo of Russia’s state paradigm, based
upon the concept of sovereignty, contributing to a better understanding of the
metamorphoses of global politics and governance. p //
/
/
/
(
)
4 Voennaia doktrina Rossiiskoi Federatsii (Russia’s Military Doctrine). Kremlin. 2014, December 19. http://static.kremlin.ru/
media/events/files/41d527556bec8deb3530.pdf (accessed 20.02.2022) 3 Kontseptsiia vneshnei politiki Rossiiskoi Federatsii (The Concept of Russian Foreign Policy). Kremlin. 2016, November 30.
http://www.kremlin.ru/acts/bank/41451 (accessed 20.02.2022) 3 Kontseptsiia vneshnei politiki Rossiiskoi Federatsii (The Concept of Russian Foreign Policy). Kremlin. 2016, November 30.
http://www.kremlin.ru/acts/bank/41451 (accessed 20.02.2022)
4 Voennaia doktrina Rossiiskoi Federatsii (Russia’s Military Doctrine). Kremlin. 2014, December 19. http://static.kremlin.ru/
media/events/files/41d527556bec8deb3530 pdf (accessed 20 02 2022) Kontseptsiia vneshnei politiki Rossiiskoi Federatsii (The Concept of Russian Foreign Policy). Kremlin. 2016, November 30.
http://www.kremlin.ru/acts/bank/41451 (accessed 20.02.2022)
4 Voennaia doktrina Rossiiskoi Federatsii (Russia’s Military Doctrine). Kremlin. 2014, December 19. http://static.kremlin.ru/
media/events/files/41d527556bec8deb3530.pdf (accessed 20.02.2022) Security dimension of sovereignty The current state of international relations and development of the global
order is characterized by growing tensions, conflicts and rivalry following from
competition between the US, as the declining hegemon of the unipolar system
that emerged after the end of the Cold War, and the non-Western powers, first
and foremost China and Russia. The objective decline in Western states’ poten-
tial and the concurrent shift of the political and economic center to the East
arouse defiance in the West. From the Russian perspective, the existing system
of international relations and security is unfair, discriminating against the rival
emerging powers.3 Irrespective of the frictions in Russia’s near neighborhood
(Ukraine, Belarus, the Caucasus or Central Asia) and a long-term confronta-
tional policy from the West including large-scale sanctions, the Russian leader-
ship does not expect a full-fledged war against the country.4 The perception of threats plays a serious role in the leadership’s deci-
sion-making. These threats are formulated in the Military Doctrine as a sub-
stantial part of the state’s strategic planning. The current version was adopted in
2014. The most serious external threat is seen in NATO, its expansion and vest- 2022, Vol. 1, No. 1 65 Ladislav Zemánek ing the organization with global functions regardless of the mandate of the UN.5
The tension between Russia and NATO is a permanent feature of international
politics, which especially endangers European countries. While the Russian side
is not against further enlargement of the EU in principle, perceiving the Euro-
pean integration project as legitimate and analogous to Eurasian integration in
the post-Soviet space and even a model for the EAEU in a sense, it has rejected
US-led NATO policies and activities for a long period (Sergunin, 2016, p. 154,
162). The origins of the current controversies lie in the Gorbachev era (Gvozdev
& Marsh, 2014, p. 78). Boris Yeltsin allowed for expansion only if Russia itself
was included (Gvozdev & Marsh, 2014, p. 211), thus anticipating future projects
of a single security area comprising North America, Europe and Russia (Dmitry
Medvedev) or common military systems (Vladimir Putin). From a comparative
perspective, Russia respects the development of the EU while disapproving of
NATO. It follows that the main obstacle for security and stability between Rus-
sia and Europe lies in Washington’s hegemonic interests discursively justified by
a concept of the Euro-Atlantic community. 5 Ibid.
6 Voennaia doktrina Rossiiskoi Federatsii (Russia’s Military Doctrine). Kremlin. 2014, December 19. http://static.kremlin.ru/
media/events/files/41d527556bec8deb3530.pdf (accessed 20.02.2022)
7 Ibid. 5 Ibid. Governance and Politics Security dimension of sovereignty The second major threat is seen in the destabilization of individual coun-
tries and whole regions.6 Undoubtedly, the Russian strategists have the practice
of color revolutions in mind. This point can also be interpreted as a criticism of
the export of liberal democracy which is connected with Western exception-
alism and the false self-perception of this model as a universal one that is to
be established globally disregarding specific local conditions. Whereas the first
defined threat regards the military practice, the second one applies to the po-
litical practice carried out by the Western countries. Another risk is seen in the
deployment of military contingents on the territories of the adjacent or allied
countries as well as in the expansion of missile systems, undermining agree-
ments on the non-proliferation of nuclear weapons, development of the global
strike concept or militarization of outer space.7 The current Military Doctrine anticipates that nuclear weaponry will safe-
guard the role of deterrence and prevention of an outbreak of full-fledged as well Governance and Politics Governance and Politics 66 Political Science / Country in Focus UDC: 323, 327, 369.032 as limited military conflicts. Therefore, the Russian Federation has been inter-
ested in the maintenance of the Strategic Arms Reduction Treaty (New START)
as the cornerstone of the stability of the international regime of nuclear arms. The Treaty was signed in Prague in 2010 as a sequel of quite a long history of
negotiations between Moscow and Washington in the field of non-proliferation,
control and reduction of strategic weapons, which started in the 1960s with the
multilateral Partial Test Ban Treaty (PTBT), Nuclear Non-Proliferation Treaty
(NPT) and the bilateral Interim Agreement on Offensive Arms (SALT I) from
1972. There have been several bilateral agreements since then. The collapse of
the USSR did not make Russia-US treaties irrelevant, as the Russian Federation
took over a substantial part of the Soviet military arsenal. Even though overall
Russia’s role diminished in the newly emerged unipolar world dominated by
the US as a global hegemon in the 1990s, the country remained a military great
power. As such, Russia has played a crucial role in the balance and stability of
international security, endeavoring to preserve the status quo. However, the security regime was put in danger during Donald Trump’s
presidency. (
)
9 Sanger, D. E., & Broad, W. J. U.S. to Tell Russia It Is Leaving Landmark I.N.F. Treaty. New York Times. 2019, October 19.
URL: https://www.nytimes.com/2018/10/19/us/politics/russia-nuclear-arms-treaty-trump-administration.html (accessed
20.02.2022) 8 Marcus, J. INF Nuclear Treaty: US Pulls out of Cold War-era Pact with Russia. BBC, 2019, August 02. URL: https://www.bbc.
com/news/world-us-canada-49198565 (accessed 20.02.2022) 8 Marcus, J. INF Nuclear Treaty: US Pulls out of Cold War-era Pact with Russia. BBC, 2019, August 02. URL: https://www.bbc.
com/news/world-us-canada-49198565 (accessed 20.02.2022)
9 Sanger, D. E., & Broad, W. J. U.S. to Tell Russia It Is Leaving Landmark I.N.F. Treaty. New York Times. 2019, October 19.
URL: https://www.nytimes.com/2018/10/19/us/politics/russia-nuclear-arms-treaty-trump-administration.html (accessed
20.02.2022) 2022, Vol. 1, No. 1 8 Marcus, J. INF Nuclear Treaty: US Pulls out of Cold War-era Pact with Russia. BBC, 2019, August 02. URL: htt
com/news/world us canada 49198565 (accessed 20 02 2022) Security dimension of sovereignty From the beginning, Trump took a stand against the bilateral agree-
ments in the field, similarly as in the case of the multilateral Joint Comprehensive
Plan of Action, regarding the Iranian nuclear program. In 2019, the US with-
drew from the Intermediate-Range Nuclear Forces Treaty (INF Treaty) signed by
Mikhail Gorbachev and Ronald Reagan in 1987. This step provoked concerns
about a situation when no arms control limits would be in force. Washington ac-
cused Russia of breaching the treaty, which Moscow denied.8 The real reason be-
hind the US attack on the INF Treaty was, nevertheless, connected with growing
tensions with China, with the allegations against Russia’s “transgressions” being
rather a pretext than anything else. As China was not included in the treaty, it
was free to develop and deploy intermediate-range nuclear missiles.9 Trump attempted to establish new security regimes instead of the existing
ones, which were considered disadvantageous for the US as putting other rele- 2022, Vol. 1, No. 1 67 Ladislav Zemánek vant actors, first and foremost China, aside. It was not only a question of the INF
Treaty but also of the New START. The latter was to expire in February 2021. Under the circumstances when the US withdrew from the INF Treaty and added
new conditions to agree with the prolongation, including a redefinition of the
agreement and engaging China, it seemed very unlikely that the New START
would be extended. All the more so that Washington refused Moscow’s sugges-
tions, for instance, temporary extension without any preconditions.10 When the
current US President Joe Biden assumed the presidency, it was clear that there
would be no quick substantial turn in the relationship with Russia. Standing as
a candidate, Biden wrote about the need for countering “Putin’s kleptocratic au-
thoritarian system”.11 In his first presidential speech dedicated to foreign affairs,
Biden accused Russia of aggression including interference in the US election,
conducting cyber-attacks or poisoning its citizens12. In spite of the confronta-
tional policy, the new administration avoided the imminent threat of the arms
race, supporting the prolongation of the New START, thus manifesting the US
readiness to find common ground at least as far as most risk issues are con-
cerned. The treaty would probably not have been extended until 2026 without
Biden becoming President. The Russian leadership has put a strong emphasis on the use of nuclear arms
with regard to the deterrence policy. )
12 Biden, J. R. Remarks by President Biden on America’s Place in the World. White House, 2021, February 04. URL: https://
www.whitehouse.gov/briefing-room/speeches-remarks/2021/02/04/remarks-by-president-biden-on-americas-place-in-
the-world/ (accessed 20.02.2022); Crane, M. Biden Agrees Putin Is a Killer, Says He’ll Pay for Meddling. Bloomberg, 2021,
March 17. URL https://www.bloomberg.com/news/articles/2021-03-17/biden-agrees-putin-is-killer-says-he-ll-pay-price-
for-meddling (accessed 20.02.2022)
13 Osnovy gosudarstvennoi politiki Rossiiskoi Federatsii v oblasti iadernogo sderzhivaniia (Foundations of State Policy of the
Russian Federation in the Area of Nuclear Deterrence). Kremlin. 2020, June 02. URL: http://static.kremlin.ru/media/events/i 10 TASS. 2020, October 16. URL: https://tass.ru/politika/9738037 (accessed 20.02.2022) ,
p //
/p
/
(
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11 Biden, J. R. Why America Must Lead Again. Rescuing U.S. Foreign Policy After Trump. Foreign Affairs, 2020, March/
April. URL: https://www.foreignaffairs.com/articles/united-states/2020-01-23/why-america-must-lead-again (accessed
20.02.2022) 10 TASS. 2020, October 16. URL: https://tass.ru/politika/9738037 (accessed 20.02.2022)
11 Biden, J. R. Why America Must Lead Again. Rescuing U.S. Foreign Policy After Trump. Foreign Affairs, 2020, March/
April. URL: https://www.foreignaffairs.com/articles/united-states/2020-01-23/why-america-must-lead-again (accessed
20.02.2022)
12 Biden, J. R. Remarks by President Biden on America’s Place in the World. White House, 2021, February 04. URL: https://
www.whitehouse.gov/briefing-room/speeches-remarks/2021/02/04/remarks-by-president-biden-on-americas-place-in-
the-world/ (accessed 20.02.2022); Crane, M. Biden Agrees Putin Is a Killer, Says He’ll Pay for Meddling. Bloomberg, 2021,
March 17. URL https://www.bloomberg.com/news/articles/2021-03-17/biden-agrees-putin-is-killer-says-he-ll-pay-price-
for-meddling (accessed 20.02.2022)
13 Osnovy gosudarstvennoi politiki Rossiiskoi Federatsii v oblasti iadernogo sderzhivaniia (Foundations of State Policy of the
Russian Federation in the Area of Nuclear Deterrence). Kremlin. 2020, June 02. URL: http://static.kremlin.ru/media/events/
files/ru/IluTKhAiabLzOBjIfBSvu4q3bcl7AXd7.pdf (accessed 20.02.2022) g (
)
13 Osnovy gosudarstvennoi politiki Rossiiskoi Federatsii v oblasti iadernogo sderzhivaniia (Foundations of State Policy of the
Russian Federation in the Area of Nuclear Deterrence). Kremlin. 2020, June 02. URL: http://static.kremlin.ru/media/events/
files/ru/IluTKhAiabLzOBjIfBSvu4q3bcl7AXd7.pdf (accessed 20.02.2022) Governance and Politics Security dimension of sovereignty President Putin signed a strategic docu-
ment dedicated to this area which was made public for the first time ever in
2020. In the new Foundations of State Policy of the Russian Federation in the
Area of Nuclear Deterrence, the nuclear arsenal is considered a crucial element
for Russia’s sovereignty.13 The strategy postulates a strictly defensive character
of the latter. At the same time it allows the active use of nuclear arms but solely
in the case of nuclear attack against the country or its allies, as well as in case Governance and Politics 68 Political Science / Country in Focus UDC: 323, 327, 369.032 of any aggression endangering the existence of the Russian state.14 Similarly, ar-
ticle 22 of the Military Doctrine enables employing the Armed Forces in the
case of aggression against Russia or allies, in the case of the decision of the UN
Security Council or other collective security bodies and, last but not least, in
the case of need for protection of Russian citizens abroad.15 Article 31 expands
the legitimacy of the external military intervention if this is carried out in line
with principles and norms of international law, international agreements and
Russian legislation. These provisions might be used to active military actions
abroad, making the defensive posture more offensive. Russia’s action readiness was demonstrated especially in Syria and Crimea
in the last years. Russia has been endeavoring to strengthen its security, protect
the sovereignty and multiply military potential through cooperation with other
partners, first and foremost within the Collective Security Treaty Organization
(CSTO), the Commonwealth of Independent States (CIS) and the Shanghai
Cooperation Organization (SCO). The allies sensu stricto are countries partic-
ipating in the CSTO which is a military alliance, unlike two other multilateral
institutions. The asset of the SCO lies in the fact that transcends the limited
post-Soviet area including a wide array of Eurasian countries and provides the
states with an opportunity to interconnect and converge national development
strategies as well as grand initiatives as the Belt and Road Initiative or the Eura-
sian Economic Union (Kaczmarski, 2017, p. 1036). The significance of the SCO
consists in the fact that the member states generate more than 20% of the global
GDP, accounting for virtually 42% of the world’s population. 14 Ibid.
15 Voennaia doktrina Rossiiskoi Federatsii (Russia’s Military Doctrine). Kremlin. 2014, December 19. http://static.kremlin.ru/
media/events/files/41d527556bec8deb3530.pdf (accessed 20.02.2022) 14 Ibid. Security dimension of sovereignty It is the most im-
portant non-Western regional organization which can contribute considerably
to reshaping the global order according to the reformist agenda of the partici-
pants (Zemánek, 2020, p. 202). Naturally, the leading role has been played by the engines of Eurasian inte-
gration – Russia and China. Moscow found common ground with Beijing as far
as the fight against terrorism, separatism and extremism are concerned (Chi-
na calls these threats “The Three Evils”). Security and military cooperation be- 2022, Vol. 1, No. 1 69 Ladislav Zemánek tween Russia and China have been deepening, especially after 2014. Both major
powers reject the idea of establishment of the alliance, thus clearly demonstrat-
ing an innovative attitude towards interstate relations and cooperation, based
on openness, overlapping interests and heterogeneous integration model, the
basis of which lies in different speed and levels of integration in individual areas,
sometimes even going beyond the traditional alliance (Lukin, 2018b, p. 128). Both countries are aware of the Western hegemonic ambitions, export of liberal
democracy and destabilizing actions in relation to the countries with different
values and political systems. The SCO, therefore, serves to the deterrence of
the Western quasi-universalism and gradual transformation of the international
order to be based on polycentrism, equality, inclusiveness and the principles of
peaceful coexistence, the prerequisite of which is the very existence of different
models and a plurality of development as well as modernization patterns. Rus-
sia’s sovereignty can be protected in a more effective way through the above-
mentioned institutions. Paradoxically, engagement in supranational organiza-
tions and integration, thus openness and not introversion, may become the way
towards stronger sovereignty and national security for Russia. 16 The 2021 National Security Strategy leaves the term “strategic partnership” of the 2015 NSS out, replacing it with “win-win
international cooperation”. Comprehensive sovereignty: an internal perspective The principle of sovereignty is enshrined in strategic documents in a wide
array of fields. It applies not only to national security, external relations or econ-
omy but also to the energy sector, information security or cultural policy. Its
significance, therefore, cannot be overestimated, sovereignty being a quintes-
sence of the current Russian state and hegemonic paradigm. Russia’s concept
of sovereignty can be interpreted as based on the threefold principle of strate-
gic autonomy/independence, strategic stability and strategic partnership.16 These
categories define both internal and external dimensions of the existence of the
state. The National Security Strategy (hereinafter referred to as NSS) follows the
tradition of patrimonialism and etatism (see: Pipes, 1974, p. 24) when defining
the need for strengthening the power of the state to accomplish social security. Governance and Politics Governance and Politics 70 Political Science / Country in Focus UDC: 323, 327, 369.032 Such an attitude preserves the crucial role of the power vertical and its respon-
sibility for addressing people’s interests. The strong role of the vertical is also to
secure protection against external interference which is frequently thematized
in different contexts by the NSS. An emphasis is put on the threat posed by
foreign intelligence services and other foreign organizations, terrorist, extrem-
ist and radical forces undermining the sovereignty, territorial integrity, political
and social stability or traditional religious and moral Russian values. Besides,
the strong state vertical is to protect citizens against external propaganda as well
as the one carried out by “inner enemies”.17 Such a strategy clearly demonstrates
that the elites consider the strong state, its apparatus and institutions as a key to
maintaining the country’s sovereignty. Economic sovereignty is an inseparable part of the political one. The impor-
tant point is a crucial role ascribed to the state – analogous to its role in social
development. A strong role of the state in the economic processes is a constant
of Russian history (Tsygankov, 2014, p. 7). Neither the free market nor the We-
berian protestant ethic has been characteristic of Russian society. On the contra-
ry, economy was perceived as a secondary social institution (always subjugated
either to religious principles or political and social goals) in the pre-revolution-
ary epoch. Even the history of the Russian socialist and communist movement
is suffused with the adoration of the state and its role, despite the Marxist theory
of the economic base and social superstructure. i
18 Not by coincidence, the first Soviet People’s Commissar for Education Anatolii Lunacharskii propounded a specific theory
of bogostroitel’stvo, “God-Building”, emphasizing that socialism is the most religious of all religions. See: Lunacharskii
(1908). 17 Strategiia natsional’noi bezopasnosti Rossiiskoi Federatsii (National Security Strategy). Kremlin. 2015, December 31. URL:
http://static.kremlin.ru/media/acts/files/0001201512310038.pdf (accessed 20.02.2022)ii Comprehensive sovereignty: an internal perspective The Bolsheviks did not win
because of the economic conditions – they win because of their political will,
credo and quasi-religious zeal.18 The Soviet era further deepened the embedded
Russian mistrust of market mechanisms and entrepreneurship. Russia’s civiliza-
tional trajectory confirms the observations presented by social anthropologist
Chris Hann, according to whom the Eurasian societies have been characteristic
of “inclusive embeddedness”, the economy being subordinated to broader social
goals and needs (Hann, 2016, p. 4). Similarly, the Soviet system was nothing
absolutely extraneous, but at least partially followed older traditions and devel-
opmental tendencies based on the strong role played by the Russian state. 2022, Vol. 1, No. 1 71 Ladislav Zemánek The principles of central planning, the state’s control over the economic ac-
tivities, priority of political goals as well as the dominant position of the state
in the modernization and reforms were presented in different forms both in the
pre-revolutionary Russia and the Soviet Union. One can conclude that the So-
viet experiment radicalized older patterns, reinterpreting them in a modernist
way. However, a different perspective also existed. Reformists Sergei Witte and
Pyotr Stolypin started to pave the way for Russia with the middle class, more
inclusive and participatory model with wider space for individual activities and
market mechanisms. In post-Soviet Russia, Dmitry Medvedev followed this
line. Amidst the economic recession in 2009, he formulated a modernization
program in which the Russian President harshly criticized economic backward-
ness, commodity dependence, low productivity and innovations or fragility of
democratic institutions. Medvedev rejected paternalism and historic forms of
Russian modernization connected with Peter I and the Bolsheviks. The new
program was to develop Russian democracy and establish a new, post-industri-
al economy.19 The liberal ethos of the three mentioned reformists did not deny
local peculiarities or conservative moral values arising from the Orthodoxy but
represented a more open, more liberal model of Russia’s modernization as well
as external behavior. Both at the beginning of the 20th century and in the 2010s, this reformist
shift was interrupted due to the external factors – the Great War, which was
joined by the Russian Empire with an utter reluctance, and the color revolu-
tion in Ukraine in 2014, the subsequent anti-Russian campaign launched by the
Western countries and deteriorating relations. Vladimir Putin has accentuated
different elements, but the policies throughout his terms have not been incom-
patible with the reformist modernization agenda essentially. 19 Medvedev, D. Go Russia! Kremlin. 2009, September 10. URL: http://en.kremlin.ru/events/president/news/5413 (accessed
20.02.2022) 20 According to President Putin, the Internet and media should be regulated not only by the positive legal norms but also
by moral, natural law. Putin Calls for Internet Bound by Moral Rules, Criticizes Opposition Rallies. Reuters. 2021, March 04.
URL: https://www.reuters.com/article/us-russia-internet-idUSKCN2AW2D4 (accessed 20.02.2022); Priniat zakon o «suver-
ennom internete» (Adopting Sovereign Internet Law). Gosudarstvennaia Duma. 2019, April 16. URL: http://duma.gov.ru/
news/44551/ (accessed 20.02.2022)i 22 Russia to Launch Own TikTok Developed With Putin’s Alleged Daughter. AFP. 2020, December 23. URL: https://www.
themoscowtimes.com/2020/12/23/russia-to-launch-own-tiktok-developed-with-putins-alleged-daughter-a72467
(ac-
cessed 20.02.2022); Rossiiskii analog “Vikipedii” oboidetsia biudzhetu RF pochti v 2 mldr rublei (Russian analogous Wikipe-
dia will cost 2 bln rubles). Interfax. 2019, September 26. URL: https://www.interfax.ru/russia/677964 (accessed 20.02.2022) n, Q. China, Russia to Form Closer Partnership on 5G in Defiance of US. Global Times. 2020, August 24. URL: https
w.globaltimes.cn/content/1198654.shtml (accessed 20.02.2022) 2022, Vol. 1, No. 1 Comprehensive sovereignty: an internal perspective Putin’s program
has also been aimed at integration in the global economy, acceleration of the
economic growth, progressive development of the national economy, support
for the business environment, and the political system development. The agen-
da, nevertheless, gradually became more securitized and the key goal was not
“mild” Westernization and building the Greater Europe anymore but protec- /en.kremlin.ru/events/president/news/5413 (accessed Governance and Politics Governance and Politics 72 UDC: 323, 327, 369.032 Political Science / Country in Focus tion of sovereignty and Russian peculiarity, deepening of autonomy in all nec-
essary fields and Eurasian integration where possible. Modernization, regional
and interregional integration or sovereign opening up was not abandoned but
modified under adverse external conditions and growing frictions worldwide
(Sakwa, 2021, p. 5-6). Safeguarding of sovereignty was accompanied by stronger emphasis on
self-sufficiency and development of own systems independent of the global ones
(which creates a comfortable position in the cases of crises or conflicts) in the
post-2014 period. It applies to the payment system, interbank financial telecom-
munication system, navigation satellite system, 5G systems or the Internet. Pro-
tection of the Russian Internet against external threats should be accomplished
on the basis of the act passed in 2019 in response to the US National Cyber Strat-
egy which declared the ambition to spread US particular interests worldwide.20
Russia’s path towards information and digital sovereignty is not isolated. It has
to be perceived in an international context when many actors seek the same
self-protection. In general, the assertion of information sovereignty is one of the
most important tasks states have in the digital era (Romashkina, 2019). Moscow
introduced numerous acts in this direction, including a data localization law
or a law regarding import substitution of foreign IT products. Foreign encryp-
tion protocols of the Russian Internet should be supplemented by the domestic
ones in the near future and the 5G networks are to be based on Russian or Chi-
nese technologies.21 One of Russia’s leading media holding Gazprom-Media has
started to prepare a Russian version of TikTok while a national alternative of
Wikipedia is also under preparation.22 Similarly, the US, the EU and China seek
digital sovereignty, trying to minimize mutual interdependence and vulnerabil-
ity. Washington banned TikTok and WeChat, preventing Huawei and ZTE from 2022, Vol. 1, No. 1 73 Ladislav Zemánek doing business in the country. 23 The Clean Network. U.S. Department of State. 2020. URL: https://2017-2021.state.gov/the-clean-network/index.html (ac-
cessed 20.02.2022)
24 Csernatoni, R. The EU’s Rise as a Defense Technological Power: From Strategic Autonomy to Technological Sovereignty.
Carnegie Europe, 2021, August 12. URL: https://carnegieeurope.eu/2021/08/12/eu-s-rise-as-defense-technological-pow-
er-from-strategic-autonomy-to-technological-sovereignty-pub-85134 (accessed 20.02.2022)
25 Epifanova, A. Digital Sovereignty on Paper: Russia’s Ambitious Laws Conflict with Its Tech Dependence. Wilson Center.
2020, October 23. URL:.https://www.wilsoncenter.org/blog-post/digital-sovereignty-paper-russias-ambitious-laws-con-
flict-its-tech-dependence (accessed 20.02.2022) 23 The Clean Network. U.S. Department of State. 2020. URL: https://2017-2021.state.gov/the-clean-network/index.html (ac-
cessed 20.02.2022) g
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25 Epifanova, A. Digital Sovereignty on Paper: Russia’s Ambitious Laws Conflict with Its Tech Dependence. Wilson Center.
2020, October 23. URL:.https://www.wilsoncenter.org/blog-post/digital-sovereignty-paper-russias-ambitious-laws-con-
flict-its-tech-dependence (accessed 20.02.2022) Governance and Politics 24 Csernatoni, R. The EU’s Rise as a Defense Technological Power: From Strategic Autonomy to Technological So
Carnegie Europe, 2021, August 12. URL: https://carnegieeurope.eu/2021/08/12/eu-s-rise-as-defense-technolo
er-from-strategic-autonomy-to-technological-sovereignty-pub-85134 (accessed 20.02.2022)l 23 The Clean Network. U.S. Department of State. 2020. URL: https://2017-2021.state.gov/the-clean-network/index.html (ac-
cessed 20.02.2022)
24 Csernatoni, R. The EU’s Rise as a Defense Technological Power: From Strategic Autonomy to Technological Sovereignty.
Carnegie Europe, 2021, August 12. URL: https://carnegieeurope.eu/2021/08/12/eu-s-rise-as-defense-technological-pow-
f
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b 85134 (
d 20 02 2022) Comprehensive sovereignty: an internal perspective Trump’s administration introduced the Clean
Network program aimed at the protection of data, privacy, security and rights
against “aggressive intrusions by malign actors”.23 The EU promotes the policy
of strategic autonomy within which cooperation with the pioneering Chinese
companies is jeopardized.24 However, Russia lags behind the other major pow-
ers in many respects, and the overall competitiveness, innovative potential and
technological development are not comparable to the US or China’s. Therefore
Russia is likely to deepen cooperation with Chinese partners to offset this con-
siderable disadvantage.25 The National Security Strategy is a cornerstone of Russia’s official state para-
digm. Unlike other strategic documents (for instance, Military Doctrine, Foreign
Policy Concept, Economic Security Strategy, Energetic Security Doctrine, Infor-
mation Security Doctrine or Foundations of State Cultural Policy), it deals with
all these areas, synthesizing them into a comprehensive whole. Dmitry Trenin
considers the 2021 NSS “the most important Kremlin strategy statement” and
“a manifesto” for the present era characteristic of a deep confrontation with the
Western world (Trenin, 2021). Indeed, the two last versions of the NSS were
formulated under different circumstances. The preceding one was issued in re-
sponse to the Ukrainian crisis, sudden deterioration of relations with the West,
the pivot to the East and strong uncertainty about the impacts of the pres-
sure and sanctions on the Russian economy and society as such. The last years
showed that the country was able to withstand, transforming economic pro-
cesses, diversifying external ties and deepening integration within the Greater
Eurasia (Lukin & Novikov, 2021, p. 52-53). The current NSS reflects the further
deterioration of relations with Europe and the US, missing them out completely. Similarly, prospects of cooperation with NATO are omitted, thus signaling that
Moscow does not expect any substantial progress in the relations with the West
in the upcoming years. 74 UDC: 323, 327, 369.032 Political Science / Country in Focus In this regard, the 2021 Strategy is much more skeptical and explicit as far
as the confrontational actions of the US, its allies as well as transnational cor-
porations are concerned. 26 Strategiia natsional’noi bezopasnosti Rossiiskoi Federatsii. Kremlin (National Security Strategy 2021). 2021, July 02. URL:
http://static.kremlin.ru/media/events/files/ru/QZw6hSk5z9gWq0plD1ZzmR5cER0g5tZC.pdf (accessed 20.02.2022)
27 U.S. – Russia Presidential Joint Statement on Strategic Stability. Kremlin 2021, June 16. URL: http://en.kremlin.ru/supple-
ment/5658 (accessed 20.02.2022)
28 Strategiia natsional’noi bezopasnosti Rossiiskoi Federatsii (National Security Strategy). Kremlin. 2015, December 31. URL:
http://static.kremlin.ru/media/acts/files/0001201512310038.pdf (accessed 20.02.2022)
29 Strategiia natsional’noi bezopasnosti Rossiiskoi Federatsii. Kremlin (National Security Strategy 2021). 2021, July 02. URL:
http://static.kremlin.ru/media/events/files/ru/QZw6hSk5z9gWq0plD1ZzmR5cER0g5tZC.pdf (accessed 20.02.2022) 2022, Vol. 1, No. 1 /
(
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tegiia natsional’noi bezopasnosti Rossiiskoi Federatsii (National Security Strategy). Kremlin. 2015, December 31. U
://static.kremlin.ru/media/acts/files/0001201512310038.pdf (accessed 20.02.2022) 26 Strategiia natsional’noi bezopasnosti Rossiiskoi Federatsii. Kremlin (National Security Strategy 2021). 2021, July 02. URL:
http://static.kremlin.ru/media/events/files/ru/QZw6hSk5z9gWq0plD1ZzmR5cER0g5tZC.pdf (accessed 20.02.2022)
27
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tegiia natsional’noi bezopasnosti Rossiiskoi Federatsii. Kremlin (National Security Strategy 2021). 2021, July 02. U
://static.kremlin.ru/media/events/files/ru/QZw6hSk5z9gWq0plD1ZzmR5cER0g5tZC.pdf (accessed 20.02.2022) /
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ussia Presidential Joint Statement on Strategic Stability. Kremlin 2021, June 16. URL: http://en.kremlin.ru/supple-
658 (accessed 20.02.2022) tegiia natsional’noi bezopasnosti Rossiiskoi Federatsii. Kremlin (National Security Strategy 2021). 2021, July 02. U
//
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d 20 02 2022) Strategiia natsional noi bezopasnosti Rossiiskoi Federatsii (National Security Strategy). Kremlin. 2015, December 31. URL:
http://static.kremlin.ru/media/acts/files/0001201512310038.pdf (accessed 20.02.2022)
29 Strategiia natsional’noi bezopasnosti Rossiiskoi Federatsii. Kremlin (National Security Strategy 2021). 2021, July 02. URL:
http://static.kremlin.ru/media/events/files/ru/QZw6hSk5z9gWq0plD1ZzmR5cER0g5tZC.pdf (accessed 20.02.2022) Comprehensive sovereignty: an internal perspective The illiberal wording becomes obvious when the cul-
tural Westernization is called a fatal threat for Russia and the absolutization
of freedom of individuals, egoism and immorality is criticized as proof of the
West’s decline while emphasizing the significance of the traditional values and
norms as well as religious foundations of societies.26 Such rhetoric corresponds
with Putin’s assessment of today’s Western liberalism. In an interview given to
the Financial Times in 2019, he refused the hegemonic ambitions of the liberal
elites, castigating them for their contempt for the rights of the majority, assaults
on the traditional values and silencing opponents. Russian President expressed
his belief that biblical values are more universal than liberal ones.27 The 2021
NSS thus embodies the conservative worldview of the Russian leadership, ar-
ticulating and shaping the hegemonic discourse, which will be reflected in the
adjustments of the partial doctrines in the following months and years. The conservative character of the ruling paradigm (Diesen, 2021, p. 205)
is confirmed by the definition of the traditional Russian values, put forward by
the 2015 NSS, with the priority of the spiritual over the material at the top and
followed by the protection of human life, rights and freedoms, family, creative
labor, service to the motherland, the norms of morality, humanism, charity, fair-
ness, mutual assistance, collectivism, unity of Russia’s nations and the continuity
of the Russian history. This conservative discourse is complemented by a strict
refusal of external interference undermining cultural sovereignty. The Russian
state stipulates the right to protect citizens against external expansion in terms
of ideology and values, as well as to control the information sphere.28 The 2021
Strategy rearranges the enumeration of the Russian values only minimally.29 The establishment and reinforcement of sovereignty in different spheres
of the life of the state and society brings multiple risks, especially in terms of 2022, Vol. 1, No. 1 75 Ladislav Zemánek individual rights and freedoms. The dominant conservative paradigm under
Vladimir Putin is nevertheless moderate. Potential radicalization is dependent
mainly on the objective external pressure and subjective sense of danger. This
combination is risky if taking the Russian tradition of patrimonialism, strong
state, and collectivism into consideration. It could result in a substantial closure
of the country, the building of a fortress and isolationism. In that case, Rus-
sia might become a real international threat – which is not absolutely at this
moment. Comprehensive sovereignty: an internal perspective On the contrary, it is the anti-Russian narrative together with actual
political steps aimed against Russia which forces the country to the adoption of
defensive, partially hostile stance. Governance and Politics Comprehensive sovereignty: an external perspective Russia’s concept of sovereignty includes the principles of autonomy and
independence, strategic stability and partnership. Whereas the autonomy and
independence reflect predominantly the internal dimension, the stability and
partnership concern the external one – the external relations as well as a vision
of and practical steps towards reform of the global system in line with the gen-
eral normative outlook on how the system should be designated and structured
in the 21st century. In this chapter, we will turn to the external dimension, going
through the strategic framework defined by the National Security Strategies and
subsequently focusing on the relations with the US and China. It is worth noticing that both the 2015 and 2021 NSS do not position the
relations with the West (the US and Europe) on the top of priorities. It has a
symbolic significance connected with the deep disillusionment with the West-
ern policies in the last decades, turn to the East and renunciation of Westerni-
zation. It shows that the West is not in the spotlight anymore, thus underlining
the objective reality of multipolarization and the rise of the East. Taking this
development into account, the secondary position of the US and Europe within
the NSS exceeds the genuinely symbolic meaning and expresses the rational and
pragmatic choice made in response to the transformation of the global order,
economy and politics. Not surprisingly, therefore, the 2015 NSS emphasized the
multilateral cooperation within BRICS, SCO, APEC and G20, highlighting the
relationship with China and India. Besides, stress was laid on the near neigh- Governance and Politics 76 Political Science / Country in Focus UDC: 323, 327, 369.032 borhood, the CIS and integration processes in Eurasia, first and foremost, the
Eurasian Economic Union (EAEU). As far as Europe is concerned, the NSS
mentioned the support for win-win cooperation with the European states and
the EU, harmonization of integration processes in Europe and on the post-So-
viet territory (EAEU) as well as the establishment of joint collective security
architecture in the Euro-Atlantic region.30 These were long-term goals pursued
by the Russian establishment. Their inclusion in the 2015 NSS can be perceived
as proof of continuity and transparency of Russia’s foreign policy if we become
aware of the context of the sharp deterioration in the relations with the West. Irrespective of obvious Washington’s involvement in the coup d’état in Ukraine,
Moscow reaffirmed its readiness to develop the bilateral partnership. 30 Strategiia natsional’noi bezopasnosti Rossiiskoi Federatsii (National Security Strategy). Kremlin. 2015, December 31. URL:
http://static.kremlin.ru/media/acts/files/0001201512310038.pdf (accessed 20.02.2022)
31 Ibid Ibid.
32 Strategiia natsional’noi bezopasnosti Rossiiskoi Federatsii. Kremlin (National Security Strategy 2021). 2021, July 02. URL:
http://static.kremlin.ru/media/events/files/ru/QZw6hSk5z9gWq0plD1ZzmR5cER0g5tZC.pdf (accessed 20.02.2022) 30 Strategiia natsional’noi bezopasnosti Rossiiskoi Federatsii (National Security Strategy). Kremlin. 2015, December 31. URL:
http://static.kremlin.ru/media/acts/files/0001201512310038.pdf (accessed 20.02.2022)
31 Ibid.
32 Strategiia natsional’noi bezopasnosti Rossiiskoi Federatsii. Kremlin (National Security Strategy 2021). 2021, July 02. URL:
http://static.kremlin.ru/media/events/files/ru/QZw6hSk5z9gWq0plD1ZzmR5cER0g5tZC.pdf (accessed 20.02.2022) Comprehensive sovereignty: an external perspective Moreover,
it admitted the development of relations with NATO on the condition that the
latter respects international law and Russia’s interests.31 The wording of the 2021 NSS is different. It contains the keystones of the
long-term Russian strategy such as maintenance of the central coordination
role of the UN and the UN Security Council, deepening cooperation within
CIS, CSTO, SCO, BRICS, EAEU or ASEAN. However, no explicit mentions of
Europe, the US and NATO are made. It is especially the part dedicated to ex-
ternal affairs which distinguishes the present strategy from the former. And it is
sharper in criticism. The world is characterized as a place where contradictions
and conflicts have been intensifying and where the declining powers of the West
assault on others, violate principles and norms of the international law as well
as international treaties, breach sovereignty, undermine trust among individual
actors and stability of the system, impose sanctions and attempt to revise the ex-
isting rules. Obviously, Russia endeavors to present itself as a crucial stabilizing
element and protector of the status quo. In this regard, the NSS addresses the
need for reinforcement of coherence and resilience of the international system
and its legal base, strengthening of international peace and collective security,
prevention of military conflicts and, last but not least, – strategic stability.32 2022, Vol. 1, No. 1 77 Ladislav Zemánek Russia’s self-perception as a protector of the status quo and concurrent accu-
sation against the declining Western actors of revisionism is not new, since it has
appeared in recent articles published by the highest representatives, especially
Minister of Foreign Affairs Sergei Lavrov. In the article The World at a Cross-
roads and a System of International Relations for the Future from 2019, Lavrov
subjected the West and its liberal democratic ideology to criticism. Lambasting
the West for endeavors to usurp the fruits of globalization to the detriment of the
“rest”, he describes the Western countries as hypocrites, for the narrative based
on freedom, democracy and human rights entails inequality, injustice, selfish-
ness and exceptionalism (Lavrov, 2019, p. 10). More or less explicitly, Minister
follows the concepts of multiple modernities, multipolarity and multilateralism
which fit the reality of the transforming global order. From the Russian perspec-
tive, the West invented the concept of a rules-based order which is revisionist
in its essence. 33 Ibid, p. 11.
34 Sanger, D. E. Biden Defines His Underlying Challenge with China: ‘Prove Democracy Works’. New York Times. 2021, March
26. URL: https://www.nytimes.com/2021/03/26/us/politics/biden-china-democracy.html (accessed 20.02.2022)
35 President Biden to Convene Leaders’ Summit for Democracy. The White House. 2021, August 11. URL: https://www.white-
house.gov/briefing-room/statements-releases/2021/08/11/president-biden-to-convene-leaders-summit-for-democra-
cy/ (accessed 20.02.2022) Comprehensive sovereignty: an external perspective Lavrov characterizes it as a calculated, selective combination of
rules, unilaterally employed with the aim to circumvent multilateral, collective
decision-making and international legal instruments and processes based on
the UN Charter as a core of the post-war order.33 The rules-based order introduces a division between liberal democracies
and “authoritarian powers”, “autocracies”, between allies and systemic rivals,
friends and enemies. Inevitably, it leads to confrontation, conflicts and insta-
bility of the international system. The Western concept is opposite to pragma-
tism, openness and pluralism, being another sequel of Western universalism. Moreover, it is utterly moralistic, introducing the ethical categories of good and
evil into international politics, making it very ideologized. The US President is
trying to establish an “alliance of democracies” to confront “autocracies”. At his
first news conference after the election, Biden spoke about a “battle” between
these two artificially invented camps.34 Later on, the US highest representative
announced the plan to organize a summit of the “democratic states” to counter
global authoritarianism.35 The Biden administration develops continuously the Governance and Politics 78 Political Science / Country in Focus UDC: 323, 327, 369.032 confrontational narrative created during Donald Trump’s presidency, albeit in
a more sophisticated, ostensibly more diplomatic and moderate way. Biden´s
“alliance of democracies” is a reformulation of Trump’s and Pompeo’s antinomy
between the “free world” and “new tyranny”, i.e. China and its kind.36 Continu-
ity in external policy between these two seemingly contradictory Presidents is
indisputable, even though Biden pursues a more multilateral, more collective,
more inclusive attitude. This attitude is, nevertheless, conditioned by adherence
to the “democratic” camp, to the universalist and hegemonic Western paradigm. The others are excluded. That is why the UN-based order ceases being con-
venient for the West since the objective power decline results in an inability to
control the system. And that is why the Western actors are seeking to replace the
UN-based order with the rules-based one. The West’s attempts to marginalize the UN and weaken collective deci-
sion-making irrespective of socioeconomic, political, ideological or cultural
differences are obvious, so the Russian accusations of revisionism are justifiable. On the other hand, however, the post-1991 world was dominated by the West
with the US on the top, being greatly unipolar, and these dominating actors – be
they states, transnational corporations or other non-state subjects – are interest-
ed in the preservation of the model and their hegemony. g
/
/
(
)
37 In this article, I put aside the difference between reformism and revisionism as it is not substantial for the reasoning (com-
pare with: Sergunin, 2016, p. 25, 27, 32-35). Revisionism is simply meant as the effort to change the status quo. 36 Pompeo, M. R. Communist China and the Free World’s Future. U.S. Department of State 2020, July 23. URL: https://2017-2021.
state.gov/communist-china-and-the-free-worlds-future-2/index.html (accessed 20.02.2022)f Comprehensive sovereignty: an external perspective The reality is therefore
more complex and the role of the West is ambiguous, being typical of contra-
dictory tendencies towards the maintenance of the status quo and revisionism.37 The revisionist tendencies can be seen in the US withdrawal from the An-
ti-Ballistic Missile Treaty (ABM Treaty) in 2002, Joint Comprehensive Plan of
Action (JCPOA), the UN Human Rights Council and UNESCO in 2018, In-
termediate-Range Nuclear Forces Treaty (INF Treaty) in 2019, Paris Agreement
and Treaty on Open Skies in 2020. The US position towards the Comprehensive
Nuclear Test Ban Treaty (CTBT) is also questionable as Washington is reluctant
to ratify it. Similarly, the destiny of the Treaty on Measures for the Further Re-
duction and Limitation of Strategic Offensive Arms (New START) was extreme-
ly uncertain for a long period. Many of these steps disrupted the foundations 2022, Vol. 1, No. 1 79 Ladislav Zemánek of the system, gradually forming after World War II amidst the systemic con-
frontation between and coexistence of the two camps. The US withdrawals thus
undermined relative stability and transparency brought about by the bilateral
agreements between the US and the USSR. The Russian Federation assumed ob-
ligations and inherited armaments from the Soviet period and as such, it contin-
ues to be one of the pillars of international security architecture with the UN Se-
curity Council as its core. In this regard, it is Washington that has attempted to
reverse the long-term status quo. The US have ignored the UN Security Council
resolutions (for instance regarding the settlement of the Israeli-Palestinian con-
flict), launched unilateral military operations without authorization by this UN
body and carried out multiple measures aimed against other countries – extra-
territorial use of the US legislation, spying on their closest allies, abuse of the
status of the USD as the main means of payment, introduction of protectionist
barriers and, last but not least, imposition of sanctions. These practices have
become an integral part of the US-led rules-based order. The imposition of sanctions is an increasingly frequent practice used by lib-
eral democracies against others. Given the intensifying frictions and transfor-
mation of the global order, continuation and deepening of this tendency are to
be expected in the following period. Governance and Politics Comprehensive sovereignty: an external perspective It is worth noting that in spite of the strong
pressure, the impacts on the Russian economy have been relatively low com-
pared to other external factors such as the drop in oil prices, global pandemic
and related economic slump. The data published by the International Monetary
Fund follows that the role of sanctions accounted only for -0.2% in terms of
GDP growth. At the same time, low macroeconomic influence is accompanied
by high risks for individual businesses and projects (Timofeev, 2021, p. 4). In
his report Sanctions Against Russia: A Look into 2021, Ivan Timofeev, therefore,
comes to the conclusion that the best long-term answer for Russia is to lower
dependency on the Western structures through concurrent development of the
national payment system and a single payment system within the EAEU and
the SCO, de-dollarization of the Russian economy as well as the development
of international/regional mechanisms of counter-sanctions (Timofeev, 2021,
p. 19). The significance of the latter is highlighted by the fact that it is Chi-
na and Russia together with Iran that is the most frequent targets. Adaptation
on the Western restrictive regimes thus could be made more effective through Governance and Politics 80 UDC: 323, 327, 369.032 Political Science / Country in Focus the SCO, especially after the Islamic Republic becomes a full member of the
organization.38 Not only sanctions show that the discourse dividing countries into “democ-
racies” and “autocracies” is dangerous. Instead, the division into those who ad-
vocate unipolarity or multipolarity is relevant. We can hardly avoid delimita-
tion, as the world is full of antagonism, contradictions, divergent interests and
conflicts. As long the sphere of the political exists, dividing lines are inevitable. The difference between friends and foes, allies and challengers remains. As Carl
Schmitt (2015) put it, the political world is pluriverse in its essence, since the
existence of genuine universality requires complete depoliticization, hence the
elimination of the state as a social institution. At this stage of development, the
elimination of the state or withering away of the state – if we use the Marxist
term coined by Friedrich Engels in his Anti-Dühring – is not only unfeasible but
also undesirable. The opponents of Western hegemonism and unipolarity need
to have a strong state to break those structures that hamper the development to-
wards a more democratic, fairer, more equal, more inclusive model. 38 SCO Opposes External Meddling, Builds Shield against Outside Forces. Global Times. 2021, September 17. URL: https://
www.globaltimes.cn/page/202109/1234595.shtml (accessed 20.02.2022) Comprehensive sovereignty: an external perspective Naturally, it
raises resistance from the declining powers which have resorted to the narrative
of democracies versus autocracies, rules-based order versus a “law-of-the-jun-
gle-based” order. Such a picture is false, serving merely the particular interests
of the weakening hegemons. Joe Biden gave up the isolationist America First imperative, replacing it with
“Democracies First”. Of course, the US only has liberal democracies in mind. The internationalist agenda, however, remains particularist, albeit presented as
universalist and objectively valid. The US President has revived ties with the
allies, making some initial concessions (Nord Stream 2), but continues estab-
lishing a “global democratic phalanx”, as an American scholar called one of the
pillars of the Biden doctrine (Brands, 2021). Biden makes use of the existing,
frequently overwhelming structures like Group of Seven, the Quad (Quadrilat-
eral Security Dialogue), the Indo-Pacific Strategy (“A free and open Indo-Pacif-
ic”), América Crece, T-12 (League of Digital Democracies), D-10 (Democracies
10) and the Clean Network, or is developing new ones such as B3W initiative 2022, Vol. 1, No. 1 81 Ladislav Zemánek (Build Back Better World), the Summit for Democracy, AUKUS or Trade and
Technology Council (TTC) to advance hegemonic interests and contain “au-
tocracies”. The first leader-level summit of the Quad was held in March 2021.39
The Biden administration tries to give a new impetus to this grouping with the
aim to incorporate it into the “global phalanx” and employ it against China. The
G7 summit in Carbis Bay in June 2021 presented the B3W initiative to provide
an alternative to the Belt and Road Initiative (BRI) in relation to the “strategic
competition” with Beijing. The ambitious infrastructure project is focused on
low- and middle-income countries from the Americas to Africa, Indo-Pacif-
ic and beyond. Representatives of the largest liberal democracies agreed on an
investment worth at least 40 trillion USD. Both the ideologic background and
financial sources come predominantly from the US. The idea can be interpreted
as an expanded and updated version of the New Silk Road Initiative announced
by the Secretary of State Hillary Clinton in 2011. It also builds on the Blue Dot
Network (BDN) launched in 2019. 39 Quad Leaders’ Joint Statement: “The Spirit of the Quad”. The White House. 2021, March 12. URL: https://www.whitehouse.
gov/briefing-room/statements-releases/2021/03/12/quad-leaders-joint-statement-the-spirit-of-the-quad/
(accessed
20.02.2022) 39 Quad Leaders’ Joint Statement: “The Spirit of the Quad”. The White House. 2021, March 12. URL: https://www.whitehouse.
gov/briefing-room/statements-releases/2021/03/12/quad-leaders-joint-statement-the-spirit-of-the-quad/
(accessed
20.02.2022)
40 President Biden and G7 Leaders Launch Build Back Better World (B3W) Partnership. The White House. 2021c, June 12.
URL: https://www.whitehouse.gov/briefing-room/statements-releases/2021/06/12/fact-sheet-president-biden-and-g7-
leaders-launch-build-back-better-world-b3w-partnership/ (accessed 20.02.2022)
41 The Czech Republic expressed an interest in the mediation of the meeting in Prague, but the plan was thwarted as a result
of an unprecedented anti-Russian campaign in the country based on the allegation of Russia’s “terrorist act” on Czech
territory in 2014. For greater detail see Zemánek (2021a; 2021b). p
p
41 The Czech Republic expressed an interest in the mediation of the meeting in Prague, but the plan was thwarted as a result
of an unprecedented anti-Russian campaign in the country based on the allegation of Russia’s “terrorist act” on Czech
territory in 2014. For greater detail see Zemánek (2021a; 2021b). Governance and Politics )
40 President Biden and G7 Leaders Launch Build Back Better World (B3W) Partnership. The White House. 2021
URL: https://www.whitehouse.gov/briefing-room/statements-releases/2021/06/12/fact-sheet-president-bide
leaders-launch-build-back-better-world-b3w-partnership/ (accessed 20.02.2022) )
40 President Biden and G7 Leaders Launch Build Back Better World (B3W) Partnership. The White House. 2021c, June 12.
URL: https://www.whitehouse.gov/briefing-room/statements-releases/2021/06/12/fact-sheet-president-biden-and-g7-
leaders-launch-build-back-better-world-b3w-partnership/ (accessed 20.02.2022)
41 The Czech Republic expressed an interest in the mediation of the meeting in Prague, but the plan was thwarted as a result )
40 President Biden and G7 Leaders Launch Build Back Better World (B3W) Partnership. The White House. 2021c, June 12.
URL: https://www.whitehouse.gov/briefing-room/statements-releases/2021/06/12/fact-sheet-president-biden-and-g7-
leaders-launch-build-back-better-world-b3w-partnership/ (accessed 20.02.2022)
41 The Czech Republic expressed an interest in the mediation of the meeting in Prague, but the plan was thwarted as a result
of an unprecedented anti-Russian campaign in the country based on the allegation of Russia’s “terrorist act” on Czech
territory in 2014. For greater detail see Zemánek (2021a; 2021b). Comprehensive sovereignty: an external perspective Washington as a leading force of the B3W
plans to engage the Development Finance Corporation, the Agency for Inter-
national Development (USAID), the Export-Import Bank (EXIM), the Millen-
nium Challenge Corporation, the US Trade and Development Agency or the
Transaction Advisory Fund.40 Generally, the US-led global initiative is in line
with the Strategic Competition Act of 2021, one of the priorities of which is to
counter the “malign influence of the Communist Party of China” worldwide,
thus following the anti-Chinese discourse and policies of Trump’s era with its
zero-sum-game logic. What, then, is Russia’s position amid the “strategic com-
petition” between Washington and Beijing declared by the US political class? R
i
US Chi
t i
l
Th fi t
it f Bid
d P ti
i
G Russia-US-China triangle. The first summit of Biden and Putin in Geneva,
following the public accusations made by the US President41, could be interpret-
ed as a step towards stabilization of the volatile and deteriorating relations. The 82 Political Science / Country in Focus UDC: 323, 327, 369.032 goal was not a reset of the relationship, and created a more realistic context. As
Dmitry Suslov (2021) aptly expressed it, the realistic aim was to lay foundations
for a “managed confrontation”. Moscow welcomed this initiative irrespective of
the recent US actions perceived as hostile – sanctions, the expulsion of diplo-
mats, hesitation in prolongation of the New START, forming a front against
“autocracies”, engagement in Belarus, military provocations, labeling Russia’s
President as a “killer”, accusations of attacks against other countries (the Czech
Republic in 2014 in particular) and so on and so forth. The reasons behind Bid-
en’s efforts are likely to be connected with China, as the latter has become the
cardinal rival for Washington, replacing Russia.42 Therefore, Biden is motivated
to seek sustainability in their relations with Moscow, notwithstanding deeply
divergent interests and values. It seems to be a sequel to Trump’s vision to revive
cooperation with Russia against China which failed primarily due to historical
experience, and the mental and ideological inertia of the US establishment. In both cases, nevertheless, it is still the same zero-sum-game perspective,
calculated cooperation with one country against another. Whereas Washington
took side with China several times in the last century, such a symbiosis never
existed with Russia – with the exception of hints in the 1990s. 42 Annual Threat Assessment of the US Intelligence Community. Office of the Director of National Intelligence. 2021, April 09.
URL: https://www.dni.gov/files/ODNI/documents/assessments/ATA-2021-Unclassified-Report.pdf (accessed 20.02.2022) Threat Assessment of the US Intelligence Community. Office of the Director of National Intelligence. 2021, April 09.
tps://www.dni.gov/files/ODNI/documents/assessments/ATA-2021-Unclassified-Report.pdf (accessed 20.02.2022) Comprehensive sovereignty: an external perspective The US support-
ed Japan financially against Russia in the Russo-Japanese War at the beginning
of the 20th century. They refused to recognize the USSR until 1933 as the last
of the major powers, even though the rule of the Soviet regime had been a fait
accompli long before this. The first bilateral agreement was concluded no earlier
than 1964. In spite of the balance of power based on nuclear deterrence, both
hegemons waged a wide array of proxy wars against each other. The US also
imposed sanctions on the Soviet Union, for example in response to the Soviet
invasion of Afghanistan. The unique possibility to incorporate the new Russia
into the Euro-Atlantic community after 1991 miscarried predominantly due to
the West’s arrogant belief of superiority and hegemonism (NATO expansion
eastwards, support for separatism in Russia, disrespect for Russia’s specific con-
ditions, interests and needs). Given these experiences, it is hard to expect that
any kind of “Russiamerica” (akin to “Chinamerica”) will emerge in the future 2022, Vol. 1, No. 1 83 Ladislav Zemánek and oppose anyone, unless other emerging non-Western actors adopt a radi-
cal anti-Russian attitude. The Biden-Putin summit was merely aimed to find
an elementary level of understanding and communication which had been lost
because of the extreme politicization of the Russian question in the US under
Donald Trump. Russia became one of the main factors in the domestic political
struggle. It made a constructive policy towards Moscow impossible. Unlike the
preceding President, Joe Biden is not accused of having ties with the Kremlin
in the public discourse, which paradoxically may facilitate mutual interactions
(Kortunov, 2021, p. 7-10). Regardless of discrepancies, the Geneva summit can be interpreted in terms
of starting talks about “strategic stability”.43 The US side has made a constructive
step when agreeing with the extension of the New START which remains in force
until 2026. Both major powers could also find a way of cooperation in the field
of cybersecurity, especially cybercrime, and at least define rules of cyberwarfare. Another area of possible cooperation concerns the nuclear programs of Iran
and North Korea, as well as climate change and green transition (Suslov, 2021). The latter became easier after the US rejoined the Paris Agreement. The war on
terrorism was an exception where Russia and the US managed to intensify co-
operation during Trump’s presidency (Shakirov, 2020, p. 8). This tendency could
continue under Biden. 43 U.S. – Russia Presidential Joint Statement on Strategic Stability. Kremlin 2021, June 16. URL: http://en.kremlin.ru/supple-
ment/5658 (accessed 20.02.2022) Comprehensive sovereignty: an external perspective Similarly, both powers will be confronted with a move
towards militarization and exploitation of outer space and new regions such as
the Arctic, which will require the establishment of rules and a legal framework
(Shakirov, 2020, p. 10). The dynamic development, disruptions and innovations
in many fields (cyberspace, the Arctic, outer space) will push both Moscow and
Washington into interacting and seeking pragmatic solutions. However, the
confrontation will be a harsh reality. Russia belongs to the “autocratic camp” and
the unprecedented level of cooperation and understanding between Moscow
and Beijing is already raising concerns among the liberal democracies. Some
experts, therefore, warn about the West’s possible attempts to undermine the
Sino-Russian strategic partnership, taking the side of either the Russians or the
Chinese (Kortunov, 2021, p. 27). Others even call upon the Western political Governance and Politics Governance and Politics 84 UDC: 323, 327, 369.032 Political Science / Country in Focus elites to rupture the “axis of the autocratic powers” (Kendall-Taylor & Shullman,
2021). Still, such a scenario is merely wishful thinking. Although the 2021 NSS is very concise as far as the relationship with China
is concerned44, the real state of affairs is much more vivid. Possibly, the lacon-
ism of the NSS is intended to weaken the assertions, according to which the
Sino-Russian “axis” is a de facto alliance, and to emphasize Russia’s multi-vec-
tor external orientation, excluding superiority of partnership with one coun-
try. Whatever the reasons, the fact is that Sino-Russian relations have reached
the highest level in history. The legal framework of the bilateral interactions
is defined by the Treaty of Good Neighborliness and Friendly Cooperation con-
cluded in 2001. On the occasion of the 20th anniversary of the signing of this
fundamental document, both sides issued a Joint Statement45 that reflects the
unprecedented development of the comprehensive partnership, demonstrating
its specific, innovative nature, being an example of a new type of interstate rela-
tions in our era. The document characterizes bilateral ties as a “comprehensive partnership”,
“strategic interaction”, “international relations of a new type” and a “model of
harmonious coexistence” based on the “comprehensive consideration of the
partner´s interests and non-interference in each other´s internal affairs”. p //
/
/
/i
/
/Q
g
q p
g
p
(
)
45 Joint Statement of the Russian Federation and the People’s Republic of China on the Twentieth Anniversary of the Treaty
of Good Neighborliness and Friendly Cooperation between the Russian Federation and the People’s Republic of China.
Kremlin. 2021, June 28. URL: http://static.kremlin.ru/media/events/files/en/Bo3RF3JzGDvMAPjHBQAuSemVPWTEvb3c.pdf
(accessed 20.02.2022)
46 Ibid 44 Strategiia natsional’noi bezopasnosti Rossiiskoi Federatsii. Kremlin (National Security Strategy 2021). 2021, July 02. URL:
http://static kremlin ru/media/events/files/ru/QZw6hSk5z9gWq0plD1ZzmR5cER0g5tZC pdf (accessed 20 02 2022) (
46 Ibid. 2022, Vol. 1, No. 1 44 Strategiia natsional’noi bezopasnosti Rossiiskoi Federatsii. Kremlin (National Security Strategy 2021). 2021 Comprehensive sovereignty: an external perspective Refus-
ing the concept of a military or political alliance of the past, both actors stress
that the partnership is not directed against third sides.46 Undoubtedly, we are
witnesses of a peculiar phenomenon that can anticipate new forms of interac-
tions in the reformed, polycentric, regions-based order. After all, China is a pi-
oneer of such a shift, if we take into consideration the nature and specific forms
of the Belt and Road Initiative, 16+1 (Central and Eastern Europe + China)
and other analogic formats in different regions. Unlike these, the Sino-Russian
relationship is bilateral and its crucial significance is connected with objective
factors, as well as the fact that both major powers are the engines of the Eurasian
integration and pillars of the emerging new order. 2022, Vol. 1, No. 1 85 Ladislav Zemánek The June Joint Statement47 defines the overall direction and priorities of the
Sino-Russian partnership for the ongoing period. (1) The major emphasis is put
on Putin-Xi diplomacy as the cornerstone and guarantee of successful develop-
ment. Mutual meetings at leaders´ level are very frequent and regular. The same
applies to the lower levels (PMs, ministers or state agencies). (2) Military and
military technology cooperation are in second place. It regards not only joint
exercises, military exchanges or purchases of armaments but also the reduction
of armed forces in the border areas. Both China and Russia have expanded the
military cooperation and potential through multilateral platforms, particular-
ly the SCO. (3) The economic and trade ties are seen as a substantial element
of the social development and improvement of the people´s living standards. The goals are set as follows: (i) to increase the volume of bilateral trade; (ii) to
strengthen energy cooperation; (iii) to deepen financial interaction, support the
expansion of mutual payments in national currencies in trade; (iv) to strengthen
cooperation in the field of industry, information and communication technol-
ogies, space and aviation; (v) to enhance cooperation in the scientific and tech-
nological innovations; (vi) to ensure protection and enforcement of intellectual
property rights; (vii) to increase cooperation in agro-industry; (viii) to deepen
interactions in the field of transport; (ix) to strengthen cooperation within the
Northern Sea Route (or the so-called Polar Silk Road), promoting sustainable
development of the Arctic; (x) to enhance interregional ties. 47 Ibid. Comprehensive sovereignty: an external perspective (4) The expansion
of interregional ties is of the utmost importance given the obvious tendency
towards regionalization as an accompanying phenomenon of globalization at
the current stage of development that is typical of the ambivalent logic of inte-
gration and autonomatization, openness and introversion. The Eurasian mac-
ro-region is the most dynamic space in this regard as it includes several grand
integration processes, starting with the Belt and Road Initiative, the Shanghai
Cooperation Organization and the Eurasian Economic Union subsumed into
the overarching vision of the Greater Eurasian Partnership (GEP), and ending
with the European Union in the Western part of Eurasia. The abovementioned projects (with the exception of the EU) are crucial to
redefining the international order embodying the new paradigm as a perspec The abovementioned projects (with the exception of the EU) are crucial to
redefining the international order, embodying the new paradigm as a perspec- 47 Ibid. Governance and Politics 86 Political Science / Country in Focus UDC: 323, 327, 369.032 tive of the emancipating and rising non-Western world, seeking full-fledged
recognition, equity and participation (Zemánek, 2020, p. 200). Not by coinci-
dence, the GEP is mentioned in the recent Russian NSS for the first time ever.48
Agreement on the basic values and principles of the international order between
Russia and China is crucial as it multiplies the relevance, attraction and influence
of the new paradigm. The Joint Statement replicates the wording of the Russian
strategic documents. The mutual understanding can be demonstrated in Russia’s
explicit recognition of the China-proposed concept of building a community of
shared future for humanity and China’s concurrent recognition of the concept
of multipolar global order pursued by Russia for a long period.49 Both major
powers have the identical perception of the main risks in terms of the interna-
tional stability and security – unilateralism and undermining of the UN-based
legal framework (the withdrawal from several arms control agreements made by
the US), development of the US missile defense system, building high-precision
non-nuclear weapons, militarization and the arms race in outer space, or the so-
called Three Evils of terrorism, separatism and extremism. http://static.kremlin.ru/media/events/files/ru/QZw6hSk5z9gWq0plD1ZzmR5cER0g5tZC.pdf (accessed 20.02.2022)
49 Joint Statement of the Russian Federation and the People’s Republic of China on the Twentieth Anniversary of the Treaty
of Good Neighborliness and Friendly Cooperation between the Russian Federation and the People’s Republic of China.
Kremlin. 2021, June 28. URL: http://static.kremlin.ru/media/events/files/en/Bo3RF3JzGDvMAPjHBQAuSemVPWTEvb3c.pdf
(accessed 20.02.2022)
50 Ibid Ibid.
51 Joint Statement by the Foreign Ministers of China and Russia on Certain Aspects of Global Governance in Modern Condi-
tions. The Ministry of Foreign Affairs of the Russian Federation. 2021, March 23. URL: https://www.mid.ru/en/foreign_pol-
icy/news/-/asset_publisher/cKNonkJE02Bw/content/id/4647776 (accessed 20.02.2022) 48 Strategiia natsional’noi bezopasnosti Rossiiskoi Federatsii. Kremlin (National Security Strategy 2021). 2021, July 02. URL:
http://static kremlin ru/media/events/files/ru/QZw6hSk5z9gWq0plD1ZzmR5cER0g5tZC pdf (accessed 20 02 2022) (
50 Ibid. Comprehensive sovereignty: an external perspective Moscow and Beijing
make a commitment to pursue multilateralism and keep global stability through
developing regional partnerships and integration as a step towards the commu-
nity with a shared future for mankind, protecting security through the establish-
ment of a global information security system (China’s Global Initiative on Data
Security as an alternative to the US-led Clean Network) with the sovereign states’
right to regulate the national segment of the Internet, as well as promoting a
multilateral and open trading system with the WTO as its core.50 Despite being labelled “autocratic”, both Russia and China adhere to democ-
racy and human rights. The cause of controversies between these countries and
the West rests in the simple fact that Russia and China admit the existence of
multiple democracies and conceptions of human rights.51 They are more open, 2022, Vol. 1, No. 1 87 Ladislav Zemánek plural and tolerant than the West with its monism and moralism, hampering
pragmatic cooperation to the benefit of all members of the global community. The gradual building of the Greater Eurasian Partnership comprising manifold
integration processes, development patterns as well as political models, and
based on both common interests and shared principles, creates a new norma-
tive framework, significantly influenced by Russian perspective as enshrined in
the basic national strategic documents. The rising Eurasian community poses
a great challenge to the European Union that has been caught in a trap of Eu-
ro-Atlanticism. It inevitably complicates relations with Russia and China.h The EU and Russia: seeking a new modus vivendi. The EU elites decided to
follow the path of strategic autonomy. While commenced in the field of the de-
fense industry, the concept has gradually spread to other fields including econ-
omy, technology, politics, research and development or education. The concept
became a part of the Common Security and Defense Policy (CSDP) in 2013, before
the split with Moscow and in the times of the record mutual trade exchange and
deepening relations (Shkolyar, 2021). After the crisis around Ukraine erupted,
the strategic autonomy expanded into the external policies, being present in the
2016 EU Global Strategy. It is a similar process compared to that in Russia where
the concept of sovereignty started to be employed in different fields including
economy, power industry, information or culture, thus not limiting itself to the
foreign policy or military. 52 Borrell, J. B. Why European Strategic Autonomy Matters. European External Action Service. 2020, December 03. URL:
https://eeas.europa.eu/headquarters/headquarters-homepage/89865/why-european-strategic-autonomy-matters_en
(accessed 20.02.2022) Comprehensive sovereignty: an external perspective Securitization of the individual fields is accompanied
by the introduction of the concept of sovereignty into new contexts. The same
applies to strategic autonomy in the EU. Josep Borrell, the High Representa-
tive of the European Union for Foreign Affairs and Security Policy, published
an article Why European Strategic Autonomy Matters in which he explains the
reasons for this strategy. Borrell explicitly admits that strategic autonomy is a
“process of political survival” given the objective tendency of decline in the Eu-
ropean significance and influence in the world.52 It is therefore connected with
the protection of the “European way of life” (irrespective of the controversial
and obscure nature of this term), development of the independent position and Governance and Politics Governance and Politics 88 UDC: 323, 327, 369.032 Political Science / Country in Focus distinctive “mission” within the global community and, last but not least, with
the economic base. Face to face with Trump’s protectionism, unilateralism, growing interna-
tional instability and rivalry, as well as the coronavirus pandemic, the EU elites
noticed the Union’s vulnerability and weak points of interdependence. From this
perspective, strategic autonomy can be interpreted as a positive effort to make
the EU one of the poles in the multipolar world, globalized and regionalized
at the same time. Donald Trump’s America First policy, China’s dual circula-
tion and Russia’s comprehensive sovereignty are of a similar nature even though
the individual characteristics vary. Within the EU’s strategic autonomy, Brus-
sels endeavors to boost the international role of the euro, regulate and control
foreign investment (through screening mechanisms) or critical infrastructure
(including 5G, energy sector or health service) as well as increase the military
capabilities. It has launched the European Development Fund (EDF), Perma-
nent Structured Cooperation (PESCO), European Battery Alliance (EBA), Eu-
ropean Raw Material Alliance (ERMA), Future Combat Air System (FCAS) and
many other projects in different fields. The EU aspires to deepen self-sufficien-
cy in some regards, particularly those having strategic significance, to increase
domestic production and decrease the role of import where the latter collides
with the Union’s interests. 53 A Globally Connected Europe: Council Approves Conclusions. European Council. 2021, July 12. URL: https://www.con-
silium.europa.eu/en/press/press-releases/2021/07/12/a-globally-connected-europe-council-approves-conclusions/ (ac-
cessed 20.02.2022) 55 Emmott, R., & Siebold, S. After G7 Pledge, EU Seeks to Rival China’s ‘Belt and Road’ with Own Infrastructure Plan. Reuters,
2021, July 12. URL: https://www.reuters.com/world/china/after-g7-pledge-eu-seeks-rival-chinas-belt-road-with-own-in-
frastructure-plan-2021-07-12/ (accessed 20.02.2022) 54 Global Gateway: up to €300 billion for the European Union’s strategy to boost sustainable links around the world. Europe-
an Commission. 2021, December 01. URL: https://ec.europa.eu/commission/presscorner/detail/en/ip_21_6433 (accessed
20.02.2022) 53 A Globally Connected Europe: Council Approves Conclusions. European Council. 2021, July 12. URL: https://www.con-
silium.europa.eu/en/press/press-releases/2021/07/12/a-globally-connected-europe-council-approves-conclusions/ (ac-
cessed 20.02.2022)
54 Global Gateway: up to €300 billion for the European Union’s strategy to boost sustainable links around the world. Europe-
an Commission. 2021, December 01. URL: https://ec.europa.eu/commission/presscorner/detail/en/ip_21_6433 (accessed
20.02.2022)
55 Emmott, R., & Siebold, S. After G7 Pledge, EU Seeks to Rival China’s ‘Belt and Road’ with Own Infrastructure Plan. Reuters,
2021, July 12. URL: https://www.reuters.com/world/china/after-g7-pledge-eu-seeks-rival-chinas-belt-road-with-own-in-
frastructure-plan-2021-07-12/ (accessed 20.02.2022) Comprehensive sovereignty: an external perspective In response to the results of the G7 summit in Carbis
Bay and following the European Commission’s Asian strategy called Connecting
Europe and Asia – Building Blocks for an EU Strategy announced in 2018, the
Council of the EU approved the basic framework for the initiative A Globally
Connected Europe, a rival project of the BRI.53 It subsequently materialized in
the project of the Global Gateway that explicitly declares the goal to expand
the “EU’s democratic values”.54 The new global infrastructure plan is, however,
prepared in close cooperation with Washington, as German Foreign Minister
Heiko Maas avowed, and builds on partnerships with India and Japan.55 It will 2022, Vol. 1, No. 1 89 Ladislav Zemánek therefore be predisposed to become misused for the US-led confrontation with
“autocracies”. The gradual integration of national military capability and formation of the
EU army can also be subsumed into the strategic autonomy. Integration in this
field poses serious risks in terms of the individual member states, at the same
time, however, it can result in emancipation from Washington and NATO or
a substantial transformation of the North Atlantic Alliance. Obviously, Borrell
understands the importance of the strategic autonomy and its potential, never-
theless, being still unable to overcome the Euro-Atlantic paradigm as demon-
strated by the following words: “[N]o one disputes the vital character of the
transatlantic relationship and no one advocates the development of a fully au-
tonomous European force outside NATO, which remains the only viable frame-
work to ensure the territorial defense of Europe”.56 In fact, the opposite is true. If the EU wants to position itself as an independent center of power, if it aspires
to a sovereign policy, if the highest representatives are serious about the need
for the EU to assume responsibility for itself, the Euro-Atlantic concept must be
abandoned. The EU will not play an independent role in the world if remains
subordinated to the US. Until the Union does not emancipate from Washing-
ton, integration of the Eurasian macro-region, a common space from Lisbon to
Vladivostok and Jakarta free of confrontation and dividing lines will be a mere
vision. Notwithstanding Russia’s pivot to the East, Moscow still reiterates its read-
iness to deepen cooperation with the EU. Formal ties began in 1997 when the
Partnership and Cooperation Agreement (PCA) was concluded, establishing a
general framework of the EU-Russian political and economic relations. 56 Borrell, J. B. Why European Strategic Autonomy Matters. European External Action Service. 2020, December 03. URL:
https://eeas.europa.eu/headquarters/headquarters-homepage/89865/why-european-strategic-autonomy-matters_en
(accessed 20.02.2022) 57 EU and Russia Launch New Partnership for Modernization. European Commission. 2010, June 01. URL: https://ec.europa.
eu/commission/presscorner/detail/en/IP_10_649 (accessed 20.02.2022) eu/commission/presscorner/detail/en/IP_10_649 (accessed 20.02.2022)
58 Lavrov, S. Foreign Minister Sergey Lavrov’s Remarks at a Conference on Relations between Russia and the European Un-
ion. The Ministry of Foreign Affairs of the Russian Federation. 2021a, May 31. URL: https://www.mid.ru/en/foreign_policy/
news/-/asset_publisher/cKNonkJE02Bw/content/id/4759042 (accessed 20.02.2022)
59 EU-Russia Relations: Commission and High Representative Propose the Way forward. European Commission. 2021, June
16. URL: https://ec.europa.eu/commission/presscorner/detail/en/ip_21_3010 (accessed 20.02.2022)
60 Joint Communication to the European Parliament, the European Council and the Council on EU-Russia relations – Push
Back, Constrain and Engage. European Commission. 2021, June 16. URL: https://ec.europa.eu/info/sites/default/files/
joint-communication-eu-russia-relations.pdf (accessed 20.02.2022) 2022, Vol. 1, No. 1 57 EU and Russia Launch New Partnership for Modernization. European Commission. 2010, June 01. URL: https://ec.europa.
eu/commission/presscorner/detail/en/IP_10_649 (accessed 20.02.2022)
58 Lavrov, S. Foreign Minister Sergey Lavrov’s Remarks at a Conference on Relations between Russia and the European Un-
i
Th Mi i t
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i
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31 URL htt
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id
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i
li
/ news/-/asset_publisher/cKNonkJE02Bw/content/id/4759042 (accessed 20.02.2022)
59 EU-Russia Relations: Commission and High Representative Propose the Way forward. European Commission. 2021, June
16. URL: https://ec.europa.eu/commission/presscorner/detail/en/ip_21_3010 (accessed 20.02.2022)
60 Joint Communication to the European Parliament, the European Council and the Council on EU-Russia relations – Push
Back, Constrain and Engage. European Commission. 2021, June 16. URL: https://ec.europa.eu/info/sites/default/files/
joint-communication-eu-russia-relations.pdf (accessed 20.02.2022) 58 Lavrov, S. Foreign Minister Sergey Lavrov’s Remarks at a Conference on Relations between Russia and the Eur
ion. The Ministry of Foreign Affairs of the Russian Federation. 2021a, May 31. URL: https://www.mid.ru/en/forei
news/-/asset_publisher/cKNonkJE02Bw/content/id/4759042 (accessed 20.02.2022) Ibid.
62 Lavrov, S. Foreign Minister Sergey Lavrov’s Remarks at a Conference on Relations between Russia and the European Un-
ion. The Ministry of Foreign Affairs of the Russian Federation. 2021a, May 31. URL: https://www.mid.ru/en/foreign_policy/
news/-/asset_publisher/cKNonkJE02Bw/content/id/4759042 (accessed 20.02.2022)
63 Putin, V. Stat’ia Vladimira Putina «Byt’ otkrytymi, nesmotria na proshloe» (Putin: Be Open, Regardless of the Past). Kremlin.
2021, June 22. URL: http://kremlin.ru/events/president/news/65899 (accessed 20.02.2022)
64 Lavrov, S. Foreign Minister Sergey Lavrov’s Remarks at a Conference on Relations between Russia and the European Un-
ion. The Ministry of Foreign Affairs of the Russian Federation. 2021a, May 31. URL: https://www.mid.ru/en/foreign_policy/
news/-/asset_publisher/cKNonkJE02Bw/content/id/4759042 (accessed 20.02.2022) Comprehensive sovereignty: an external perspective In 2003,
both sides agreed on four “common spaces”, strengthening the strategic partner-
ship. It included the creation of common economic space, cooperation in the
field of both internal and external security and justice, research, education and
culture. In 2010, Moscow and Brussels launched a new Partnership for Modern-
ization on the 25th EU-Russian summit in Rostov-on-Don, addressing common Governance and Politics Governance and Politics 90 Political Science / Country in Focus UDC: 323, 327, 369.032 challenges and problems, for instance, those emerging from the global econom-
ic crisis.57 Recently, Foreign Minister Lavrov reminded the progress made by
both actors prior to 2014 as well as Russia’s proactiveness, when putting forward
the European Security Treaty to establish a common security area, the idea of
a common energy complex, the joint committee on foreign policy and secu-
rity, cooperation in crisis management or visa-free regime, the latter being a
step from realization. Mutual interaction was increasingly intensive at different
levels, including the political one. Permanent Partnership Council as the main
working body of the EU-Russia cooperation was active, joint summits were held
every six months, the Russian Government and the European Commission held
a joint session once a year.58 The gradual building of Greater Europe was a reality. However, this auspicious development failed in 2014. The EU suspended mutual
initiatives, imposed sanctions and joined the US confrontational policy. Since
2016, the EU’s approach to Russia is guided by five principles: (1) full imple-
mentation of the Minsk agreements; (2) strengthening the Eastern Partnership,
involving other countries from the region; (3) building Union’s resilience in the
field of energy security, hybrid threats or strategic communication; (4) selec-
tive engagement with Moscow in chosen areas; (5) promoting people-to-people
contacts and supporting Russian “civil society”.59 The official discourse vis-à-vis
Russia remains hostile. The European Council arranged the first strategic debate
on Russia in May 2021. The result was not a plan how to improve the relation-
ship but a simple condemnation of the “illegal, provocative and disruptive Rus-
sian activities against the EU, its Member States and beyond”.60 The political confrontation does not correspond with other ways of inter-
action whatsoever. Irrespective of the restrictive measures, Russia is the EU’s
fifth-largest trade partner while the EU remains Russia’s biggest trade partner. The EU also accounts for up to 75% of all foreign direct investment in the coun- 2022, Vol. 1, No. Governance and Politics 61 Ibid. Comprehensive sovereignty: an external perspective 1 91 Ladislav Zemánek try. Similar is the situation in the energy sector. Almost two thirds of Russia’s oil
exports, two thirds of the gas exports and approximately half of its coal exports
go to the EU. At the same time, up to 26% of EU oil imports and 40% of gas
imports come from Russia.61 The economic interdependence has thus remained
deep. The mutual interactions and exchange would even intensify in case of po-
litical normalization. The Russian side has also repeatedly raised the question of
linking the EAEU with the EU, but Brussels conditions the debate by normali-
zation.62 The European (and Western) vector is absent in the 2021 NSS, but the Rus-
sian leadership have sent messages that Moscow is interested in stable ties and
revival of the strategic partnership. Vladimir Putin published an article in the
German newspaper Die Zeit in June 2021. The Russian President speaks out
in favor of “constructive interdependence”, “comprehensive partnership”, “com-
mon space of equal cooperation, security and prosperity from the Atlantic to
the Pacific”, thus reaffirming the continuity of the basic principles and goals of
Russia’s European policy regardless of the shift after 2014. Putin emphasizes
Russia’s cultural and historical affinity with Europe, expressing his long-term
belief that the partnership is to be grounded in the close relationship between
Moscow and Berlin.63 Sergei Lavrov presented a somewhat more skeptical atti-
tude in December 2020, asserting that even though the EU could play a role of
an independent pole in the multipolar global system, it has allegedly given up
this ambition as shown by German and French recent policies following the
concept of “rules-based multilateralism”.64 Multilateralism as such is positive (as
opposed to Trump’s unilateralism) but the Western notion of it is exclusive and
permeated with pseudo-universalism, moralism and superiority. In a sense, it is
an expanded unilateralism as it does not accept “the others” on equal terms. The
EU’s multilateralism is conditioned with adherence to the currently proclaimed Governance and Politics 92 Political Science / Country in Focus UDC: 323, 327, 369.032 European (Western) values and rules, to liberal democracy.65 The development
of the rules-based order (RBO) can undermine the UN-based international sys-
tem that is multilateral and inclusive. The RBO is liberal in its essence and de-
nies the equal existence of different patterns and models, whether they are po-
litical, economic or cultural. (
)
70 News Conference Following Russian-Hungarian Talks. Kremlin. 2022, February 01. URL: http://en.kremlin.ru/events/presi-
dent/news/67690 (accessed 20.02.2022) 65 Joint Communication to the European Parliament, the European Council and the Council on Strengthening the EU´s Con-
tribution to Rules-Based Multilateralism. European Commission. (2021, February 17. URL: https://eeas.europa.eu/sites/de-
fault/files/en_strategy_on_strengthening_the_eus_contribution_to_rules-based_multilateralism.pdf (accessed 20.02.2022)
66 Lavrov, S. (2021). O prave, pravakh i pravilakh (On Law, Rights and Rules). Global Affairs, June 28. URL: https://globalaffairs.
ru/articles/o-prave-pravah-i-pravilah/ (accessed 20.02.2022) g
g
69 Siebold, S., & Emmott, R. France and Germany Drop Russia Summit Plan after EU’s East Objects. Reuters. 2021, June 25.
URL https://www.reuters.com/world/europe/france-germany-drop-plans-russia-summit-after-eu-outcry-2021-06-25/
(accessed 20.02.2022) /
/
p
p
p
/ (
)
67 Rettman, A. Macron: EU Sanctions on Russia Do not Work. EU Observer. 2021, May 26. URL: https://euobse
world/151946 (accessed 20.02.2022) 68 Merkel, Macron: ‘Russia Is a Big Challenge for Us’. Deutsche Welle. 2021, June 18. URL: https://www.dw.com/
macron-russia-is-a-big-challenge-for-us/a-57960118 (accessed 20.02.2022) 65 Joint Communication to the European Parliament, the European Council and the Council on Strengthening the EU´s Con-
tribution to Rules-Based Multilateralism. European Commission. (2021, February 17. URL: https://eeas.europa.eu/sites/de-
fault/files/en_strategy_on_strengthening_the_eus_contribution_to_rules-based_multilateralism.pdf (accessed 20.02.2022)
66 Lavrov, S. (2021). O prave, pravakh i pravilakh (On Law, Rights and Rules). Global Affairs, June 28. URL: https://globalaffairs.
ru/articles/o-prave-pravah-i-pravilah/ (accessed 20.02.2022)
67 Rettman, A. Macron: EU Sanctions on Russia Do not Work. EU Observer. 2021, May 26. URL: https://euobserver.com/
world/151946 (accessed 20.02.2022)
68 Merkel, Macron: ‘Russia Is a Big Challenge for Us’. Deutsche Welle. 2021, June 18. URL: https://www.dw.com/en/merkel-
macron-russia-is-a-big-challenge-for-us/a-57960118 (accessed 20.02.2022)
69 Siebold, S., & Emmott, R. France and Germany Drop Russia Summit Plan after EU’s East Objects. Reuters. 2021, June 25.
URL https://www.reuters.com/world/europe/france-germany-drop-plans-russia-summit-after-eu-outcry-2021-06-25/
(accessed 20.02.2022)
70 News Conference Following Russian-Hungarian Talks. Kremlin. 2022, February 01. URL: http://en.kremlin.ru/events/presi-
dent/news/67690 (accessed 20.02.2022) Comprehensive sovereignty: an external perspective These reasons lie behind Lavrov’s critical remarks. In another article, the Foreign Minister relates the EU’s reinterpretation of mul-
tilateralism to the need for an ideological justification in maintaining the West´s
declining power in the transforming world order.66 Russia wants the EU to be a pole in a multipolar system. Not a few political
actors in Europe are interested in equal and win-win cooperation with its biggest
neighbor, but the decision-making processes continue to be dominated by ad-
herents of Euro-Atlanticism who obstruct normalization and a restart of mutual
relations. Signs of a more constructive, rational and pragmatic policy can, nev-
ertheless, be observed throughout the continent. French President Emmanuel
Macron stated in May 2021 that anti-Russian sanctions were not working, call-
ing upon the European Commission to rethink its confrontational position.67
Shortly after, Macron and Chancellor Angela Merkel declared that maintaining
dialogue with Russia was crucial in terms of security and stability.68 Both leaders
subsequently put forward the idea to convene the EU-Russia summit. However,
the initiative was foiled by the Baltic states and Poland.69 Hungary, Greece, Italy
and Portugal belong to countries that are in favor of normalization, or that are
even developing relations with Moscow. Amidst tensions at the beginning of
February 2022, Viktor Orbán made an official visit to Russia, showing an ex-
ample of a constructive, win-win relationship despite Hungary’s membership
in NATO and the EU.70 Viktor Orbán’s “illiberal democracy” with multi-vector 2022, Vol. 1, No. 1 93 Ladislav Zemánek external orientation and concurrent affiliation to Western structures is an in-
spiring model. Furthermore, the prospective successful fulfilment of the Nord
Stream 2 gas pipeline would be a symbol of win-win bilateral cooperation with
Russia despite US sanctions. It seems that – under the current circumstances –
bilateral interactions represent a more viable path compared to the EU-Russia
level, which is extremely politicized. Increasing bilateral relations could subse-
quently affect the collective decisions and actions. f
Neither the EU nor Russia will be able to overcome the essential discrep-
ancies in the years to come. The relationship will likely become a “cold partner-
ship” (Fischer & Timofeev, 2020). It corresponds with the EU’s selective engage-
ment policy presented by the Joint Communication on the EU’s relations with
Russia in June 2021 in accordance with the five principles guiding the Union’s
Russian strategy. Comprehensive sovereignty: an external perspective Josep Borrell summarized it as follows: “Our ambition should
be to explore paths that could help change the current dynamics gradually into a
more predictable and stable relationship. The EU will simultaneously push back,
constrain and engage with Russia, based on a strong common understanding
of Russia’s aims and an approach of principled pragmatism”.71 The dialectics of
confrontation, containment, deterrence, sanctioning on one hand and moder-
ate selective cooperation on the other seem to be the reality of the EU-Russia
interactions of the coming years, whether this is called a “cold partnership” or
“managed confrontation”. 71 EU-Russia Relations: Commission and High Representative Propose the Way forward. European Commission. 2021, June
16. URL: https://ec.europa.eu/commission/presscorner/detail/en/ip_21_3010 (accessed 20.02.2022) Conclusion Interactions between Russia and “the rest” can be characterized in terms
of binary alternation of openness (and endeavor to follow and adopt external
developmental patterns: political, economic, social or cultural) and introversion. Prior to 2014, Russia succeeded in integrating into the international structures
in many respects but rather as an independent center with its own interests,
goals and pretenses, not as a minor, marginal partner of the dominating West-
ern powers. It gradually sobered up from the initial enthusiasm for the West 94 Governance and Politics Governance and Politics Political Science / Country in Focus UDC: 323, 327, 369.032 and the temptation of liberal democracy. Face to face with the NATO expan-
sion eastwards, Western support for Chechen Islamists, separatist forces within
Russia, anti-Russian forces in the post-Soviet countries, experiences with wars
in Afghanistan, Iraq and Yugoslavia, the role of the Western countries in the
color revolutions, weakening of the central role of the United Nations, stagna-
tion of the West after the 2008 economic crisis and – last but not least –cultur-
al wars and progressivist tendencies in the West, Russia revised its conceptual
framework, put an end to Westernization, turned to the East and rejected the
Western developmental pattern as an example to follow (Bratersky, 2014, p. 54-
55, 60-61). The final reversal happened in 2014 in connection with the crisis
in Ukraine. The vision of Russia’s integration in Euro-Atlantic security systems
including NATO did not take place either. If Yeltsin, Putin or Medvedev (and
Gorbachev before them) hoped for the West to recognize Russia as an equal,
full-fledged, important and sovereign member of a single Western community,
they must have been deeply disappointed (Kotkin, 2001). Russia opened un-
precedentedly to the West but the latter remained closed. What is Russia like today, then? The new, still emergent, post-Soviet Rus-
sia is lacking a comprehensive ideology. It avoids extremes, rethinking its posi-
tion and role in the international order. It rejected the radical openness of the
1990s, refusing to be an isolated fortress. It can rather be characterized as find-
ing a middle path. The absence of a strong, high-profiled ideology need not be a
shortcoming for the time being. On the contrary, pragmatism enables flexibility
and synthesis of heterogeneous elements. The new Russia has brought an end to
dogmatism, messianism and seeking utopia, be it religious (Holy Rus’, the Third
Rome) or quasi-religious (Communism) (Lukin, 2018a, p. 2022, Vol. 1, No. 1 Conclusion 150). The recent state
is close to the Chinese principle of seeking truth from facts. The new Russia
wants to revive or safeguard positive traditions and social patterns while adopt-
ing external elements where useful or necessary. The ruling elites have realized
that building a strong state is an objective need and a rational choice for their
country. Historical experiences teach them that Russia cannot get along without
this social institute, as otherwise the society falls into chaos, as happened in the
smutnoe vremia (Time of Troubles) of the early 17th century, after the fall of the
monarchy in 1917 or in the 1990s. Andrei Tsygankov (2014, p. viii-ix) observes
that a strong state has been advocated by socialists, liberals, conservatives as 95 Ladislav Zemánek well as Eurasianists, and so the question is not whether a strong state will be
established in Russia or not – the only question is what the Russian strong state
will be like. While combining traditional etatism or patrimonialism, social-lib-
eral economic policies with conservatism in terms of values, Russia develops a
“managed” or “illiberal” democracy internally. From the external point of view,
Russia cultivates pragmatic relations with different countries, regions and organ-
izations, putting an emphasis on the principles of peaceful coexistence. It wants
to be engaged in global processes and institutions, pursuing integration but at
the same time insists on independence, autonomy, non-interference. Learning
lessons from both the past experiences and the present objective developmental
tendencies, the Russian leadership has arrived at comprehensive sovereignty and
stability which is the leading principle of official policies in post-2014 Russia. fi
In search of a new model of the global order, maintaining some fundamen-
tals of the present one, particularly the central role of the UN, while rejecting
others with the unipolar US-based hegemonism at the top, Russia collides and
clashes with revisionist attempts of the Western actors who oppose the idea of
global polycentric architecture, being imbued with cold-war mentality, confron-
tational notion of a permanent struggle against “autocrats of the world”, and
superiority of liberal democratic model as well as Western values. These char-
acteristics are embodied in the concept of the “rules-based international order”
that substitutes the international law and principles emerging from the UN for
particularist moral categories and haphazardly introduced rules. Such pressure
inevitably provokes resistance among the “rest”. 72 Antonov, A., & Qin, G. Russian and Chinese Ambassadors: Respecting People’s Democratic Rights. National Interest. 2021,
November 26. URL: https://nationalinterest.org/feature/russian-and-chinese-ambassadors-respecting-people’s-demo-
cratic-rights-197165 (accessed 20.02.2022) Conclusion The concurrent containment
of Russia and China has also resulted in an unprecedented level of mutual un-
derstanding and cooperation between the Eurasian major powers. Both coun-
tries’ ambassadors in Washington called for a “harmonious coexistence between
countries with different social systems, ideologies, histories, cultures, and devel-
opment levels” as a basis of what China calls the “community with a shared fu-
ture for mankind”72 in November 2021. Against the Western hegemonism, these
Eurasian major powers uphold the legitimacy of pluriversum and diversity, in- Governance and Politics Governance and Politics 96 Political Science / Country in Focus UDC: 323, 327, 369.032 cluding multiple modernities or democracies. This lower-level declaration was
reaffirmed by the joint statement of President Xi Jinping and Vladimir Putin “on
international relations entering a new era and global sustainable development”,
made on the occasion of the 24th Olympic Winter Games in Beijing in Febru-
ary 2022. The document can be perceived as a manifesto against hegemonism
and militarism as seen in the ongoing NATO expansion or the establishment of
AUKUS, thus promoting cooperation and solidarity instead of confrontation
and divisions.73 Euro-Atlanticism will be a source of frictions and one of the major obstacles
to a peaceful move forward, global restructuring, and the establishment of a
common security area including both Europe and Russia. Unless Washington
gives up its claims to control the European subcontinent, a sustainable solution
of conflicts between Russia and Europe will not be reached. And genuine nego-
tiations about Moscow’s security concerns and national interests are also unlike-
ly to be held, as was shown by the intensified interactions and crisis bargaining
in the first weeks of 2022, in which the US and Joe Biden’s himself played an
inauspicious role.74 Russia’s security requirements aimed at a multilateral, com-
prehensive security settlement in Europe were denied.75 A new impetus to Euro-Atlanticism was given by Biden’s electoral victo-
ry. The US President managed to revive the mutual partnership and Western
multilateralism based on liberal principles and embodied by the rules-based
order. Washington made necessary concessions to overcome the mistrust that
emerged under Donald Trump and amid scandals concerning US spying on
their European allies.76 The EU elites, however, cannot forget the experience
with Biden’s predecessor who exposed the allies to serious uncertainty. Trump’s
unilateralism and “America First” policy contributed to the development and
legitimacy of the strategic autonomy in the EU. Paradoxically, Trumpism played 2022, Vol. 76 Henley, J. Denmark Helped US Spy on Angela Merkel and European Allies – Report. The Guardian, 2021, May 31. URL:
https://www.theguardian.com/world/2021/may/31/denmark-helped-us-spy-on-angela-merkel-and-european-allies-re-
port (accessed 20.02.2022) 73 Joint Statement of the Russian Federation and the People’s Republic of China on the International Relations Entering a
New Era and the Global Sustainable Development. Kremlin. 2022, February 04. URL: http://en.kremlin.ru/supplement/5770
74 Tsygankov, A. P. Liberal Messianism and the Ukraine Crisis Have Turned Joe Biden into a Russia Hawk. Canadian Dimen-
sion. 2022, February 17. https://canadiandimension.com/articles/view/liberal-messianism-and-the-ukraine-crisis-have-
turned-joe-biden-into-a-russia-hawk (accessed 20.02.2022) 73 Joint Statement of the Russian Federation and the People’s Republic of China on the International Relations Entering a
New Era and the Global Sustainable Development. Kremlin. 2022, February 04. URL: http://en.kremlin.ru/supplement/5770
74 Tsygankov, A. P. Liberal Messianism and the Ukraine Crisis Have Turned Joe Biden into a Russia Hawk. Canadian Dimen-
sion. 2022, February 17. https://canadiandimension.com/articles/view/liberal-messianism-and-the-ukraine-crisis-have-
turned-joe-biden-into-a-russia-hawk (accessed 20.02.2022)
75 Obrashchenie Prezidenta Rossiiskoi Federatsii (Presidential Address). Kremlin. 2022, February 21. URL: http://kremlin.ru/
events/president/news/67828 (accessed 20.02.2022)
76 Henley, J. Denmark Helped US Spy on Angela Merkel and European Allies – Report. The Guardian, 2021, May 31. URL:
https://www.theguardian.com/world/2021/may/31/denmark-helped-us-spy-on-angela-merkel-and-european-allies-re-
port (accessed 20.02.2022) 73 Joint Statement of the Russian Federation and the People’s Republic of China on the International Relations Entering a
New Era and the Global Sustainable Development. Kremlin. 2022, February 04. URL: http://en.kremlin.ru/supplement/5770 73 Joint Statement of the Russian Federation and the People’s Republic of China on the International Relations Entering a
New Era and the Global Sustainable Development. Kremlin. 2022, February 04. URL: http://en.kremlin.ru/supplement/5770
74 Tsygankov, A. P. Liberal Messianism and the Ukraine Crisis Have Turned Joe Biden into a Russia Hawk. Canadian Dimen-
sion 2022 February 17 https://canadiandimension com/articles/view/liberal messianism and the ukraine crisis have chenie Prezidenta Rossiiskoi Federatsii (Presidential Address). Kremlin. 2022, February 21. URL: http://kremlin.ru/
president/news/67828 (accessed 20.02.2022) Conclusion 1, No. 1 97 Ladislav Zemánek a progressive role, and made the “autonomization” of individual countries or
regions (the US, the EU, China, Russia) inevitable. All these actors have set out
a path of development for mobilizing and focusing on domestic sources and po-
tential, and deepening integration with their close partners, thus reaffirming the
tendency towards regionalization within globalization. The EU’s strategic au-
tonomy, Russia’s comprehensive concept of sovereignty and China’s dual circu-
lation can be interpreted as a manifestation of the transformation of the global
order towards polycentrism. As such, it may avert a slide into a new, China-US
bipolarity, even though bipolarity coincides with the West’s predisposition to
a dualist or Manichaean way of thinking (Zhao & Kortunov, 2020). This shift
entails the risk of deglobalization, particularism and conflicts, but I expect that
given the existing long-term tendencies and trajectories the scenario of glocali-
zation and pragmatic polycentrism will prevail eventually. References Brands, H. (2021). The Emerging Biden Doctrine: Democracy, Autocracy, and the Defining
Clash of Our Time. Foreign Affairs, June 29. URL: https://www.foreignaffairs.com/articles/unit-
ed-states/2021-06-29/emerging-biden-doctrine (accessed: 14.01.2022)f Bratersky, M. (2014). Transformation of Russia’s Foreign Policy. Russia in Global Affairs, 12(2), 54–
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& Littlefield. Fischer, S., & Timofeev, I. (2020). Alternative futures of EU-Russia relations in 2030. EUREN, No-
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https://eu-russia-expertnetwork.eu/en/sabine-fischer-ivan-timofeev-alternative-fu-
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Belt, One Road Initiative. Europe-Asia Studies, 69 (7), 1027-1046. DOI: 10.1080/09668136.2017.1373270 Kendall-Taylor, A., & Shullman, O. (2021). China and Russia’s Dangerous Convergence. How to
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sia). Russian International Affairs Council. URL: https://russiancouncil.ru/papers/Biden-Russia-Paper60. pdf (in Russian) (accessed: 14.01.2022) Kotkin, S. (2001). Armageddon Averted: The Soviet Collapse, 1970-2000. Oxford University Press. Lavrov, S. V. (2019). The World at a Crossroads and a System of International Relations for the
Future. Russia in Global Affairs, 17(4), 8–18. Lukin, A. (2018a). Putin’s Political Regime and Its Alternatives. Strategic Analysis, 42(2), 134-153. DOI: 10.1080/09700161.2018.1439331 98 Governance and Politics Governance and Politics UDC: 323, 327, 369.032 Political Science / Country in Focus Lukin, A. (2018b). China and Russia. The New Rapprochement. Polity Press. Lukin, A., & Novikov, D. (2021). Greater Eurasia: From Geopolitical Pole to International Society? In
G. Diesen, & A. Lukin (Eds.), The Return of Eurasia. Continuity and Change. Palgrave Macmillan, 33–78. Lunacharskii, A. V. (1908). Religiia i sotsializm. 2 V. Shipovnik. Pipes, R. (1974). Russia under the Old Regime. Charles Scribner’s Sons. Romashkina, N. (2019). Informatsionnyi suverenitet ili pochemu Rossii nuzhna strategiia infor-
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Center, July 06. URL: https://carnegiemoscow.org/commentary/84893 (accessed: 14.01.2022) Tsygankov, A. P. (2014). The Strong State in Russia: Development and Crisis. Oxford University Press. Zemánek, L. (2020). Belt & Road Initiative and Russia: From Mistrust Towards Cooperation. Human
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siagate-contemporary-mccarthyism-in-practice/ (accessed: 14.01.2022) Zemánek, L. (2021). Chastening Experience: Cooperation with Russia Fatal. China-CEE Institute, June
03. URL: https://china-cee.eu/2021/06/03/czech-republic-external-relations-briefing-chastening-ex-
perience-cooperation-with-russia-fatal/ (accessed: 14.01.2022) Zhao, H., & Kortunov, A. (2020). The Coming Bipolarity and Its Implications: Views from China and
Russia. Russian International Affairs Council, November 23. URL: https://russiancouncil.ru/en/analyt-
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Microbiological Oropharyngeal Patterns in Patients with Different Phenotypes of Chronic Obstructive Pulmonary Disease
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Sovremennye tehnologii v medicine
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Microbiological Oropharyngeal Patterns
in Patients with Different Phenotypes
of Chronic Obstructive Pulmonary Disease DOI: 10.17691/stm2018.10.2.11
Received April 27, 2017 DOI: 10.17691/stm2018.10.2.11
Received April 27, 2017 M.A. Karnaushkina, MD, PhD, Associate Professor, Department of Hospital Therapy No.21; M.A. Karnaushkina, MD, PhD, Associate Professor, Department of Hospital Therapy No.21;
S.V. Fedosenko, MD, Assistant, Department of General Practice and Polyclinic Therapy2;
A.E. Sazonov, PhD, Senior Researcher, Central Research Laboratory2;
V.A. Petrov, Junior Researcher, Central Research Laboratory2;
D.Yu. Ovsyannikov, MD, DSc, Head of the Department of Pediatrics3;
L.M. Ogorodova, MD, DSc, Professor, Corresponding Member of the Russian Academy of Sciences,
Head of the Department of Pediatrics with the Course of Childhood Diseases, Faculty of Medicine2
1I.M. Sechenov First Moscow State Medical University, 8/2 Trubetskaya St., Moscow, 119991, Russia;
2Siberian State Medical University, 2 Moskovsky trakt, Tomsk, 634050;
3Peoples’ Friendship University of Russia, 6 Miklukho-Maklaya St., Moscow, 117198, Russia p
p
S.V. Fedosenko, MD, Assistant, Department of General Practice and Polyclin A.E. Sazonov, PhD, Senior Researcher, Central Research Labo
V.A. Petrov, Junior Researcher, Central Research Laboratory2; ,
,
y ;
D.Yu. Ovsyannikov, MD, DSc, Head of the Department of Pediatrics3; y
p
L.M. Ogorodova, MD, DSc, Professor, Corresponding Member of the Russian Academy of Sciences,
Head of the Department of Pediatrics with the Course of Childhood Diseases, Faculty of Medicine2 . Sechenov First Moscow State Medical University, 8/2 Trubetskaya St., Moscow, 119991, Russia;
berian State Medical University, 2 Moskovsky trakt, Tomsk, 634050; 1I.M. Sechenov First Moscow State Medical University, 8/2 Trubetskaya St., Moscow, 119991, Russia;
2Sib
i
St t
M di
l U i
it
2 M
k
k t
kt T
k 634050 y,
y
,
,
;
3Peoples’ Friendship University of Russia, 6 Miklukho-Maklaya St., Moscow, 117198, Russia Persistent bronchial inflammation in chronic obstructive pulmonary disease (COPD) is considered the cause of ventilation disorders
and related contamination with conditionally pathogenic microorganisms; the latter can proceed and transform into a full infection, which can
aggravate and exacerbate COPD. The aim of the study was to evaluate the relations between the oropharyngeal microbiota in patients with COPD
functional, and prognostic parameters of the disease. Materials and Methods. 64 patients with COPD were included in the study; the participants were scheduled to visit our clinic on
two occasions. In the first visit, their medical history was studied in detail and the major examination procedures were conducted. сlinical medicine
Microbiological Oropharyngeal Patterns
in Patients with Different Phenotypes
of Chronic Obstructive Pulmonary Disease
DOI: 10.17691/stm2018.10.2.11
Received April 27, 2017
M.A. Karnaushkina, MD, PhD, Associate Professor, Department of Hospital Therapy No.21;
S.V. Fedosenko, MD, Assistant, Department of General Practice and Polyclinic Therapy2;
A.E. Sazonov, PhD, Senior Researcher, Central Research Laboratory2;
V.A. Petrov, Junior Researcher, Central Research Laboratory2;
D.Yu. Ovsyannikov, MD, DSc, Head of the Department of Pediatrics3;
L.M. Ogorodova, MD, DSc, Professor, Corresponding Member of the Russian Academy of Sciences,
Head of the Department of Pediatrics with the Course of Childhood Diseases, Faculty of Medicine2
1I.M. Sechenov First Moscow State Medical University, 8/2 Trubetskaya St., Moscow, 119991, Russia;
2Siberian State Medical University, 2 Moskovsky trakt, Tomsk, 634050;
3Peoples’ Friendship University of Russia, 6 Miklukho-Maklaya St., Moscow, 117198, Russia
Persistent bronchial inflammation in chronic obstructive pulmonary disease (COPD) is considered the cause of ventilation disorders сlinical medicine
Microbiological Oropharyngeal Patterns
in Patients with Different Phenotypes
of Chronic Obstructive Pulmonary Disease
DOI: 10.17691/stm2018.10.2.11
Received April 27, 2017
M.A. Karnaushkina, MD, PhD, Associate Professor, Department of Hospital Therapy No.21;
S.V. Fedosenko, MD, Assistant, Department of General Practice and Polyclinic Therapy2;
A.E. Sazonov, PhD, Senior Researcher, Central Research Laboratory2;
V.A. Petrov, Junior Researcher, Central Research Laboratory2;
D.Yu. Ovsyannikov, MD, DSc, Head of the Department of Pediatrics3;
L.M. Ogorodova, MD, DSc, Professor, Corresponding Member of the Russian Academy of Sciences,
Head of the Department of Pediatrics with the Course of Childhood Diseases, Faculty of Medicine2
1I.M. Sechenov First Moscow State Medical University, 8/2 Trubetskaya St., Moscow, 119991, Russia;
2Siberian State Medical University, 2 Moskovsky trakt, Tomsk, 634050;
3Peoples’ Friendship University of Russia, 6 Miklukho-Maklaya St., Moscow, 117198, Russia
Persistent bronchial inflammation in chronic obstructive pulmonary disease (COPD) is considered the cause of ventilation disorders сlinical medicine
Microbiological Oropharyngeal Patterns
in Patients with Different Phenotypes
of Chronic Obstructive Pulmonary Disease
DOI: 10.17691/stm2018.10.2.11
Received April 27, 2017
M.A. Karnaushkina, MD, PhD, Associate Professor, Department of Hospital Therapy No.21;
S.V. Fedosenko, MD, Assistant, Department of General Practice and Polyclinic Therapy2;
A.E. Sazonov, PhD, Senior Researcher, Central Research Laboratory2;
V.A. Petrov, Junior Researcher, Central Research Laboratory2;
D.Yu. Ovsyannikov, MD, DSc, Head of the Department of Pediatrics3;
L.M. Ogorodova, MD, DSc, Professor, Corresponding Member of the Russian Academy of Sciences,
Head of the Department of Pediatrics with the Course of Childhood Diseases, Faculty of Medicine2
1I.M. Sechenov First Moscow State Medical University, 8/2 Trubetskaya St., Moscow, 119991, Russia;
2Siberian State Medical University, 2 Moskovsky trakt, Tomsk, 634050;
3Peoples’ Friendship University of Russia, 6 Miklukho-Maklaya St., Moscow, 117198, Russia
P
i t
t b
hi l i fl
ti
i
h
i
b t
ti
l
di
(COPD) i
id
d th
f
til ti
di
d сlinical medicine сlinical medicine Microbiological Oropharyngeal Patterns
in Patients with Different Phenotypes
of Chronic Obstructive Pulmonary Disease Those
included an assessment of the respiratory function, the 6-minute walk test, the degree of dyspnea by the Medical Research Council scale,
body plethysmography, the diffusion capacity of the lungs, and a chest CT scan. The second visit took place 12 months after the first one to
assess the changes in the course of the disease. The result was considered negative if, in the second examination, the patient‘s condition
was found more severe. Oropharyngeal samples of all patients were sequenced to identify the V3–V4 variable sites of the 16S rRNA gene. Results. It is found that the microbiological oropharyngeal patterns in COPD patients depend on the source of micro-aspiration. In
addition, the changes in the oropharyngeal microbiota correlate with the severity and prognosis of the disease, as well as the patient
phenotype. Based on the data obtained by sequencing parts of the 16S rRNA gene, the role of oropharyngeal microbiota in determining the
course and prognosis of COPD has been elucidated. Conclusion. The presented clinical and functional characteristics associated with oropharyngeal microbiota indicate that micro-
aspirations from other body compartments not only affect the composition of oropharyngeal microbiota in patients with COPD but also have
an important prognostic significance. Key words: COPD; oropharyngeal microbiota; sequencing; COPD phenotypes; severity. Key words: COPD; oropharyngeal microbiota; sequencing; COPD phenotypes; severity. Corresponding author: Maria A. Karnaushkina, e-mail: kar3745@yandex.ru Introduction The authors suggested
their research would shed light on the causes of chronic
pulmonary disorders. However, none of the published
studies analyzed the respiratory microbiota in respect
to the disease progression and its clinical/functional
parameters [8–11]. This may be due to the fact that,
firstly, COPD is a clinically heterogeneous disease, and
secondly, determination of the microbiota composition
using classical microbiological methods does not provide
a complete picture of its composition.i bronchodilation test and the pulmonary diffusion index
(body plethysmography). The study was performed
using a MasterScreen Body instrumentation (Erich
Jaeger, Germany). The obtained data were compared
to the reference values calculated by the generally
accepted formulas recommended by the European
Respiratory Society and American Thoracic Society. All
patients underwent computed tomography scan of the
chest (chest CT) with a Somatom Sensation 40 spiral CT
scanner (Siemens, Germany). Densitometry of the lung
tissue and bronchial structure was performed using the
Pulmo CT program (Siemens, Germany). During the first
visit, oropharyngeal swabs were taken from all patients. Based on the clinical and functional parameters
obtained at the examination, the patients were divided
into groups according to three phenotypes of COPD:
“chronic bronchitis” (n=30), “emphysema” (n=14), and
“mixed” (n=20). This stratification was based on the
emphysema and bronchitis phenotypes as proposed by
Burrows (1966) and Dornhorst (1955) and modified by us. In this study, we used the phenotypic classification
of COPD based on clinical and functional differences
between
the
patients. As
compared
with
the
determination of the forced expiratory volume (FEV1)
alone, the proposed parameter better reflects the
severity and prognosis of the disease. Here, the
composition of microbiota was determined using
the methods of molecular-genetic identification of
microorganisms [1, 4], which significantly changed our
knowledge about the oropharyngeal microbiome. The
obtained results enabled us to propose a prognostic
model to predict the severity of COPD based on the
taxonomic composition of the oropharyngeal microbiota. The phenotype of “chronic bronchitis” describes
patients whose major clinical symptom (aside from
exacerbation periods) was cough with mucopurulent
sputum. At auscultation, harsh breathing sounds and
rales were detected; the chest CT showed signs of
bronchitis, the diffusion lung capacity was normal. The “emphysema” phenotype refers to patients
whose major clinical symptom (aside from exacerbation
periods) was shortness of breath; cough was mild and
unproductive. Introduction At auscultation, a weakened vesicular
breath with wheezing episodes was heard; the
characteristic signs and a decreased diffusion capacity
were present in the CT scans. The aim of the study was to evaluate the relations
between the oropharyngeal microbiota in patients
with COPD and the clinical, functional, and prognostic
parameters of the disease. Patients, who showed no obvious signs of bronchitis
or emphysema during non-exacerbation periods, were
included in the “mixed” phenotype group. Introduction contamination of the bronchial tree in COPD has been
implicated in the increasing risk of COPD transformation
into an infectious process with its exacerbations and
disease progression [3, 4]. However, the involvement
of the respiratory microbiota associated with the COPD
phenotype in this process is still debatable. The development of ventilation disorders in chronic
obstructive pulmonary disease (COPD) is manifested
by a progressive decrease in airflow associated with an
abnormal inflammatory response of the lungs to harmful
particles [1, 2], including microorganisms [1]. At the
same time, chronic bronchopulmonary inflammation and
a decreased airflow create conditions for contamination
of the respiratory tract by opportunistic and pathogenic
flora that change the composition of the respiratory
microbiota [1, 3]. In recent publications, the bacterial Thus, Chambers et al. [5] demonstrated that the
microbiota in the respiratory tract of patients with
predominantly severe COPD significantly differed from
the bacterial composition of the airways in healthy
individuals. In the COPD patients, there were less
Bacteroides spp. and an increased representation Corresponding author: Maria A. Karnaushkina, e-mail: kar3745@yandex.ru СТМ ∫ 2018 — vol. 10, No.2 101 СТМ ∫ 2018 — vol. 10, No.2 101 Microbiological Oropharyngeal Patterns in Patients with Different Phenotypes of COPD сlinical medicine of Firmicutes and Proteobacteria. Similar results
were obtained by Charlson et al. [6]. It has been also
suggested that the abnormal changes in microbiota
could be initiated at the perinatal period. Thus,
Górecka D. and Puścińska E. [7] analyzed the
composition of the respiratory tract microbiota in a
newborn and its correlation with baby’s nutrition, and the
use of antibiotics by the mother. The authors suggested
their research would shed light on the causes of chronic
pulmonary disorders. However, none of the published
studies analyzed the respiratory microbiota in respect
to the disease progression and its clinical/functional
parameters [8–11]. This may be due to the fact that,
firstly, COPD is a clinically heterogeneous disease, and
secondly, determination of the microbiota composition
using classical microbiological methods does not provide
a complete picture of its composition.i of Firmicutes and Proteobacteria. Similar results
were obtained by Charlson et al. [6]. It has been also
suggested that the abnormal changes in microbiota
could be initiated at the perinatal period. Thus,
Górecka D. and Puścińska E. [7] analyzed the
composition of the respiratory tract microbiota in a
newborn and its correlation with baby’s nutrition, and the
use of antibiotics by the mother. M.A. Karnaushkina, S.V. Fedosenko, A.E. Sazonov, V.A. Petrov, D.Yu. Ovsyannikov, L.M. Ogorodova Materials and Methods this
du
wit
pat
CO
and
mo
res
dat
Comparative characte
with different phenoty
(Me [Q1; Q3])
Parameters
Exacerbations
for 12 months
Existence of comorbid
disorders (n/%)
“Triple” therapy, per year
(n/%)
The number
of antibacterial courses,
per year
BODE index (scores) were tested for the presence of the
and the V3–V4 sites sequences
16S Metagenomic Sequencing Lib
protocol recommended by Illumina (U
sequencer. A taxonomic classificat
out according to the sequence data
rRNA gene of Greengenes v. 13.5 wi
Bayesian classifier and the QIIME (Qu
into Microbial Ecology) software. T
differences between the phenotype-
specified groups of samples at the
level of the operating taxonomic
unit (OTU), a method based on
linear
regression
analysis
and
adjusted to the metagenome data
by the metagenomeSeq package
was used [12]. To detect clusters
of
microorganisms,
the
CoNet
plugin version 1.1.beta [13] of the
Cytoscape software was utilized. Correlations between the amounts
of
individual
microorganisms
were
determined
according
to
the Spearman method, only the
correlations with a coefficient of
0.05 or higher were considered. To quantify the impact of the
oropharyngeal microbiota on COPD
progression, a prognostic model was
developed using the caret package,
where
the
recursive
elimination
of variables with a 10-fold cross-
validation was applied. The composition of oropharyngeal microbiota in
patients with different clinical and functional types of
COPD was dominated by the Streptococcus, Veillonella,
and Prevotella genera of microorganisms (10% and
more) in all phenotypes of the disease (Figure 1). Our
results on these “highly represented taxa” agree with the
data reported by others [14–17]. Comparative characteristics of patients (the extended examination group)
with different phenotypes of chronic obstructive pulmonary disease
(Me [Q1; Q3])
Parameters
COPD phenotypes (n=64)
p1–2
p1–3
p2–3
1. “Chronic
bronchitis” (n=30)
2. “Emphysema”
(n=14)
3. Materials and Methods During the second visit, which took place 12 months
after the first one, all patients underwent a clinical
and functional examination, an assessment of the
exacerbation rate, an evaluation of dyspnea by the MRC
scale, a determination of exercise tolerance and a test
of external respiration using a bronchodilator probe. We analyzed the dynamics of COPD during the past 12
months of observation, taking into account the severity
of the symptoms, the post-bronchodilation value of
FEV1 and the risk of exacerbations (GOLD 2015). The
dynamics of the disease was considered negative if, at
the second examination, the patient presented with a
more severe course of COPD than he did on the first
visit. The oropharyngeal samples were not taken during
the second visit. This prospective non-interventional study included 64
patients with COPD. The average age was 57 (50–63)
years. All patients had confirmed COPD according to the
GOLD 2015 criteria [1, 2]; the diagnosis was made at
least 12 months before the enrollment, and their smoking
index was higher than 10 packs/year. For 4 weeks prior
to the enrollment, they received no antibiotic therapy. The study was conducted in accordance with the
Helsinki Declaration (2013) and approved by the local
Ethics Committee of the Siberian State Medical
University (protocol No.1927 of 28.03.2011). An informed
consent has been obtained from each patient. The study included two patient visits. During the
first visit, all patients were clinically examined and their
relevant medical history was carefully studied (below we
will refer to these patients as the extended examination
group). Tolerance to exercise was determined using
a 6-minute walk test. The degree of dyspnea was
assessed by the MRC (Medical Research Council)
scores; the external respiration was evaluated using the The Statistica 10.0 software package was used for
statistical processing of the medical history data and
the clinical/functional indices. When comparing the
qualitative characteristics, the criterion χ2 was applied. To calculate the difference of the means between non-
paired samples, the Mann–Whitney U test was used. The qualitative data are presented as absolute or 102 СТМ ∫ 2018 — vol. 10, No.2 M.A. Karnaushkina, S.V. Fedosenko, A.E. Sazonov, V.A. Petrov, D.Yu. Ovsyannikov, L.M. Ogorodova сlinical medicine relative (%) values. Materials and Methods “Mixed”
(n=20)
Exacerbations
for 12 months
2.0 [1.0; 2.0]
2.0 [1.6; 2.0]
5.0 [3.8; 5.0]
p1–2>0.05
p1–3<0.05
p2–3<0.0001
Existence of comorbid
disorders (n/%)
7.0/23.3
3.0/21.4
16.0/80.0
p1–2>0.05
p1–3<0.001
p2–3<0.001
“Triple” therapy, per year
(n/%)
1.0/3.3
1.0/7.1
9.0/45.0
p1–2<0.05
p1–3<0.0001
p2–3<0.0001
The number
of antibacterial courses,
per year
2.0 [1.0; 3.0]
1.0 [1.0; 1.8]
3.0 [1.8; 5.0]
p1–2<0.05
p1–3<0.05
p2–3<0.0001
BODE index (scores)
2.0 [1.0; 2.0]
2.0 [1.0; 2.8]
5.0 [4.0; 6.0]
p1–2>0.05
p1–3<0.05
p2–3<0.05
MRC (scores)
2.0 [1.0; 2.0]
2.0 [1.0; 2.8]
3.0 [2.0; 4.0]
p1–2>0.05
p1–3<0.0001
p2–3=0.015
6-minute walk test (m)
500 [426; 518]
380 [348; 398]
308 [224; 376]
p1–2<0.05
p1–3<0.05
p2–3<0.0001
FEV1 after bronchodilation
(% of reference value)
68.5 [59.0; 71.0]
68.0 [58.0; 75.0]
48.0 [45.0; 49.0]
p1–2>0.05
p1–3<0.05
p2–3<0.05
SpO2 after 6-minute walk
(%)
98.0 [97.0; 99.0]
96.0 [95.0; 99.0]
94.0 [93.0; 95.0]
p1–2>0.05
p1–3<0.05
p2–3<0.05 Comparative characteristics of patients (the extended examination group)
with different phenotypes of chronic obstructive pulmonary disease
(Me [Q1; Q3]) Materials and Methods The quantitative data are presented
as follows: those with a non-normal distribution — as Me
[Q25; Q75]; and those with the normal distribution —
as M±SD, where M is the arithmetic mean, SD is the
standard deviation. The normality of distributions of
was tested with the χ2 criterion. The difference of the
means was considered statistically significant at p<0.05. The Spearman coefficient was used for the correlation
analysis. The strength of the correlation was denoted
as strong at ±0.7 to ±1; average at ±0.3 to ±0.699; and
weak at 0 to ±0.299. with the two other phenotypes, “chronic bronchitis” and
“emphysema”, patients with the “mixed” phenotype
had more exacerbations per year and a higher value
of the BODE index. The FEV1 values for the “mixed”
phenotype were significantly lower than those for the two
other phenotypes (see the Table). The analyzed data indicated that patients with the
“mixed” COPD phenotype were treated with inhalations
recommended for patients with severe and extremely
severe COPD: long-acting anticholinergic drugs +
long-acting β2-agonists + inhaled glucocorticoids. In
this group, the number of antibacterial therapy courses
during the year was significantly higher as compared
with the two other groups. Samples of oropharyngeal swabs of all patients
were tested for the presence of the 16S rRNA gene
and the V3–V4 sites sequences according to the
16S Metagenomic Sequencing Library Preparation
protocol recommended by Illumina (USA) for the MiSeq
sequencer. A taxonomic classification was carried
out according to the sequence database of the 16S
rRNA gene of Greengenes v. 13.5 with the help of the
Bayesian classifier and the QIIME (Quantitative Insights
into Microbial Ecology) software. To find taxonomic
differences between the phenotype-
specified groups of samples at the
level of the operating taxonomic
unit (OTU), a method based on
linear
regression
analysis
and
adjusted to the metagenome data
by the metagenomeSeq package
was used [12]. To detect clusters
of
microorganisms,
the
CoNet
plugin version 1.1.beta [13] of the
Cytoscape software was utilized. Correlations between the amounts
of
individual
microorganisms
were
determined
according
to
the Spearman method, only the
correlations with a coefficient of
0.05 or higher were considered. To quantify the impact of the
oropharyngeal microbiota on COPD
progression, a prognostic model was
developed using the caret package,
where
the
recursive
elimination
of variables with a 10-fold cross-
validation was applied. Results and Discussion In this study, a comparative
intergroup analysis of the clinical
and functional parameters of patients
with different COPD phenotypes
was performed. As a result, clinically
important data were obtained to
suggest
that
patients
with
the
“mixed” phenotype of COPD had
the most severe manifestations of
the disease. Thus, in comparison СТМ ∫ 2018 — vol. 10, No.2 103
Microbiological Oropharyngeal Patterns in Patients with Different Phenotypes of COPD СТМ ∫ 2018 — vol. 10, No.2 103 Microbiological Oropharyngeal Patterns in Patients with Different Phenotypes of COPD сlinical medicine The metagenomeSeq-assisted analysis of low-
represented taxa (less than 10% of the identified
microbiome) revealed a predominance of bacterial
commensals in patients with the phenotype of
emphysema. Among those, the Firmicutes bacteria
(Acidaminococcus LogFC 1.865; Anaerovibrio LogFC
2.653; Leuconostoc LogFC 1.727; Oceanobacillus
LogFC 1.107; Phascolarctobacterium LogFC 2.915;
Tetragenococcus LogFC 1.513), the Actinobacteria
Actinobacteria,
Proteobacteria,
Bacteroidetes,
Firmicutes, Bacteroides, mainly due to a higher content
of opportunistic bacteria of Haemophilus (LogFC 8.662),
Actinomyces (LogFC 9.813), Lactobacillus (LogFC
4.758), Leptotrichia (LogFC 10.556), Porphyromonas
(LogFC 5.896), Bacillus (LogFC 3.161), Gemella (LogFC
8.303) (p<0.001). Also in these patients, a considerable
amount of opportunistic bacteria in the oropharynx
was found: Prevotella melaninogenica species (LogFC
The “chronic bronchitis” phenotype
The “emphysema” phenotype
Streptococcus
29%
Granulicatella
2%
Rothia
2%
Actinomyces
3%
Porphyromonas
3%
Fusobacterium
4%
Haemophilus
4%
Leptotrichia
4%
Veillonella
19%
Veillonella
19%
other
4%
Prevotella
19%
Prevotella
19%
Streptococcus
22%
other
5%
Haemophilus
3%
Porphyromonas
3%
Fusobacterium
5%
campylobacter
2%
Actinomyces
4%
Neisseria
4%
Leptotrichia
6%
The “mixed” phenotype
other
3%
Rothia
3%
Streptococcus
31%
Porphyromonas
2%
Granulicatella
2%
Fusobacterium
2%
Leptotrichia
3%
Neisseria
3%
Actinomyces
3%
Haemophilus
4%
Veillonella
17%
Prevotella
21%
Figure 1. Prevalent genera of microorganisms, comprising
95% of the metagenome of the oropharyngeal microbiota in
patients with chronic obstructive pulmonary disease (the
extended examination group) The “emphysema” phenotype
Veillonella
19%
Prevotella
19%
Streptococcus
22%
other
5%
Haemophilus
3%
Porphyromonas
3%
Fusobacterium
5%
campylobacter
2%
Actinomyces
4%
Neisseria
4%
Leptotrichia
6% The “chronic bronchitis” phenotype
Streptococcus
29%
Granulicatella
2%
Rothia
2%
Actinomyces
3%
Porphyromonas
3%
Fusobacterium
4%
Haemophilus
4%
Leptotrichia
4%
Veillonella
19%
other
4%
Prevotella
19% Prevotella
19% Veillonella
19% The “mixed” phenotype
other
3%
Rothia
3%
Streptococcus
31%
Porphyromonas
2%
Granulicatella
2%
Fusobacterium
2%
Leptotrichia
3%
Neisseria
3%
Actinomyces
3%
Haemophilus
4%
Veillonella
17%
Prevotella
21% Figure 1. M.A. Karnaushkina, S.V. Fedosenko, A.E. Sazonov, V.A. Petrov, D.Yu. Ovsyannikov, L.M. Ogorodova Results and Discussion To identify the respiratory microbiota patterns
associated
with
specific
clinical
and
functional
characteristics of the COPD patients, the Spearman’s
correlation analysis was performed. In the “chronic bronchitis” phenotype patients,
a cluster of conditionally pathogenic bacteria was
found; within the cluster, a strong positive correlation
between the different microbial taxa was identified:
Fusobacterium + Prevotella + Mycoplasma + Bulleidia
(r=0.701; p<0.05). In the COPD patients with the “emphysema”
phenotype, a cluster of normal oropharyngeal microbiota
of the Firmicutes type was found. In addition, an
Actinobacteria cluster of opportunistic bacteria of the
Enterococcus gastrointestinal type and strong positive
intra-cluster correlations was also present (r>0.08,
p<0.001 and r>0.08, p<0.05, respectively). In the “mixed” phenotype, two clusters of opportunistic
oropharyngeal bacteria with a strong positive correlation
were found: Porphyromonas + Fusobacterium +
Prevotella (r=0.832, p<0.05) and Streptococcus + Rothia
(r=0.761, p<0.05). High levels of bacteria typical for the gastrointestinal
microbiota
were
found
in
oropharyngeal
swabs
from patients with the “emphysema” phenotype;
that included a cluster of normal oropharyngeal
microflora (such as Firmicutes) and Actinobacteria +
Enterococcus (opportunistic gastrointestinal microbiota). Published [20, 21] studies have demonstrated that
gastroesophageal reflux disease (GERD) is associated
with chronic bronchopulmonary diseases, including
COPD, thus aggravating the course of the main
disease. In our study, GERD was found in 11 patients Thus, the patients with COPD showed three patterns
of oropharyngeal microbiota composition. In patients
with the “chronic bronchitis” phenotype, opportunistic
bacteria were mostly represented. In patients with the
“emphysema” phenotype, the oropharyngeal normobiota
and
intestinal
microbiota
(including
conditionally-
pathogenic bacteria) were dominant. In patients with the
“mixed” phenotype, a pattern with a low species diversity
and a predominance of gastrointestinal opportunistic
microbiota (Firmicutes), in combination
with the opportunistic bacteria cluster
(Porphyromonas
+
Prevotella
+
Fusobacterium) was found. Figure 2. Multiple correlations between different components of the
oropharyngeal microbiota in patients with different prognoses of chronic
obstructive pulmonary disease
The data includes variables with a Spearman correlation value of ≥0.05 at p<0.05
after introducing the Benjamin–Hochberg correction for multiple comparisons. The blue diamond on the chart shows the optimal sub-selection of variables,
which allows to classification of the patients with an accuracy of 76% In this study, it was attempted, for the
first time, to develop a prognostic model
based on the clinical and functional
phenotypes
of
COPD
and
their
respective
microbiological
patterns. Results and Discussion Prevalent genera of microorganisms, comprising
95% of the metagenome of the oropharyngeal microbiota in
patients with chronic obstructive pulmonary disease (the
extended examination group) Prevotella
21% Actinobacteria,
Proteobacteria,
Bacteroidetes,
Firmicutes, Bacteroides, mainly due to a higher content
of opportunistic bacteria of Haemophilus (LogFC 8.662),
Actinomyces (LogFC 9.813), Lactobacillus (LogFC
4.758), Leptotrichia (LogFC 10.556), Porphyromonas
(LogFC 5.896), Bacillus (LogFC 3.161), Gemella (LogFC
8.303) (p<0.001). Also in these patients, a considerable
amount of opportunistic bacteria in the oropharynx
was found: Prevotella melaninogenica species (LogFC
11.738), Rothia mucilaginosa (LogFC 9.257), and
Veillonella parvula (LogFC 10.631) (p<0.001). The metagenomeSeq-assisted analysis of low-
represented taxa (less than 10% of the identified
microbiome) revealed a predominance of bacterial
commensals in patients with the phenotype of
emphysema. Among those, the Firmicutes bacteria
(Acidaminococcus LogFC 1.865; Anaerovibrio LogFC
2.653; Leuconostoc LogFC 1.727; Oceanobacillus
LogFC 1.107; Phascolarctobacterium LogFC 2.915;
Tetragenococcus LogFC 1.513), the Actinobacteria
bacteria (Micrococcus LogFC 1.002; Dermacoccus
LogFC
2.191),
and
the
Proteobacteria
bacteria
(Cardiobacterium
LogFC
1.002;
Novosphingobium
LogFC 1.561) (p<0.001). Samples of oropharyngeal swabs from patients with
COPD with the “mixed” phenotype showed the prevailing
representation of the Firmicutes and Bacteroides
bacteria (p<0.001) with a less diverse variety of species
in comparison with the two other phenotypes. Analysis of the low-represented taxa in patients
with the “chronic bronchitis” phenotype revealed a
significant predominance of bacteria of Fusobacterium, 104 СТМ ∫ 2018 — vol. 10, No.2 M.A. Karnaushkina, S.V. Fedosenko, A.E. Sazonov, V.A. Petrov, D.Yu. Ovsyannikov, L.M. Ogorodova сlinical medicine oropharyngeal microbiota depends on the source of
micro-aspiration (gastrointestinal tract, nasopharynx,
oral cavity). Numerous studies [15, 18, 19] demonstrated
the role of opportunistic oropharyngeal bacteria in
the development of pneumonia and exacerbations of
chronic bronchopulmonary diseases. These bacteria
enter the lower respiratory tract as a result of micro-
aspiration from the upper respiratory tract. According to
our study, 40% of patients with the “chronic bronchitis”
phenotype and 70% with the “mixed” phenotype
suffered from tonsillitis, sinusitis or periodontitis. In
these patients, we detected the formation of clusters of
oropharyngeal opportunistic bacteria: Fusobacterium +
Prevotella + Mycoplasma + Bulleidia, Porphyromonas +
Prevotella + Fusobacterium and Porphyromonas +
Prevotella + Streptococcus minor. This finding supports
the hypothesis that the aspirated oropharyngeal
microflora transforms the composition of the airways
microbiome in patients with the “mixed” and “chronic
bronchitis” phenotypes of COPD. References 1. Chuchalin
A.G.,
Avdeev
S.N.,
Aysanov
Z.R.,
Belevskiy A.S., Leshchenko I.V., Meshcheryakova N.N.,
Ovcharenko S.I., Shmelev E.I. Russian respiratory society. Federal guidelines on diagnosis and treatment of chronic
obstructive pulmonary disease. Russian Pulmonology 2014; 3:
15–36, https://doi.org/10.18093/0869-0189-2014-0-3-15-54. 2. Global Initiative for Chronic Obstructive Lung Disease. Global strategy for diagnosis, management, and prevention of
COPD. Updated 2015. 3. Fedosenko S.V., Ogorodova L.M., Popenko A.S.,
Petrov V.A., Tyakht A.V., Saltykova I.V., Kamaltynova E.M.,
Deev I.A., Kulikov E.S., Kirillova N.A., Govorun V.M.,
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quantitative characteristics of content of oro-pharyngeal
microbiota of patients with chronic obstructive disease of lungs. Lechenie i profilaktika 2015; 2(14): 92–99. The presented clinical and functional characteristics
of COPD patients and their correlations with the
composition of the oropharyngeal microbiota allow us
to propose that micro-aspiration from different body
compartments affects not only the oropharyngeal
microbiota but also contributes to the phenotypic
characterization and clinical prognosis of COPD. At
present, it is difficult to ascertain, what are the primary
factors determining the clinical and functional phenotype
of COPD. They can be either the phenotypic traits of
the disease (e.g., the impact of GERD) or a disorder
of the upper respiratory tract or the composition of the
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Frank I., Yadav A., Bushman F.D., Collman R.G. Topographical
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963, https://doi.org/10.1164/rccm.201104-0655oc. As shown in the present study, the low species
diversity of the oropharyngeal microbiota in patients with
COPD of the “mixed” phenotype may favor progression
and aggravation of the disease. 7. Górecka D., Puścińska E. Microbiome of the lung. Pneumonol Alergol Pol 2014; 82(6): 481–485, https://doi. org/10.5603/PiAP.2014.0063. g
8. Park H., Shin J.W., Park S.G., Kim W. Microbial
communities in the upper respiratory tract of patients with
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One 2014; 9(10): e109710, https://doi.org/10.1371/journal. pone.0109710. Results and Discussion Our analysis indicates that an increase
in the oropharyngeal Moraxellaceae,
Peptococcaceae,
Eubacteriaceae,
Enterococcaceae, Staphylococcaceae,
and
Actinomycetaceae
bacterial
families is an unfavorable prognostic
factor
in
terms
of
the
disease
progression. In the validation test, this
model showed a sensitivity of 0.67 and
specificity of 1, a prognosis accuracy of
75% with an area under the ROS curve
of 0.72 (Figure 2). Figure 2. Multiple correlations between different components of the
oropharyngeal microbiota in patients with different prognoses of chronic
obstructive pulmonary disease Thus, it can be argued that certain
changes
in
the
composition
of
oropharyngeal microbiota are significant
for the progression of COPD. The data includes variables with a Spearman correlation value of ≥0.05 at p<0.05
after introducing the Benjamin–Hochberg correction for multiple comparisons. The blue diamond on the chart shows the optimal sub-selection of variables,
which allows to classification of the patients with an accuracy of 76% To interpret the obtained results, we
assumed that the composition of the СТМ ∫ 2018 — vol. 10, No.2 105 Microbiological Oropharyngeal Patterns in Patients with Different Phenotypes of COPD сlinical medicine (78.6%) with the phenotype of “emphysema”, in
15 patients (75%) with the “mixed” phenotype, and
only 6 patients (20%) with the phenotype of “chronic
bronchitis”. It is known [14–17] that the presence of
the Porphyromonas and Prevotellgenera pathogenic
bacteria detected in patients with the “mixed” phenotype
is clinically significant because this is associated with
purulent inflammatory processes. Likewise, the impact
of the respiratory tract opportunistic microflora on the
pulmonary tissue is enhanced by the gastrointestinal
microbiota that enters the airways. The low diversity of
bacterial species in this COPD phenotype decreases
their resistance to pathogens [17] and can severely
complicate the course of COPD in patients with the
“mixed” phenotype. (78.6%) with the phenotype of “emphysema”, in
15 patients (75%) with the “mixed” phenotype, and
only 6 patients (20%) with the phenotype of “chronic
bronchitis”. It is known [14–17] that the presence of
the Porphyromonas and Prevotellgenera pathogenic
bacteria detected in patients with the “mixed” phenotype
is clinically significant because this is associated with
purulent inflammatory processes. Likewise, the impact
of the respiratory tract opportunistic microflora on the
pulmonary tissue is enhanced by the gastrointestinal
microbiota that enters the airways. The low diversity of
bacterial species in this COPD phenotype decreases
their resistance to pathogens [17] and can severely
complicate the course of COPD in patients with the
“mixed” phenotype. Conclusion Each phenotype of chronic obstructive pulmonary
disease with its specific clinical and functional features
is characterized by a specific pattern of oropharyngeal
microbiota, which determines the severity and prognosis
of the disease. The bacterial composition of these
patterns depends on the source of micro-aspiration
(upper gastrointestinal tract or nasopharynx). In addition,
the composition of the oropharyngeal microbiota is
determined by the severity of the disease and by its
phenotype. This phenotype determines the course of the
disease in a patient with chronic obstructive pulmonary
disease. 9. Molyneaux P.L., Mallia P., Cox M.J., Footitt J.,
Willis-Owen S.A., Homola D., Trujillo-Torralbo M.B., Elkin S.,
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rhinovirus exacerbation of chronic obstructive pulmonary
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chronic obstructive pulmonary disease. Thorax 2010; 65(1):
85–90, https://doi.org/10.1136/thx.2008.112888. Financial support. The study was supported by a
grant from the Federal target program “Research and
development in priority areas for the development of
the scientific and technological complex in Russia for
2014–2020” (GC No.14.604.21.0075, unique identifier
RFMEFI60414X0075).li 12. Paulson J.N., Olson N.D., Wagner J., Talukder H.,
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throughput
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Gevers D., Raes J., Huttenhower C. Microbial co-occurrence
relationships in the human microbiome. PLoS Comput Biol 2012;
8(7): e1002606, https://doi.org/10.1371/journal.pcbi.1002606. Conflict of interest. The authors confirm the absence
of financial and other conflicting interests that could
influence their work. 14. Pragman A.A., Kim H.B., Reilly C.S., Wendt C., 106 СТМ ∫ 2018 — vol. 10, No.2 M.A. Karnaushkina, S.V. Fedosenko, A.E. Sazonov, V.A. Petrov, D.Yu. Ovsyannikov, L.M. Ogorodova сlinical medicine Isaacson R.E. The lung microbiome in moderate and severe
chronic obstructive pulmonary disease. PloS One 2012; 7(10):
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rccm.201111-2075oc. СТМ ∫ 2018 — vol. 10, No.2 107 18. Ghannoum M.A., Jurevic R.J., Mukherjee P.K., Cui F., Conclusion 19. Hsiao W.W., Li K.L., Liu Z., Jones C., Fraser-
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20. Pei Z., Yang L., Peek R.M., Jr Levine S.M., Pride D.T.,
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https://doi.org/10.3748/wjg.v11.i46.7277. 16. Sin D., Sze M., Hogg J. Bacterial microbiome of lungs
in COPD. Int J Chron Obstruct Pulmon Dis 2014; 9: 229–238,
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Lynch S.V. Airway microbiome dynamics in exacerbations of
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52(8): 2813–2823, https://doi.org/10.1128/jcm.00035-14. 18. Ghannoum M.A., Jurevic R.J., Mukherjee P.K., Cui F., СТМ ∫ 2018 — vol. 10, No.2 107
Microbiological Oropharyngeal Patterns in Patients with Different Phenotypes of COPD СТМ ∫ 2018 — vol. 10, No.2 107 Microbiological Oropharyngeal Patterns in Patients with Different Phenotypes of COPD
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English
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How Quickly Do Proteins Fold and Unfold, and What Structural Parameters Correlate with These Values?
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Biomolecules
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cc-by
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Received: 27 December 2019; Accepted: 26 January 2020; Published: 29 January 2020 Abstract: The correlations between the logarithm of the unfolding rate of 108 proteins and their
structural parameters were calculated. We showed that there is a good correlation between the
logarithm of folding and unfolding rates (0.79) and protein stability and unfolding rate (0.79). Thus,
the faster the protein folds, the faster it unfolds. Folding and unfolding rates are higher for the
proteins with two-state kinetics, in comparison with the proteins with multi-state kinetics. At the same
time, two-state bacterial proteins folds and unfolds two orders of magnitude faster than two-state
eukaryotic proteins, and multi-state bacterial proteins folds and unfolds slower than multi-state
eukaryotic proteins. Despite the fact that the folding rates of thermophilic and mesophilic proteins are
close, the unfolding rates of thermophilic proteins is about two orders of magnitude lower than for
mesophilic proteins. The correlation between unfolding rate and stability of thermophilic proteins is
high (0.90). We also found that the unfolding rate correlates with such structural parameters as: size
of the protein, radius of the cross-section, logarithm of absolute contact order, and radius of gyration. This information will be useful for engineering and designing new proteins with desired properties. Keywords:
protein unfolding;
unfolding rates;
bacterial proteins;
eukaryotic proteins;
thermophile; mesophile Anna V. Glyakina 1,2 and Oxana V. Galzitskaya 1,3,* 1
Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region 142290, Russia;
glyakina@rambler.ru
2
Institute of Mathematical Problems of Biology, Russian Academy of Sciences, Keldysh Institute of Applied
Mathematics, Russian Academy of Sciences, Pushchino, Moscow Region 142290, Russia
3
Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences,
Pushchino, Moscow Region 142290, Russia
*
Correspondence: ogalzit@vega.protres.ru; Tel.: +7-903-675-0156 Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region 142290, Russia; 1
Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region 142290, Russia
glyakina@rambler.ru 1
Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region 142290, Russia;
glyakina@rambler.ru
2
Institute of Mathematical Problems of Biology, Russian Academy of Sciences, Keldysh Institute of Applied
Mathematics, Russian Academy of Sciences, Pushchino, Moscow Region 142290, Russia
3
Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences,
Pushchino, Moscow Region 142290, Russia
*
Correspondence: ogalzit@vega.protres.ru; Tel.: +7-903-675-0156 *
Correspondence: ogalzit@vega.protres.ru; Tel.: +7-903-675-0156 Received: 27 December 2019; Accepted: 26 January 2020; Published: 29 January 2020 Biomolecules 2020, 10, 197; doi:10.3390/biom10020197 How Quickly Do Proteins Fold and Unfold, and What
Structural Parameters Correlate with These Values? Anna V. Glyakina 1,2 and Oxana V. Galzitskaya 1,3,* biomolecules biomolecules biomolecules biomolecules 1. Introduction The problem of predicting folding rates (kf) for proteins with two-state and multi-state kinetics is
still important and extensively studied [1–20]. Many articles are devoted to the study of protein folding
rates and their correlation with various structural parameters [2,6–8,16]. In 1998, a relative contact
order (rCO) parameter was suggested, which is the average distance along the sequence between all
pairs of contacting residues, normalized to the size of the protein (number of amino acid residues,
further protein length). This parameter reflects the topological complexity of the protein chain. It was
shown that the rCO correlates well (correlation coefficient is 0.81) with the logarithm of the folding
rate for 12 two-state proteins [2]. Subsequent studies have shown that there is no correlation between
rCO and logarithm of the folding rate of proteins [6,7,16]. It turned out that only absolute contact
order (AbsCO, contact order multiplied by protein length) correlated with the logarithm of the folding
rate (the correlation coefficient is −0.77) [16]. It was found that the structural parameters, depending
on the protein length (L), correlated well with the logarithm of the folding rate [16]. In the set of
papers [13,15,16,21,22], the authors considered the different structural parameters of protein globule
compactness: radius of gyration (Rg); normalized radius of gyration (Rg/Rg*, where Rg* is the radius Biomolecules 2020, 10, 197; doi:10.3390/biom10020197 www.mdpi.com/journal/biomolecules 2 of 14 Biomolecules 2020, 10, 197 of gyration of the ball with the uniform density and the same volume); compactness as the relationship
between the solvent accessible surface area to the solvent accessible surface of sphere with the same
volume (Sasa/S*); and the relationship between the solvent accessible volume to the solvent accessible
surface (Vasa/Sasa). The solvent accessible volume (Vasa) is the volume surrounded by the solvent
accessible surface (Sasa). The radius of the cross-section (Vasa/Sasa) is proportional but not equal to
the radius of the smallest cross-section of the protein globule [16]. It was demonstrated that Vasa/Sasa
correlates good with the logarithm of the folding rate (the correlation coefficient is 0.74) [16]. We have
shown that the less compact the proteins, the faster they fold. Therefore, α-structural proteins are less
compact and the most rapidly folding proteins, while α/β-structural proteins are the most compact
and the most slowly folding proteins [15]. We have previously shown that bacterial proteins with two-state kinetics (further, bacterial
two-state proteins) fold and unfold faster than two-state eukaryotic proteins [23]. 1. Introduction It turned out that the
multi-state eukaryotic proteins fold and unfold faster than bacterial ones. It has been shown that there is a “golden triangle” that limits the possible range of the folding
rates for single-domain globular proteins of different sizes and stability [24]. This triangle was based
only on the biological and physical limitations. In addition, knowledge of the protein unfolding rates is also necessary to estimate their stability. Much less work has been devoted to the study of the relationship between the unfolding rates and
structural parameters. It has been shown that the logarithm of the unfolding and folding rates correlate
strongly with the AbsCO and the long-range order (parameter defined by contacts between two
residues that are close in space and far in the sequence) [3], with correlation coefficient values of
0.75 or higher. The logarithm of the unfolding rate correlated better with thermodynamic stability,
in comparison with the folding rate [19]. Early, it has been shown that structural parameters L, ln(AbsCO), Vasa/Sasa, and Rg were in a good
correlation with the logarithm of the protein folding rate [16]. The aim of the study was broader than the search for connections between protein unfolding rates
and various structural parameters, such as L, ln(AbsCO), Vasa/Sasa, and Rg. We were trying to find
out which parameters are most important for the prediction of the unfolding rates for proteins from
different organisms. 3.1. Unfolding Rates of 108 Proteins
3.1. Unfolding Rates of 108 Proteins (B) The average values of the logarithm of the unfolding and folding rates
for 108 proteins. Structural parameters that better correlate with the logarithm of the unfolding rates
are outlined in a rectangle. Figure 1. (A) Logarithm of the unfolding and folding rates for the three domains of chicken brain
alpha-spectrin proteins. (B) The average values of the logarithm of the unfolding and folding rates for
108 proteins. Structural parameters that better correlate with the logarithm of the unfolding rates are
outlined in a rectangle. The correlation between the logarithm of the unfolding and folding rates is 0.79 for all proteins. Moreover, this correlation is better for two-state (0.78) than for multi-state proteins (0.73). The
separation of 108 proteins by structural classes (α, β, α/β, and α + β) revealed that correlation
between the logarithm of the folding and unfolding rates is better for proteins from α and β (0.78
and 0.75) classes, in comparison with the proteins from α/β and α + β classes (0.59 and 0.60). Moreover, two-state proteins make the largest contribution to this correlation (see Table 1). The correlation between the logarithm of the unfolding and folding rates is 0.79 for all proteins. Moreover, this correlation is better for two-state (0.78) than for multi-state proteins (0.73). The separation
of 108 proteins by structural classes (α, β, α/β and α + β) revealed that correlation between the logarithm
of the folding and unfolding rates is better for proteins from α and β (0.78 and 0.75) classes, in comparison
with the proteins from α/β and α + β classes (0.59 and 0.60). Moreover, two-state proteins make the
largest contribution to this correlation (see Table 1). ,
p
g
(
)
The proteins from α class folds and unfolds faster, while proteins from α/β class folds and
unfolds slowly, in comparison with proteins from other structural classes (average logarithm of
folding rates: 8.49 ± 0.64 for α, 3.42 ± 0.63 for β, −0.02 ± 0.85 for α/β, and 4.71 ± 0.53 for α+β; average
logarithm of unfolding rates: 2.03 ± 1.03 for α, −4.51 ± 1.12 for β, −8.34 ± 1.64 for α/β, and −4.76 ± 0.97
for α + β; see also Supplementary Table S2). 3.1. Unfolding Rates of 108 Proteins
3.1. Unfolding Rates of 108 Proteins The goal of this work was to find the relationship between protein unfolding rates and protein
structural parameters. For three spectrin domains: R15, R16, and R17, we observed that the faster
the protein folds, the faster it unfolds, and vice versa. Domain R15 folds and unfolds faster than
its homologues, R16 and R17 (see Figure 1A). In the case of these spectrin repeats, the folding and
unfolding rates may be associated with the mechanical stability of the proteins. Previously, it has been
shown that domain R15 is less mechanically stable than domains R16 and R17 [26]. The discovered
correlation between the unfolding and folding rates suggests that the statement that the faster the
protein folds, the faster it unfolds, and vice versa, is also true for a dataset consisting of 108 proteins
(Figure 1B). The goal of this work was to find the relationship between protein unfolding rates and protein
structural parameters. For three spectrin domains: R15, R16, and R17, we observed that the faster the
protein folds, the faster it unfolds, and vice versa. Domain R15 folds and unfolds faster than its
homologues, R16 and R17 (see Figure 1A). In the case of these spectrin repeats, the folding and
unfolding rates may be associated with the mechanical stability of the proteins. Previously, it has
been shown that domain R15 is less mechanically stable than domains R16 and R17 [26]. The
discovered correlation between the unfolding and folding rates suggests that the statement that the
faster the protein folds, the faster it unfolds, and vice versa, is also true for a dataset consisting of 108
proteins (Figure 1B). Figure 1. (A) Logarithm of the unfolding and folding rates for the three domains of chicken brain
alpha-spectrin proteins. (B) The average values of the logarithm of the unfolding and folding rates
for 108 proteins. Structural parameters that better correlate with the logarithm of the unfolding rates
are outlined in a rectangle. Figure 1. (A) Logarithm of the unfolding and folding rates for the three domains of chicken brain
alpha-spectrin proteins. (B) The average values of the logarithm of the unfolding and folding rates for
108 proteins. Structural parameters that better correlate with the logarithm of the unfolding rates are
outlined in a rectangle. Figure 1. (A) Logarithm of the unfolding and folding rates for the three domains of chicken brain
alpha-spectrin proteins. 2. Materials and Methods Parameter Rg was calculated as Rg =
q P mi(ri−RC)2
M
, where mi is the mass of the i-th atom, ri is its
Cartesian coordinates, M is the mass of the protein, and RC is the coordinate vector of the mass center
of the protein, calculated as follows: P mi(ri −RC) = 0. Biomolecules 2020, 9, x
3 of 14 Errors for data from Table 1, Table 3, Table 5, Figure 5 and Figure 9 were calculated as σ =
r Pn
i=1(xi−x)
n(n−1)
, where x is the average value of a parameter and n is a number of proteins. Errors for data from Table 1, Table 3, Table 5, Figure 5 and Figure 9 were calculated as 𝜎=
ට
∑
(௫ି௫̅)
సభ
(ିଵ) , where 𝑥̅ is the average value of a parameter and n is a number of proteins. Errors for data from Table 1, Table 3, Table 5, Figure 5 and Figure 9 were calculated as σ =
r Pn
i=1(xi−x)
n(n−1)
, where x is the average value of a parameter and n is a number of proteins. Errors for data from Table 1, Table 3, Table 5, Figure 5 and Figure 9 were calculated as 𝜎=
ට
∑
(௫ି௫̅)
సభ
(ିଵ) , where 𝑥̅ is the average value of a parameter and n is a number of proteins. r Pn
i=1(xi−x)
n(n−1)
, where x is the average value of a parameter and n is a number of proteins. ට
∑
(௫ି௫̅)
సభ
(ିଵ) , where 𝑥̅ is the average value of a parameter and n is a number of proteins. r P
i=1(xi−x)
n(n−1)
, where x is the average value of a parameter and n is a number of proteins. ට
∑
(௫௫)
సభ
(ିଵ) , where 𝑥̅ is the average value of a parameter and n is a number of proteins. 2. Materials and Methods In our study, three databases of proteins were examined. The first database consisted of 108
proteins. Among them, 38 proteins demonstrated multi-state kinetics, and 70 proteins showed
two-state kinetics. The second database consisted of 42 bacterial (29 two-state and 13 multi-state) and
53 eukaryotic (32 two-state and 21 multi-state) proteins. The third database consisted of 42 bacterial
proteins. Among them, there were 10 thermophilic (8 two-state and 2 multi-state) and 32 mesophilic
(21 two-state and 11 multi-state) proteins. Proteins that have experimentally measured folding and unfolding rates were taken into
consideration. The database of such proteins has begun to be collected since 2003. At that time,
there were only 57 proteins [7]. In 2009, there were already 84 proteins [16]. Now, this database consists
of 108 proteins [19,24]. Data on folding and unfolding rates and structural parameters of the proteins (L—length of the
protein, Vasa/Sasa, ln(AbsCO) and Rg) are in Supplementary Table S1. Parameters Vasa and Sasa were calculated using YASARA program [25]. 1
N Parameters Vasa and Sasa were calculated using YASARA program [25]. 1
N Parameter ln(AbsCO) was calculated as ln(AbsCO) = ln( 1
N
PN
k=1 ∆Lij), where N is the number of
contacts (within 6 Å) between nonhydrogen atoms in the protein and ∆Lij is the number of residues
separating the interacting pair of nonhydrogen atoms (adjacent residues are assumed to be separated
by one residue). Bi
l
l
2020 10 197
3 f 14 3 of 14 Biomolecules 2020, 10, 197 Parameter Rg was calculated as Rg =
q P mi(ri−RC)2
M
, where mi is the mass of the i-th atom, ri is its
Cartesian coordinates, M is the mass of the protein, and RC is the coordinate vector of the mass center
of the protein, calculated as follows: P mi(ri −RC) = 0. Errors for data from Table 1, Table 3, Table 5, Figure 5 and Figure 9 were calculated as σ =
r Pn
i=1(xi−x)
n(n−1)
, where x is the average value of a parameter and n is a number of proteins. Biomolecules 2020, 9, x
3 of 14
Errors for data from Table 1, Table 3, Table 5, Figure 5 and Figure 9 were calculated as 𝜎=
ට
∑
(௫ି௫̅)
సభ
(ିଵ) , where 𝑥̅ is the average value of a parameter and n is a number of proteins. 3.1. Unfolding Rates of 108 Proteins
3.1. Unfolding Rates of 108 Proteins The proteins from α class folds and unfolds faster, while proteins from α/β class folds and unfolds
slowly, in comparison with proteins from other structural classes (average logarithm of folding rates:
8.49 ± 0.64 for α, 3.42 ± 0.63 for β, −0.02 ± 0.85 for α/β, and 4.71 ± 0.53 for α + β; average logarithm of
unfolding rates: 2.03 ± 1.03 for α, −4.51 ± 1.12 for β, −8.34 ± 1.64 for α/β, and −4.76 ± 0.97 for α + β;
see also Supplementary Table S2). β
pp
y
)
Table 1. Correlations between logarithm of unfolding (ln(ku) and folding (ln(kf) rates for four
structural classes of proteins α, β, α/β and α + β. It was previously shown that L, ln(AbsCO), Vasa/Sasa, and Rg correlate well with the logarithm of
the protein folding rate [16]. Thus, it can be assumed that if these parameters correlate well with the
logarithm of the folding rate, then they will also correlate well with the logarithm of the unfolding 4 of 14 Biomolecules 2020, 10, 197 rate. In this case, four parameters were examined: L is a number of amino acid residues in protein,
ln(AbsCO) is the logarithm of the absolute contact order, Vasa/Sasa is a radius of cross-section, and Rg is
a radius of gyration. rate. In this case, four parameters were examined: L is a number of amino acid residues in protein,
ln(AbsCO) is the logarithm of the absolute contact order, Vasa/Sasa is a radius of cross-section, and Rg is
a radius of gyration. Table 1. Correlations between logarithm of unfolding (ln(ku) and folding (ln(kf) rates for four structura
classes of proteins α, β, α/β and α + β. Correlations with ln(ku)
All (Two + Multi)
Two-State
Multi-State
All
0.79* (108) **
0.78 (70) **
0.73 (38) **
α
0.78 (31)
0.82 (23)
0.79 (8)
β
0.75 (37)
0.74 (25)
0.61 (12)
α/β
0.59 (12)
-
0.55 (11)
α + β
0.60 (28)
0.63 (21)
0.13 (7)
* Correlations above 0.75 are shown in bold. ** The number of proteins is indicated in parentheses. Table 1. Correlations between logarithm of unfolding (ln(ku) and folding (ln(kf) rates for four structural
classes of proteins α, β, α/β and α + β. The values of structural parameters considered in this paper (L, ln(AbsCO), Vasa/Sasa and Rg)
are lower for two-state proteins than for multi-state proteins: 78 ± 5 vs. 3.1. Unfolding Rates of 108 Proteins
3.1. Unfolding Rates of 108 Proteins 130 ± 8 for L, 3.14 ± 0.05 vs. 3.59 ± 0.06 for Vasa/Sasa, 6.91 ± 0.06 vs. 7.22 ± 0.06 for ln(AbsCO), and 12.1 ± 0.3 vs. 14.2 ± 0.3 for
Rg (see Table 2). The logarithms of the folding and unfolding rates are higher for two-state proteins,
in comparison with multi-state proteins: 6.08 ± 0.50 vs. 2.51 ± 0.59 for the folding rate and −1.51 ± 0.79
vs. −6.09 ± 1.03 for the unfolding rate, respectively (see Figure 1B). Table 2. Average values of structural parameters for 108 proteins. Vasa/Sasa = radius of cross-section. Rg = radius of gyration. ln(AbsCO) = logarithm of the absolute contact order. L = length of the protein. Table 2. Average values of structural parameters for 108 proteins. Vasa/Sasa = radius of cross-section. Rg = radius of gyration. ln(AbsCO) = logarithm of the absolute contact order. L = length of the protein. Average Value
All (Two + Multi)
Two-State
Multi-State
Number of proteins
108
70
38
<L>
96 ± 5
78 ± 5
130 ± 8
<Vasa/Sasa>
3.30 ± 0.04
3.14 ± 0.05
3.59 ± 0.06
<ln(AbsCO)>
7.02 ± 0.05
6.91 ± 0.06
7.22 ± 0.06
<Rg>
12.8 ± 0.2
12.1 ± 0.3
14.2 ± 0.3 For 108 proteins, the correlations between the logarithm of the unfolding rate (ln(ku)) and structural
parameters such as L, Vasa/Sasa, ln(AbsCO), and Rg were calculated (Table 3 and Figure 2). Vasa/Sasa
and ln(AbsCO) are better correlated with the logarithm of the unfolding rate of two-state proteins. For two-state proteins, these correlations are −0.79 and −0.87, in comparison with −0.63 and −0.69 for
multi-state proteins. The correlation between Rg and the logarithm of the unfolding rate is almost
the same for two-state and multi-state proteins (−0.61 vs. −0.60, respectively). Moreover, L is better
correlated with the logarithm of the unfolding rate of multi-state proteins. Good correlation (0.79)
between the protein stability (−(lnkf −lnku)) and the logarithm of the unfolding rate has been observed. Table 3. Correlations logarithm of the unfolding rate (ln(ku)) with protein stability (−(lnkf −lnku)) and
structural parameters (L, Vasa/Sasa, ln(AbsCO) and Rg) for 108 proteins. Table 3. Correlations logarithm of the unfolding rate (ln(ku)) with protein stability (−(lnkf −lnku)) and
structural parameters (L, Vasa/Sasa, ln(AbsCO) and Rg) for 108 proteins. * Correlations above 0.75 are shown in bold. 3.1. Unfolding Rates of 108 Proteins
3.1. Unfolding Rates of 108 Proteins The exception is only for correlation between ln(ku) and L for proteins from β
class This correlation is higher for multi-state proteins (−0 86) in comparison with two-state
After the separation of 108 proteins by structural classes (α, β, α/β and α + β), we observed that
correlations between the logarithm of the unfolding rate (ln(ku)) and L, Vasa/Sasa, ln(AbsCO), and Rg
are better for proteins from α and β classes (see Table 4). These correlations are the highest for proteins
from β class (higher than 0.8). The largest contribution to these correlations made two-state proteins
(see Table 4). The exception is only for correlation between ln(ku) and L for proteins from β class. This correlation is higher for multi-state proteins (−0.86), in comparison with two-state proteins (−0.84). oteins (−0.84). T bl
4 C
l
i
b
h l
i h
f h
f ldi
(l (k )
d
l
Table 4. Correlations between the logarithm of the unfolding rate (ln(ku) and structural parameters (L,
Vasa/Sasa, ln(AbsCO) and Rg) for four structural classes of proteins (α, β, α/β and α + β). teins (−0.84). T bl
4 C
l ti
b t
th l
ith
f th
f ldi
t (l (k )
d t
t
l
t
Table 4. Correlations between the logarithm of the unfolding rate (ln(ku) and structural parameters (L,
Vasa/Sasa, ln(AbsCO) and Rg) for four structural classes of proteins (α, β, α/β and α + β). Table 4. Correlations between the logarithm of the unfolding rate (ln(ku) and structural parameters
(L, Vasa/Sasa, ln(AbsCO), and Rg) for four structural classes of proteins (α, β, α/β, and α + β). elations with
ln(ku)
All (two + multi)
Two-state
Multi-state
α
β
α/β
α+β
α
β
α/β
α+β
α
β
α/β
α
ber of proteins
31
37
12
28
23
25
1
21
8
12
11
L
−0.71
−0.84
−0.50
−0.60
−0.72
−0.84
−
−0.77
−0.61
−0.86
−0.53
−0
Vasa/Sasa
−0.78
−0.82
−0.11
−0.78
−0.84
−0.83
−
−0.83
−0.60
−0.66
−0.28
−0
n(AbsCO)
−0.80
−0.89
−0.30
−0.73
−0.85
−0.90
−
−0.83
−0.48
−0.77
−0.29
−0
Rg
−0.73
−0.83
−0.69
−0.50
−0.79
−0.84
−
−0.57
−0.41
−0.74
−0.69
−0
* Correlations above 0.75 are shown in bold. 3.1. Unfolding Rates of 108 Proteins
3.1. Unfolding Rates of 108 Proteins Correlations with ln(ku)
All (Two + Multi)
Two-State
Multi-State
Number of proteins
108
70
38
Stability (−(lnkf −lnku))
0.79
0.79
0.83
L
−0.71
−0.65
−0.71
Vasa/Sasa
−0.77
−0.79
−0.63
ln(AbsCO)
−0.84
−0.87
−0.69
Rg
−0.65
−0.61
−0.60
* Correlations above 0.75 are shown in bold. 5 of 14
5
f 14 Biomolecules 2020, 10, 197 Figure 2. Correlations of the logarithm of the unfolding rates of 108 proteins with their structural
parameters: L—length of the protein, Vasa/Sasa—radius of the cross-section, ln(AbsCO)— logarithm of
the absolute contact order, and Rg (radius of gyration). There is a line approximation of points and its
equation: orange line corresponds to two-state proteins and purple line to multi-state proteins. R2 is a
linear approximation reliability. Figure 2. Correlations of the logarithm of the unfolding rates of 108 proteins with their structural
parameters: L—length of the protein, Vasa/Sasa—radius of the cross-section, ln(AbsCO)— logarithm of
the absolute contact order, and Rg (radius of gyration). There is a line approximation of points and its
equation: orange line corresponds to two-state proteins and purple line to multi-state proteins. R2 is a
linear approximation reliability. Figure 2. Correlations of the logarithm of the unfolding rates of 108 proteins with their structural
parameters: L—length of the protein, Vasa/Sasa—radius of the cross-section, ln(AbsCO)— logarithm of
the absolute contact order, and Rg (radius of gyration). There is a line approximation of points and its
equation: orange line corresponds to two-state proteins and purple line to multi-state proteins. R2 is a
linear approximation reliability. Figure 2. Correlations of the logarithm of the unfolding rates of 108 proteins with their structural
parameters: L—length of the protein, Vasa/Sasa—radius of the cross-section, ln(AbsCO)— logarithm of
the absolute contact order, and Rg (radius of gyration). There is a line approximation of points and its
equation: orange line corresponds to two-state proteins and purple line to multi-state proteins. R2 is a
linear approximation reliability. After the separation of 108 proteins by structural classes (α, β, α/β, and α + β), we observed that
correlations between the logarithm of the unfolding rate (ln(ku)) and L, Vasa/Sasa, ln(AbsCO), and Rg
are better for proteins from α and β classes (see Table 4). These correlations are the highest for
proteins from β class (higher than 0.8). The largest contribution to these correlations made two-state
proteins (see Table 4). 3.1. Unfolding Rates of 108 Proteins
3.1. Unfolding Rates of 108 Proteins Correlations
with ln(ku)
All (Two + Multi)
Two-State
Multi-State
α
β
α/β
α + β
α
β
α/β
α + β
α
β
α/β
α + β
Number of
proteins
31
37
12
28
23
25
1
21
8
12
11
7
L
−0.71
−0.84
−0.50
−0.60
−0.72 −0.84
−
−0.77
−0.61 −0.86
−0.53
−0.20
Vasa/Sasa
−0.78
−0.82
−0.11
−0.78
−0.84 −0.83
−
−0.83
−0.60 −0.66
−0.28
−0.54
ln(AbsCO)
−0.80
−0.89
−0.30
−0.73
−0.85 −0.90
−
−0.83
−0.48 −0.77
−0.29
−0.34
Rg
−0.73
−0.83
−0.69
−0.50
−0.79 −0.84
−
−0.57
−0.41 −0.74
−0.69
−0.11
* Correlations above 0.75 are shown in bold. 69
0.50
0.79
0.84
orrelations above 0 75 are shown in bold
* Correlations above 0.75 are shown in bold. 3.2. Unfolding Rates of Bacterial and Eukaryotic Proteins
3.2. Unfolding Rates of Bacterial and Eukaryotic Proteins The average values of the logarithm of the unfolding and folding rates for bacterial and
eukaryotic two- and multi-state proteins. Structural parameters that better correlate with the logarithm
of the unfolding rate are outlined in a rectangle. Figure 3. The average values of the logarithm of the unfolding and folding rates for bacterial and
eukaryotic two- and multi-state proteins. Structural parameters that better correlate with the
logarithm of the unfolding rate are outlined in a rectangle. Figure 3. The average values of the logarithm of the unfolding and folding rates for bacterial and
eukaryotic two- and multi-state proteins. Structural parameters that better correlate with the logarithm
of the unfolding rate are outlined in a rectangle. Figure 3. The average values of the logarithm of the unfolding and folding rates for bacterial and
eukaryotic two- and multi-state proteins. Structural parameters that better correlate with the
logarithm of the unfolding rate are outlined in a rectangle. Figure 3. The average values of the logarithm of the unfolding and folding rates for bacterial and
eukaryotic two- and multi-state proteins. Structural parameters that better correlate with the logarithm
of the unfolding rate are outlined in a rectangle. Table 5. Average values of structural parameters for two-state and multi-state bacterial and
eukaryotic proteins. Table 5. Average values of structural parameters for two-state and multi-state bacterial and
eukaryotic proteins. Table 5. Average values of structural parameters for two-state and multi-state bacterial and
eukaryotic proteins. Table 5. Average values of structural parameters for two-state and multi-state bacterial and
eukaryotic proteins. 3.2. Unfolding Rates of Bacterial and Eukaryotic Proteins
3.2. Unfolding Rates of Bacterial and Eukaryotic Proteins f
g
f
y
To find the dependence of the unfolding rates on the origin of the proteins, the 42 bacterial and
53 eukaryotic proteins from our database were separately studied. Two-state bacterial proteins fold
and unfold faster than two-state eukaryotic proteins. For multi-state proteins, we observed that
To find the dependence of the unfolding rates on the origin of the proteins, the 42 bacterial and 53
eukaryotic proteins from our database were separately studied. Two-state bacterial proteins fold and
unfold faster than two-state eukaryotic proteins. For multi-state proteins, we observed that bacterial 6 of 14 Biomolecules 2020, 10, 197 proteins fold and unfold slower than eukaryotic proteins (see Figures 3 and 4). The same result was
observed when the dataset consisted of 35 bacterial and 38 eukaryotic proteins [23]. The correlation
between the logarithm of the unfolding and folding rates is 0.73 for bacterial and 0.75 for eukaryotic
proteins. Moreover, for bacterial proteins, this correlation is better for two-state (0.69) than for
multi-state proteins (0.45). For eukaryotic proteins, this correlation is better for multi-state (0.81) than
for two-state proteins (0.72). Values Vasa/Sasa, ln(AbsCO), and Rg are slightly higher for the bacterial
proteins, and this gap increases for multi-state proteins: 3.74 ± 0.07 vs. 3.47 ± 0.07 for Vasa/Sasa,
7.40 ± 0.07 vs. 7.14 ± 0.10 for ln(AbsCO), and 14.8 ± 0.5 vs. 13.9 ± 0.4 for Rg, respectively (Table 5). same result was observed when the dataset consisted of 35 bacterial and 38 eukaryotic proteins [23]. The correlation between the logarithm of the unfolding and folding rates is 0.73 for bacterial and 0.75
for eukaryotic proteins. Moreover, for bacterial proteins, this correlation is better for two-state (0.69)
than for multi-state proteins (0.45). For eukaryotic proteins, this correlation is better for multi-state
(0.81) than for two-state proteins (0.72). Values Vasa/Sasa, ln(AbsCO), and Rg are slightly higher for the
bacterial proteins, and this gap increases for multi-state proteins: 3.74 ± 0.07 vs. 3.47 ± 0.07 for
Vasa/Sasa, 7.40 ± 0.07 vs. 7.14 ± 0.10 for ln(AbsCO), and 14.8 ± 0.5 vs. 13.9 ± 0.4 for Rg, respectively
(Table 5). Figure 3. The average values of the logarithm of the unfolding and folding rates for bacterial and
eukaryotic two- and multi-state proteins. Structural parameters that better correlate with the
logarithm of the unfolding rate are outlined in a rectangle. Figure 3. 3.2. Unfolding Rates of Bacterial and Eukaryotic Proteins
3.2. Unfolding Rates of Bacterial and Eukaryotic Proteins Average Value
All (two + multi)
Two-state
Multi-state
Bacteria
Eukaryota
Bacteria
Eukaryota
Bacteria
Eukaryota
Number of proteins
42
53
29
32
13
21
<L>
107 ± 9
92 ± 5
87 ± 10
77 ± 4
152 ± 14
115 ± 10
<Vasa/Sasa>
3.37 ± 0.07
3.30 ± 0.04
3.21 ± 0.08
3.20 ± 0.05
3.74 ± 0.07
3.47 ± 0.07
<ln(AbsCO)>
7.10 ± 0.07
7.08 ± 0.06
6.97 ± 0.09
7.04 ± 0.07
7.40 ± 0.07
7.14 ± 0.10
<Rg>
13.1 ± 0.4
12.9 ± 0.3
12.3 ± 0.5
12.3 ± 0.4
14.8 ± 0.5
13.9 ± 0.4
Average Value
All (Two + Multi)
Two-State
Multi-State
Bacteria
Eukaryota
Bacteria
Eukaryota
Bacteria
Eukaryota
Number of proteins
42
53
29
32
13
21
<L>
107 ± 9
92 ± 5
87 ± 10
77 ± 4
152 ± 14
115 ± 10
<Vasa/Sasa>
3.37 ± 0.07
3.30 ± 0.04
3.21 ± 0.08
3.20 ± 0.05
3.74 ± 0.07
3.47 ± 0.07
<ln(AbsCO)>
7.10 ± 0.07
7.08 ± 0.06
6.97 ± 0.09
7.04 ± 0.07
7.40 ± 0.07
7.14 ± 0.10
<Rg>
13.1 ± 0.4
12.9 ± 0.3
12.3 ± 0.5
12.3 ± 0.4
14.8 ± 0.5
13.9 ± 0.4 Then, the correlations between the logarithm of the unfolding rate and structural parameters
for bacterial and eukaryotic proteins were investigated. The correlations between the logarithm of
the unfolding rate and L, Vasa/Sasa, and ln(AbsCO) are almost the same for all bacterial and eukaryotic
proteins: −0.67 vs. −0.68 for L, −0.72 vs. −0.69 for Vasa/Sasa, and −0.80 vs. −0.79 for ln(AbsCO),
respectively (Table 6 and Figure 4). The difference is observed only for Rg, which correlates better
with the logarithm of the unfolding rate of bacterial proteins (−0.71). If we consider these
correlations for two-state and multi-state bacterial and eukaryotic proteins separately, we get the
following picture. For two-state proteins, the correlation between the logarithm of the unfolding rate
and Vasa/Sasa is almost the same for bacterial and eukaryotic proteins (−0.75 vs. −0.77). Rg and
ln(AbsCO) better correlate with the logarithm of the unfolding rate of two-state bacterial proteins
than with eukaryotic proteins (−0.86 vs. −0.77 for ln(AbsCO) and −0.64 vs. −0.47 for Rg, respectively). L, on the contrary, correlates better with the logarithm of the unfolding rate of two-state eukaryotic
proteins (−0.61 vs. −0.75). 3.2. Unfolding Rates of Bacterial and Eukaryotic Proteins
3.2. Unfolding Rates of Bacterial and Eukaryotic Proteins For multi-state proteins, we observed the same picture as for two-state
proteins for correlations of L and Rg with the logarithm of the unfolding rate. Both Vasa/Sasa and
ln(AbsCO) correlate better with the logarithm of the unfolding rate of multi-state eukaryotic
proteins than with bacterial proteins: −0.11 vs. −0.69 for Vasa/Sasa and −0.22 vs. −0.81 for ln(AbsCO),
Then, the correlations between the logarithm of the unfolding rate and structural parameters for
bacterial and eukaryotic proteins were investigated. The correlations between the logarithm of the
unfolding rate and L, Vasa/Sasa, and ln(AbsCO) are almost the same for all bacterial and eukaryotic
proteins: −0.67 vs. −0.68 for L, −0.72 vs. −0.69 for Vasa/Sasa, and −0.80 vs. −0.79 for ln(AbsCO),
respectively (Table 6 and Figure 4). The difference is observed only for Rg, which correlates better with
the logarithm of the unfolding rate of bacterial proteins (−0.71). If we consider these correlations for
two-state and multi-state bacterial and eukaryotic proteins separately, we get the following picture. For two-state proteins, the correlation between the logarithm of the unfolding rate and Vasa/Sasa is
almost the same for bacterial and eukaryotic proteins (−0.75 vs. −0.77). Rg and ln(AbsCO) better
correlate with the logarithm of the unfolding rate of two-state bacterial proteins than with eukaryotic
proteins (−0.86 vs. −0.77 for ln(AbsCO) and −0.64 vs. −0.47 for Rg, respectively). L, on the contrary,
correlates better with the logarithm of the unfolding rate of two-state eukaryotic proteins (−0.61 vs. −0.75). For multi-state proteins, we observed the same picture as for two-state proteins for correlations
of L and Rg with the logarithm of the unfolding rate. Both Vasa/Sasa and ln(AbsCO) correlate better
with the logarithm of the unfolding rate of multi-state eukaryotic proteins than with bacterial proteins:
−0.11 vs. −0.69 for Vasa/Sasa and −0.22 vs. −0.81 for ln(AbsCO), respectively. Biomolecules 2020, 10, 197
Bi
l
l
2020 9 7 of 14
7
f 14 molecules 2020, 9, x
7 o
Figure 4. Correlations of the logarithm of the unfolding rate of the two- and multi-state bacterial and
eukaryotic proteins with structural parameters: L, Vasa/Sasa, ln(AbsCO), and Rg. There is a line
approximation of points and its equation: green line corresponds to bacterial proteins and pink line
to eukaryotic proteins. R2 is a linear approximation reliability. Figure 4. 3.3. Unfolding Rates of Proteins from Thermophilic and Mesophilic Organisms
3.3. Unfolding Rates of Proteins from Thermophilic and Mesophilic Organisms .3. Unfolding Rates of Proteins from Thermophilic and Mesophilic Organisms
.3. Unfolding Rates of Proteins from Thermophilic and Mesophilic Organisms Since a lot of attention was paid to the search for differences between thermophilic and
mesophilic proteins — in particular, folding rates — we also decided to conduct our analysis for
these proteins. All bacterial proteins were divided into thermophilic and mesophilic groups. Further
in the text, we call proteins from thermophilic organisms as thermophilic proteins and proteins from
mesophilic organisms as mesophilic proteins. The correlation between the logarithm of the
unfolding and folding rates is better for mesophilic (0.76), in comparison with thermophilic (0.73)
proteins. Moreover, for mesophilic proteins, this correlation is better for two-state (0.76) than for
multi-state proteins (0.12). For thermophilic proteins, it is hard to say something, because there are
only two proteins with multi-state kinetics. There is a correlation between stability and the logarithm
of the unfolding rate for thermophilic (0.90) and mesophilic (0.73) proteins. The logarithm of the
folding rate of thermophilic and mesophilic proteins are almost the same (4.75 ± 1.20 vs. 4.58 ± 0.79)
(Figure 6 and Table 7). Still, mesophilic proteins unfold faster than thermophilic proteins (−5.63 ±
2.31 vs. −3.27 ± 1.12). The same picture is observed for two-state thermophilic and mesophilic
proteins. Schematic “chevron” plots for thermophilic and mesophilic proteins are presented in
Since a lot of attention was paid to the search for differences between thermophilic and mesophilic
proteins — in particular, folding rates — we also decided to conduct our analysis for these proteins. All bacterial proteins were divided into thermophilic and mesophilic groups. Further in the text, we
call proteins from thermophilic organisms as thermophilic proteins and proteins from mesophilic
organisms as mesophilic proteins. The correlation between the logarithm of the unfolding and folding
rates is better for mesophilic (0.76), in comparison with thermophilic (0.73) proteins. Moreover, for
mesophilic proteins, this correlation is better for two-state (0.76) than for multi-state proteins (0.12). For thermophilic proteins, it is hard to say something, because there are only two proteins with
multi-state kinetics. There is a correlation between stability and the logarithm of the unfolding rate for
thermophilic (0.90) and mesophilic (0.73) proteins. The logarithm of the folding rate of thermophilic
and mesophilic proteins are almost the same (4.75 ± 1.20 vs. 4.58 ± 0.79) (Figure 6 and Table 7). 3.2. Unfolding Rates of Bacterial and Eukaryotic Proteins
3.2. Unfolding Rates of Bacterial and Eukaryotic Proteins Correlations of the logarithm of the unfolding rate of the two- and multi-state bacterial
and eukaryotic proteins with structural parameters: L, Vasa/Sasa, ln(AbsCO), and Rg. There is a line
approximation of points and its equation: green line corresponds to bacterial proteins and pink line to
eukaryotic proteins. R2 is a linear approximation reliability. Figure 4. Correlations of the logarithm of the unfolding rate of the two- and multi-state bacterial and
eukaryotic proteins with structural parameters: L, Vasa/Sasa, ln(AbsCO), and Rg. There is a line
approximation of points and its equation: green line corresponds to bacterial proteins and pink line
to eukaryotic proteins R2 is a linear approximation reliability
Figure 4. Correlations of the logarithm of the unfolding rate of the two- and multi-state bacterial
and eukaryotic proteins with structural parameters: L, Vasa/Sasa, ln(AbsCO), and Rg. There is a line
approximation of points and its equation: green line corresponds to bacterial proteins and pink line to
eukaryotic proteins. R2 is a linear approximation reliability. 8 of 14
8 of 14 Biomolecules 2020, 10, 197
Biomolecules 2020, 9, x Table 6. Correlations between the logarithm of the unfolding rate (ln(ku)) and structural parameters (L,
Vasa/Sasa, ln(AbsCO) and Rg) for two-state and multi-state bacterial and eukaryotic proteins. g
g
(
(
))
p
(L, Vasa/Sasa, ln(AbsCO), and Rg) for two-state and multi-state bacterial and eukaryotic proteins. Correlations with
All (two + multi)
Two−state
Multi−state Correlations with ln(ku)
All (Two + Multi)
Two-State
Multi-State
Bacteria
Eukaryota
Bacteria
Eukaryota
Bacteria
Eukaryota
Number of proteins
42
53
29
32
13
21
L
−0.67
−0.68
−0.61
−0.75
−0.43
−0.77
Vasa/Sasa
−0.72
−0.69
−0.75
−0.77
−0.11
−0.69
ln(AbsCO)
−0.80
−0.79
−0.86
−0.77
−0.22
−0.81
Rg
−0.71
−0.46
−0.64
−0.47
−0.58
−0.50
* Correlations above 0.75 are shown in bold. ln(ku)
Bacteria
Eukaryota
Bacteria
Eukaryota
Bacteria
Eukaryota
Number of proteins
42
53
29
32
13
21
L
−0.67
−0.68
−0.61
−0.75
−0.43
−0.77
Vasa/Sasa
−0.72
−0.69
−0.75
−0.77
−0.11
−0.69
ln(AbsCO)
−0.80
−0.79
−0.86
−0.77
−0.22
−0.81
Rg
−0.71
−0.46
−0.64
−0.47
−0.58
−0.50
* Correlations above 0.75 are shown in bold. * Correlations above 0.75 are shown in bold. orrelations above 0.75 are shown in bold. Amino acid composition of bacterial and eukaryotic proteins was analyzed (Figure 5). The bacterial
proteins with two-state kinetics are enriched in Ala, Gly, Lys, and Asn, compared with eukaryotic
proteins with two-state kinetics. 3.2. Unfolding Rates of Bacterial and Eukaryotic Proteins
3.2. Unfolding Rates of Bacterial and Eukaryotic Proteins The eukaryotic proteins with two-state kinetics contain more His, Leu,
Pro, Arg, Ser, and Trp, compared to the bacterial proteins with two-state kinetics (see Figure 5). Amino acid composition of bacterial and eukaryotic proteins was analyzed (Figure 5). The
bacterial proteins with two-state kinetics are enriched in Ala, Gly, Lys, and Asn, compared with
eukaryotic proteins with two-state kinetics. The eukaryotic proteins with two-state kinetics contain
more His, Leu, Pro, Arg, Ser, and Trp, compared to the bacterial proteins with two-state kinetics (see
Figure 5). Figure 5. Amino acid composition of the bacterial and eukaryotic proteins with two- and multi-state
kinetics. Figure 5. Amino acid composition of the bacterial and eukaryotic proteins with two- and
multi-state kinetics. Figure 5. Amino acid composition of the bacterial and eukaryotic proteins with two- and multi-state
kinetics. Figure 5. Amino acid composition of the bacterial and eukaryotic proteins with two- and
multi-state kinetics. 3.3. Unfolding Rates of Proteins from Thermophilic and Mesophilic Organisms
3.3. Unfolding Rates of Proteins from Thermophilic and Mesophilic Organisms Still,
mesophilic proteins unfold faster than thermophilic proteins (−5.63 ± 2.31 vs. −3.27 ± 1.12). The same
picture is observed for two-state thermophilic and mesophilic proteins. Schematic “chevron” plots for
thermophilic and mesophilic proteins are presented in Figure 7. Biomolecules 2020, 10, 197
Biomolecules 2020 9 x 9 of 14
9 of 14 9 of 14
9 of 14 Figure 6. The average values of the logarithm of the unfolding and folding rates for thermophilic and
mesophilic two- and multi-state proteins. Structural parameters that better correlate with the
logarithm of the unfolding rate are outlined in a rectangle. Figure 6. The average values of the logarithm of the unfolding and folding rates for thermophilic and
mesophilic two- and multi-state proteins. Structural parameters that better correlate with the logarithm
of the unfolding rate are outlined in a rectangle. Figure 6. The average values of the logarithm of the unfolding and folding rates for thermophilic and
mesophilic two- and multi-state proteins. Structural parameters that better correlate with the
logarithm of the unfolding rate are outlined in a rectangle. Fi
6 Th
l
f t Figure 6. The average values of the logarithm of the unfolding and folding rates for thermophilic and
mesophilic two- and multi-state proteins. Structural parameters that better correlate with the
logarithm of the unfolding rate are outlined in a rectangle. Figure 6. The average values of the logarithm of the unfolding and folding rates for thermophilic and
mesophilic two- and multi-state proteins. Structural parameters that better correlate with the logarithm
of the unfolding rate are outlined in a rectangle. Figure 6. The average values of the logarithm of the unfolding and folding rates for thermophilic and
mesophilic two- and multi-state proteins. Structural parameters that better correlate with the
logarithm of the unfolding rate are outlined in a rectangle. Figure 7. Schematic “chevron” plots of the constant of observed rate (kapp = kf + ku) versus
temperature for thermophilic and mesophilic proteins. Figure 7. Schematic “chevron” plots of the constant of observed rate (kapp = kf + ku) versus
temperature for thermophilic and mesophilic proteins. Figure 7. Schematic “chevron” plots of the constant of observed rate (kapp = kf + ku) versus temperature
for thermophilic and mesophilic proteins. T bl
7
A
l
f t
t
l
t
f
t
t t
d
lti t t
th
hili
d Figure 7. 3.3. Unfolding Rates of Proteins from Thermophilic and Mesophilic Organisms
3.3. Unfolding Rates of Proteins from Thermophilic and Mesophilic Organisms For two−state
thermophilic proteins, the correlation between all considered parameters (L, Vasa/Sasa, ln(AbsCO),
and R ) is better than for two state mesophilic proteins:
0 92 vs
0 60 for L
0 92 vs
0 78 for
<Rg>
13.3 ± 0.8
13.0 ± 0.5
12.5 ± 0.7
12.2 ± 0.6
16.2 ± 1.1
14.5 ± 0.5
Finally, the correlations of the logarithm of the unfolding rate and structural parameters for
thermophilic and mesophilic proteins were examined (Table 8 and Figure 8). L and Rg correlate
better with the logarithm of the unfolding rate of all thermophilic proteins (−0.83 vs. −0.64 for L and
−0.87 vs. −0.66 for Rg), and ln(AbsCO) correlates better with the logarithm of the unfolding rate of all
mesophilic proteins (−0.74 vs. −0.83). Vasa/Sasa correlates with the logarithm of the unfolding rate
practically the same for all thermophilic and mesophilic proteins (−0.77 vs. −0.75). For two−state
thermophilic proteins, the correlation between all considered parameters (L, Vasa/Sasa, ln(AbsCO),
and Rg) is better than for two−state mesophilic proteins: −0.92 vs. −0.60 for L, −0.92 vs. −0.78 for
Finally, the correlations of the logarithm of the unfolding rate and structural parameters for
thermophilic and mesophilic proteins were examined (Table 8 and Figure 8). L and Rg correlate better
with the logarithm of the unfolding rate of all thermophilic proteins (−0.83 vs. −0.64 for L and −0.87 vs. −0.66 for Rg), and ln(AbsCO) correlates better with the logarithm of the unfolding rate of all mesophilic
proteins (−0.74 vs. −0.83). Vasa/Sasa correlates with the logarithm of the unfolding rate practically
the same for all thermophilic and mesophilic proteins (−0.77 vs. −0.75). For two-state thermophilic
proteins, the correlation between all considered parameters (L, Vasa/Sasa, ln(AbsCO) and Rg) is better
than for two-state mesophilic proteins: −0.92 vs. −0.60 for L, −0.92 vs. −0.78 for Vasa/Sasa, −0.93 vs. −0.85 for ln(AbsCO), and −0.88 vs. −0.60 for Rg. <ln(AbsCO)>
7.13 ± 0.15
7.09 ± 0.08
7.03 ± 0.17
6.95 ± 0.11
7.54 ± 0.07
7.38 ± 0.09
<Rg>
13.3 ± 0.8
13.0 ± 0.5
12.5 ± 0.7
12.2 ± 0.6
16.2 ± 1.1
14.5 ± 0.5
Finally, the correlations of the logarithm of the unfolding rate and structural parameters for
thermophilic and mesophilic proteins were examined (Table 8 and Figure 8). L and Rg correlate
better with the logarithm of the unfolding rate of all thermophilic proteins (−0.83 vs. −0.64 for L and
−0.87 vs. 3.3. Unfolding Rates of Proteins from Thermophilic and Mesophilic Organisms
3.3. Unfolding Rates of Proteins from Thermophilic and Mesophilic Organisms Schematic “chevron” plots of the constant of observed rate (kapp = kf + ku) versus
t
t
f
th
hili
d
hili
t i
Figure 7. Schematic “chevron” plots of the constant of observed rate (kapp = kf + ku) versus
temperature for thermophilic and mesophilic proteins. Figure 7. Schematic “chevron” plots of the constant of observed rate (kapp = kf + ku) versus temperature
for thermophilic and mesophilic proteins. temperature for thermophilic and mesophilic proteins. Table 7. Average values of structural parameters for two-state and multi-state thermophilic and
Table 7. Average values of structural parameters for two-state and multi-state thermophilic and
mesophilic proteins
Table 7. Average values of structural parameters for two-state and multi-state thermophilic and
mesophilic proteins. temperature for thermophilic and mesophilic proteins. Table 7. Average values of structural parameters for two-state and multi-state thermophilic and
Table 7. Average values of structural parameters for two-state and multi-state thermophilic and
mesophilic proteins
Table 7. Average values of structural parameters for two-state and multi-state thermophilic and
mesophilic proteins. Table 7. Average values of structural parameters for two state and multi state thermophilic and
mesophilic proteins. Average
Value
All (two + multi)
Two-state
Multi-state
Thermophile
Mesophile
Thermophile
Mesophile
Thermophile
Mesophile
Number of
proteins
10
32
8
21
2
11
<L>
105 ± 18
108 ± 11
82 ± 11
89 ± 13
197 ± 22
144 ± 14
<Vasa/Sasa>
3.30 ± 0.14
3.39 ± 0.08
3.13 ± 0.10
3.23 ± 0.10
3.97 ± 0.01
3.70 ± 0.08
<ln(AbsCO)>
7.13 ± 0.15
7.09 ± 0.08
7.03 ± 0.17
6.95 ± 0.11
7.54 ± 0.07
7.38 ± 0.09
<Rg>
13.3 ± 0.8
13.0 ± 0.5
12.5 ± 0.7
12.2 ± 0.6
16.2 ± 1.1
14.5 ± 0.5
Finally, the correlations of the logarithm of the unfolding rate and structural parameters for
thermophilic and mesophilic proteins were examined (Table 8 and Figure 8). L and Rg correlate
better with the logarithm of the unfolding rate of all thermophilic proteins (−0.83 vs. −0.64 for L and
−0.87 vs. −0.66 for Rg), and ln(AbsCO) correlates better with the logarithm of the unfolding rate of all
mesophilic proteins (−0.74 vs. −0.83). Vasa/Sasa correlates with the logarithm of the unfolding rate
practically the same for all thermophilic and mesophilic proteins (−0.77 vs. −0.75). 3.3. Unfolding Rates of Proteins from Thermophilic and Mesophilic Organisms
3.3. Unfolding Rates of Proteins from Thermophilic and Mesophilic Organisms For two−state
thermophilic proteins, the correlation between all considered parameters (L, Vasa/Sasa, ln(AbsCO),
and Rg) is better than for two−state mesophilic proteins: −0 92 vs −0 60 for L −0 92 vs −0 78 for
mesophilic proteins. Average
Value
All (two + multi)
Two-state
Multi-state
Thermophile
Mesophile
Thermophile
Mesophile
Thermophile
Mesophile
Number of
proteins
10
32
8
21
2
11
<L>
105 ± 18
108 ± 11
82 ± 11
89 ± 13
197 ± 22
144 ± 14
<Vasa/Sasa>
3.30 ± 0.14
3.39 ± 0.08
3.13 ± 0.10
3.23 ± 0.10
3.97 ± 0.01
3.70 ± 0.08
<ln(AbsCO)>
7.13 ± 0.15
7.09 ± 0.08
7.03 ± 0.17
6.95 ± 0.11
7.54 ± 0.07
7.38 ± 0.09
<Rg>
13.3 ± 0.8
13.0 ± 0.5
12.5 ± 0.7
12.2 ± 0.6
16.2 ± 1.1
14.5 ± 0.5
Finally, the correlations of the logarithm of the unfolding rate and structural parameters for
thermophilic and mesophilic proteins were examined (Table 8 and Figure 8). L and Rg correlate
better with the logarithm of the unfolding rate of all thermophilic proteins (−0.83 vs. −0.64 for L and
−0.87 vs. −0.66 for Rg), and ln(AbsCO) correlates better with the logarithm of the unfolding rate of all
mesophilic proteins (−0.74 vs. −0.83). Vasa/Sasa correlates with the logarithm of the unfolding rate
practically the same for all thermophilic and mesophilic proteins (−0.77 vs. −0.75). For two−state
thermophilic proteins, the correlation between all considered parameters (L, Vasa/Sasa, ln(AbsCO),
and Rg) is better than for two−state mesophilic proteins: −0.92 vs. −0.60 for L, −0.92 vs. −0.78 for
p
p
Average Value
All (Two + Multi)
Two-State
Multi-State
Thermophile
Mesophile
Thermophile
Mesophile
Thermophile
Mesophile
Number of proteins
10
32
8
21
2
11
<L>
105 ± 18
108 ± 11
82 ± 11
89 ± 13
197 ± 22
144 ± 14
<Vasa/Sasa>
3.30 ± 0.14
3.39 ± 0.08
3.13 ± 0.10
3.23 ± 0.10
3.97 ± 0.01
3.70 ± 0.08
<ln(AbsCO)>
7.13 ± 0.15
7.09 ± 0.08
7.03 ± 0.17
6.95 ± 0.11
7.54 ± 0.07
7.38 ± 0.09
<Rg>
13.3 ± 0.8
13.0 ± 0.5
12.5 ± 0.7
12.2 ± 0.6
16.2 ± 1.1
14.5 ± 0.5
Finally, the correlations of the logarithm of the unfolding rate and structural parameters for
thermophilic and mesophilic proteins were examined (Table 8 and Figure 8). 3.3. Unfolding Rates of Proteins from Thermophilic and Mesophilic Organisms
3.3. Unfolding Rates of Proteins from Thermophilic and Mesophilic Organisms L and Rg correlate better
with the logarithm of the unfolding rate of all thermophilic proteins (−0.83 vs. −0.64 for L and −0.87 vs. −0.66 for Rg), and ln(AbsCO) correlates better with the logarithm of the unfolding rate of all mesophilic
proteins (−0.74 vs. −0.83). Vasa/Sasa correlates with the logarithm of the unfolding rate practically
the same for all thermophilic and mesophilic proteins (−0.77 vs. −0.75). For two-state thermophilic
proteins, the correlation between all considered parameters (L, Vasa/Sasa, ln(AbsCO) and Rg) is better
than for two-state mesophilic proteins: −0.92 vs. −0.60 for L, −0.92 vs. −0.78 for Vasa/Sasa, −0.93 vs. −0.85 for ln(AbsCO), and −0.88 vs. −0.60 for Rg. mesophilic proteins. 3.3. Unfolding Rates of Proteins from Thermophilic and Mesophilic Organisms
3.3. Unfolding Rates of Proteins from Thermophilic and Mesophilic Organisms Average
Value
All (two + multi)
Two-state
Multi-state
Thermophile
Mesophile
Thermophile
Mesophile
Thermophile
Mesophile
Number of
proteins
10
32
8
21
2
11
<L>
105 ± 18
108 ± 11
82 ± 11
89 ± 13
197 ± 22
144 ± 14
<Vasa/Sasa>
3.30 ± 0.14
3.39 ± 0.08
3.13 ± 0.10
3.23 ± 0.10
3.97 ± 0.01
3.70 ± 0.08
p
p
Average
Value
All (two + multi)
Two-state
Multi-state
Thermophile
Mesophile
Thermophile
Mesophile
Thermophile
Mesophile
Number of
proteins
10
32
8
21
2
11
<L>
105 ± 18
108 ± 11
82 ± 11
89 ± 13
197 ± 22
144 ± 14
<Vasa/Sasa>
3.30 ± 0.14
3.39 ± 0.08
3.13 ± 0.10
3.23 ± 0.10
3.97 ± 0.01
3.70 ± 0.08
<ln(AbsCO)>
7 13 ± 0 15
7 09 ± 0 08
7 03 ± 0 17
6 95 ± 0 11
7 54 ± 0 07
7 38 ± 0 09
Average Value
All (Two + Multi)
Two-State
Multi-State
Thermophile
Mesophile
Thermophile
Mesophile
Thermophile
Mesophile
Number of proteins
10
32
8
21
2
11
<L>
105 ± 18
108 ± 11
82 ± 11
89 ± 13
197 ± 22
144 ± 14
<Vasa/Sasa>
3.30 ± 0.14
3.39 ± 0.08
3.13 ± 0.10
3.23 ± 0.10
3.97 ± 0.01
3.70 ± 0.08
<ln(AbsCO)>
7.13 ± 0.15
7.09 ± 0.08
7.03 ± 0.17
6.95 ± 0.11
7.54 ± 0.07
7.38 ± 0.09
<Rg>
13.3 ± 0.8
13.0 ± 0.5
12.5 ± 0.7
12.2 ± 0.6
16.2 ± 1.1
14.5 ± 0.5 <ln(AbsCO)>
7.13 ± 0.15
7.09 ± 0.08
7.03 ± 0.17
6.95 ± 0.11
7.54 ± 0.07
7.38 ± 0.09
<Rg>
13.3 ± 0.8
13.0 ± 0.5
12.5 ± 0.7
12.2 ± 0.6
16.2 ± 1.1
14.5 ± 0.5
Finally, the correlations of the logarithm of the unfolding rate and structural parameters for
thermophilic and mesophilic proteins were examined (Table 8 and Figure 8). L and Rg correlate
better with the logarithm of the unfolding rate of all thermophilic proteins (−0.83 vs. −0.64 for L and
−0.87 vs. −0.66 for Rg), and ln(AbsCO) correlates better with the logarithm of the unfolding rate of all
mesophilic proteins (−0.74 vs. −0.83). Vasa/Sasa correlates with the logarithm of the unfolding rate
practically the same for all thermophilic and mesophilic proteins (−0.77 vs. −0.75). <ln(AbsCO)>
7.13 ± 0.15
7.09 ± 0.08
7.03 ± 0.17
6.95 ± 0.11
7.54 ± 0.07
7.38 ± 0.09
<Rg>
13.3 ± 0.8
13.0 ± 0.5
12.5 ± 0.7
12.2 ± 0.6
16.2 ± 1.1
14.5 ± 0.5
Finally, the correlations of the logarithm of the unfolding rate and structural parameters for
thermophilic and mesophilic proteins were examined (Table 8 and Figure 8). L and Rg correlate
better with the logarithm of the unfolding rate of all thermophilic proteins (−0.83 vs. −0.64 for L and
−0.87 vs. −0.66 for Rg), and ln(AbsCO) correlates better with the logarithm of the unfolding rate of all
mesophilic proteins (−0.74 vs. −0.83). Vasa/Sasa correlates with the logarithm of the unfolding rate
practically the same for all thermophilic and mesophilic proteins (−0.77 vs. −0.75). For two−state
thermophilic proteins, the correlation between all considered parameters (L, Vasa/Sasa, ln(AbsCO),
and R ) is better than for two state mesophilic proteins:
0 92 vs
0 60 for L
0 92 vs
0 78 for
(
)
<Rg>
13.3 ± 0.8
13.0 ± 0.5
12.5 ± 0.7
12.2 ± 0.6
16.2 ± 1.1
14.5 ± 0.5
Finally, the correlations of the logarithm of the unfolding rate and structural parameters for
thermophilic and mesophilic proteins were examined (Table 8 and Figure 8). L and Rg correlate
better with the logarithm of the unfolding rate of all thermophilic proteins (−0.83 vs. −0.64 for L and
−0.87 vs. −0.66 for Rg), and ln(AbsCO) correlates better with the logarithm of the unfolding rate of all
mesophilic proteins (−0.74 vs. −0.83). Vasa/Sasa correlates with the logarithm of the unfolding rate
practically the same for all thermophilic and mesophilic proteins (−0.77 vs. −0.75). For two−state
thermophilic proteins, the correlation between all considered parameters (L, Vasa/Sasa, ln(AbsCO),
and Rg) is better than for two−state mesophilic proteins: −0.92 vs. −0.60 for L, −0.92 vs. −0.78 for
Finally, the correlations of the logarithm of the unfolding rate and structural parameters for
thermophilic and mesophilic proteins were examined (Table 8 and Figure 8). L and Rg correlate better
with the logarithm of the unfolding rate of all thermophilic proteins (−0.83 vs. −0.64 for L and −0.87 vs.
−0.66 for Rg), and ln(AbsCO) correlates better with the logarithm of the unfolding rate of all mesophilic
proteins (−0.74 vs. −0.83). Vasa/Sasa correlates with the logarithm of the unfolding rate practically
the same for all thermophilic and mesophilic proteins (−0.77 vs. −0.75). For two-state thermophilic
proteins, the correlation between all considered parameters (L, Vasa/Sasa, ln(AbsCO) and Rg) is better
than for two-state mesophilic proteins: −0.92 vs. −0.60 for L, −0.92 vs. −0.78 for Vasa/Sasa, −0.93 vs.
−0.85 for ln(AbsCO), and −0.88 vs. −0.60 for Rg. 3.3. Unfolding Rates of Proteins from Thermophilic and Mesophilic Organisms
3.3. Unfolding Rates of Proteins from Thermophilic and Mesophilic Organisms −0.66 for Rg), and ln(AbsCO) correlates better with the logarithm of the unfolding rate of all
mesophilic proteins (−0.74 vs. −0.83). Vasa/Sasa correlates with the logarithm of the unfolding rate
practically the same for all thermophilic and mesophilic proteins (−0.77 vs. −0.75). For two−state
thermophilic proteins, the correlation between all considered parameters (L, Vasa/Sasa, ln(AbsCO),
a d R ) i
bette tha
fo t
o
tate
e o hili
otei
0 92
0 60 fo L
0 92
0 78 fo
<Rg>
13.3 ± 0.8
13.0 ± 0.5
12.5 ± 0.7
12.2 ± 0.6
16.2 ± 1.1
14.5 ± 0.5
Finally, the correlations of the logarithm of the unfolding rate and structural parameters for
thermophilic and mesophilic proteins were examined (Table 8 and Figure 8). L and Rg correlate
better with the logarithm of the unfolding rate of all thermophilic proteins (−0.83 vs. −0.64 for L and
−0.87 vs. −0.66 for Rg), and ln(AbsCO) correlates better with the logarithm of the unfolding rate of all
mesophilic proteins (−0.74 vs. −0.83). Vasa/Sasa correlates with the logarithm of the unfolding rate
practically the same for all thermophilic and mesophilic proteins (−0.77 vs. −0.75). For two−state
thermophilic proteins, the correlation between all considered parameters (L, Vasa/Sasa, ln(AbsCO),
and Rg) is better than for two−state mesophilic proteins: −0.92 vs. −0.60 for L, −0.92 vs. −0.78 for
Finally, the correlations of the logarithm of the unfolding rate and structural parameters for
thermophilic and mesophilic proteins were examined (Table 8 and Figure 8). L and Rg correlate better
with the logarithm of the unfolding rate of all thermophilic proteins (−0.83 vs. −0.64 for L and −0.87 vs. −0.66 for Rg), and ln(AbsCO) correlates better with the logarithm of the unfolding rate of all mesophilic
proteins (−0.74 vs. −0.83). Vasa/Sasa correlates with the logarithm of the unfolding rate practically
the same for all thermophilic and mesophilic proteins (−0.77 vs. −0.75). For two-state thermophilic
proteins, the correlation between all considered parameters (L, Vasa/Sasa, ln(AbsCO) and Rg) is better
than for two-state mesophilic proteins: −0.92 vs. −0.60 for L, −0.92 vs. −0.78 for Vasa/Sasa, −0.93 vs. −0.85 for ln(AbsCO), and −0.88 vs. −0.60 for Rg. Biomolecules 2020, 10, 197
Biomolecules 2020 9 x 10 of 14
10 of 14 Figure 8. 3.3. Unfolding Rates of Proteins from Thermophilic and Mesophilic Organisms
3.3. Unfolding Rates of Proteins from Thermophilic and Mesophilic Organisms Correlations of the logarithm of the unfolding rate of the two- and multi-state thermophilic
and mesophilic proteins with structural parameters: L (length of the protein), Vasa/Sasa, ln(AbsCO),
and Rg. There is a line approximation of points and its equation: red line corresponds to thermophilic
proteins and blue line to mesophilic proteins. R2 is a linear approximation reliability. Figure 8. Correlations of the logarithm of the unfolding rate of the two- and multi-state thermophilic
and mesophilic proteins with structural parameters: L (length of the protein), Vasa/Sasa, ln(AbsCO),
and Rg. There is a line approximation of points and its equation: red line corresponds to thermophilic
proteins and blue line to mesophilic proteins. R2 is a linear approximation reliability. Figure 8. Correlations of the logarithm of the unfolding rate of the two- and multi-state thermophilic
and mesophilic proteins with structural parameters: L (length of the protein), Vasa/Sasa, ln(AbsCO),
and Rg. There is a line approximation of points and its equation: red line corresponds to thermophilic
proteins and blue line to mesophilic proteins. R2 is a linear approximation reliability. Figure 8. Correlations of the logarithm of the unfolding rate of the two- and multi-state thermophilic
and mesophilic proteins with structural parameters: L (length of the protein), Vasa/Sasa, ln(AbsCO),
and Rg. There is a line approximation of points and its equation: red line corresponds to thermophilic
proteins and blue line to mesophilic proteins. R2 is a linear approximation reliability. 11 of 14
with the
ore Asp, Biomolecules 2020, 10, 197
The thermophil
mesophilic proteins Table 8. Correlations between the logarithm of the unfolding rate (ln(ku)) and structural parameters (L,
Vasa/Sasa, ln(AbsCO) and Rg) for two-state and multi-state thermophilic and mesophilic proteins. ,
,
,
p
p
p
(
g
)
bout two-state thermophilic and mesophilic proteins. These data are also consistent with those
we obtained earlier in the study of 373 pairs of structurally similar thermophilic and mesop Table 8. Correlations between the logarithm of the unfolding rate (ln(ku)) and structural parameters (L,
Vasa/Sasa, ln(AbsCO) and Rg) for two-state and multi-state thermophilic and mesophilic proteins. Correlations with
ln(ku)
All (Two + Multi)
Two-State
Multi-State
Thermophile
Mesophile
Thermophile
Mesophile
Thermophile
Mesophile
Number of proteins
10
32
8
21
2
11
L
−0.83
−0.64
−0.92
−0.60
−
−0.27
Vasa/Sasa
−0.77
−0.75
−0.92
−0.78
−
0.09
ln(AbsCO)
−0.74
−0.83
−0.93
−0.85
−
−0.35
Rg
−0.87
−0.66
−0.88
−0.60
−
−0.47
* Correlations above 0.75 are shown in bold. 3.3. Unfolding Rates of Proteins from Thermophilic and Mesophilic Organisms
3.3. Unfolding Rates of Proteins from Thermophilic and Mesophilic Organisms about two-state thermophilic and mesophilic proteins. These data are also consistent with those that
we obtained earlier in the study of 373 pairs of structurally similar thermophilic and mesophilic
proteins [27]. Table 8. Correlations between the logarithm of the unfolding rate (ln(ku)) and structural parameters
(L, Vasa/Sasa, ln(AbsCO), and Rg) for two-state and multi-state thermophilic and mesophilic proteins. Correlation
s with
ln(ku)
All (two + multi)
Two−state
Multi−state
Thermophil
e
Mesophil
e
Thermophil
e
Mesophil
e
Thermophil
e
Mesophil
e
Number of
10
32
8
21
2
11 * Correlations above 0.75 are shown in bold. e
e
e The thermophilic proteins are enriched with Lys, Arg, and Val, in comparison with the mesophilic
proteins, and enriched in Lys, Asp, Ala, and the mesophilic proteins contain more Asp, Asn, Ser, and
Thr, in comparison with the thermophilic proteins (Figure 9). The same can be said about two-state
thermophilic and mesophilic proteins. These data are also consistent with those that we obtained
earlier in the study of 373 pairs of structurally similar thermophilic and mesophilic proteins [27]. proteins
L
−0.83
−0.64
−0.92
−0.60
−
−0.27
Vasa/Sasa
−0.77
−0.75
−0.92
−0.78
−
0.09
ln(AbsCO)
−0.74
−0.83
−0.93
−0.85
−
−0.35
Rg
−0.87
−0.66
−0.88
−0.60
−
−0.47
* Correlations above 0.75 are shown in bold. Figure 9. Amino acid composition of the bacterial thermophilic and mesophilic proteins with two-
and multi-state kinetics. Figure 9. Amino acid composition of the bacterial thermophilic and mesophilic proteins with two- and
multi-state kinetics. Figure 9. Amino acid composition of the bacterial thermophilic and mesophilic proteins with two-
and multi-state kinetics. Figure 9. Amino acid composition of the bacterial thermophilic and mesophilic proteins with two- and
multi-state kinetics. 4. Discussion
4. Discussion In this paper, we tried to find parameters that are important for predicting the protein
unfolding rates. For this, the database consists of 108 proteins with known unfolding and folding
rates, and such structural parameters as L, ln(AbsCO), Vasa/Sasa, and Rg were considered. In this paper, we tried to find parameters that are important for predicting the protein unfolding
rates. For this, the database consists of 108 proteins with known unfolding and folding rates, and such
structural parameters as L, ln(AbsCO), Vasa/Sasa, and Rg were considered. rates, and such structural parameters as L, ln(AbsCO), Vasa/Sasa, and Rg were considered. The good correlation (0.79) between the logarithm of the unfolding rate and protein stability
was observed for 108 proteins. p
(
)
g
The good correlation (0.79) between the logarithm of the unfolding rate and protein stability was
observed for 108 proteins. was obse ved o
08 p otei s. First, we divided the proteins in our database into two-states and multi-states. On average, the
logarithms of the folding and unfolding rates are higher for two-state proteins, in comparison with
First, we divided the proteins in our database into two-states and multi-states. On average,
the logarithms of the folding and unfolding rates are higher for two-state proteins, in comparison with
multi-state proteins. A good correlation (not lower than 0.70) for the logarithm of the folding and 12 of 14 Biomolecules 2020, 10, 197 unfolding rates for two- and multi-state proteins was observed. It has been shown that the logarithm
of the unfolding rate of two-state proteins correlate better with Vasa/Sasa (−0.79) and ln(AbsCO) (−0.87),
and the logarithm of the unfolding rate of multi-state correlates better with L (−0.71). Then, we separately studied bacterial and eukaryotic proteins from our database. It has been shown
that two-state bacterial proteins fold and unfold faster than two-state eukaryotic proteins, and multi-state
eukaryotic proteins fold and unfold faster than multi-state bacterial proteins. The logarithm of the
unfolding rate of two-state bacterial proteins correlates better with ln(AbsCO) (−0.86) and Rg (−0.64),
and eukaryotic proteins correlate better with L (−0.75). For multi-state proteins, the following picture
is observed: the logarithm of the unfolding rate of bacterial proteins correlates better with Rg (−0.58),
and eukaryotic proteins correlate better with L (−0.77), Vasa/Sasa (−0.69), and ln(AbsCO) (−0.81). Finally, we separately studied the thermophilic and mesophilic bacterial proteins from our database. 4. Discussion
4. Discussion There is correlation of the logarithm of the unfolding rate with protein stability for thermophilic
(0.90) and mesophilic proteins (0.73). It has been shown that the logarithm of the unfolding rates
of thermophilic proteins are about two orders of magnitude lower than that of mesophilic proteins,
but the logarithm of the folding rates of thermophilic and mesophilic proteins are almost the same. The logarithm of the unfolding rate of two- and multi-state thermophilic proteins correlate better
with all considered structural parameters (L, Vasa/Sasa, ln(AbsCO) and Rg), in comparison with the
mesophilic proteins. We have tried to find out which parameters are most important for the prediction of the unfolding
rates for proteins from different structural classes (α, β, α/β and α + β); proteins of different origins
(bacterial and eukaryotic); and proteins from different organisms (thermophilic and mesophilic). 5. Conclusions Thus, it has been shown that there is a good correlation between the logarithm of the unfolding
and folding rates (0.79) and between the logarithm of the unfolding rate and proteins stabilities (0.79)
for 108 proteins. The correlation between the unfolding and folding rates is better for: two-state
(0.78), in comparison with multi-state (0.73) proteins; α and β proteins (0.78 and 0.75), in comparison
with α/β and α + β protein (0.59 and 0.60) structural classes; eukaryotic (0.75), in comparison with
bacterial (0.73) proteins; and mesophilic (0.76), in comparison with thermophilic (0.73) proteins. The structural parameter ln(AbsCO) better correlates with the logarithm of the unfolding rate for:
all 108 proteins; proteins from α and β structural classes; and bacterial, eukaryotic, and mesophilic
proteins, in comparison with other parameters (L, Vasa/Sasa and Rg). Supplementary Materials: The following are available online: http://www.mdpi.com/2218-273X/10/2/197/s1. Table S1. Logarithm of folding and unfolding rates and some structural parameters for 108 proteins. Table S2. Average values of different parameters for four structural classes of proteins (α, β, α/β and α + β). Author Contributions: Conceptualization and design of the experiments, O.V.G. and A.V.G.; software, A.V.G.;
formal analysis, A.V.G. and O.V.G.; investigation, A.V.G. and O.V.G.; data curation, A.V.G. and O.V.G.; visualization,
A.V.G.; writing—original draft preparation, A.V.G. and O.V.G.; funding acquisition, A.V.G.; supervision, O.V.G.;
and validation, A.V.G. and O.V.G. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Russian Science Foundation, grant number 19-74-10051. Funding: This research was funded by the Russian Science Foundation, grant number 19-74-10051. Conflicts of Interest: The authors declare no conflicts of interest. ln(kf)—logarithm of the folding rate, ln(ku)—logarithm of the unfolding rate, ln(kmt)—logarithm of the
mid-transition folding/unfolding rate, L—length of the protein, rCO—relative contact order, ln(AbsCO)—logarithm
of the absolute contact order, Vasa/Sasa—radius of cross-section, and Rg—radius of gyration. Abbreviations ln(kf)—logarithm of the folding rate, ln(ku)—logarithm of the unfolding rate, ln(kmt)—logarithm of the
mid-transition folding/unfolding rate, L—length of the protein, rCO—relative contact order, ln(AbsCO)—logarithm
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When null hypothesis significance testing is unsuitable for research: a reassessment
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Edited by:
Satrajit S. Ghosh,
Massachusetts Institute of
Technology, United States Edited by:
Satrajit S. Ghosh,
Massachusetts Institute of
Technology, United States
Reviewed by:
Bertrand Thirion,
Institut National de Recherche en
Informatique et en Automatique
(INRIA), France
Cyril R. Pernet,
University of Edinburgh,
United Kingdom Keywords: replication crisis, false positive findings, research methodology, null hypothesis significance testing,
Bayesian methods Reviewed by:
Bertrand Thirion,
Institut National de Recherche en
Informatique et en Automatique
(INRIA), France
Cyril R. Pernet,
University of Edinburgh,
United Kingdom Reviewed by:
Bertrand Thirion,
Institut National de Recherche en
Informatique et en Automatique
(INRIA), France
Cyril R. Pernet,
University of Edinburgh,
United Kingdom “What used to be called judgment is now called prejudice and what used to be called prejudice is
now called a null hypothesis. In the social sciences, particularly, it is dangerous nonsense (dressed
up as the “scientific method”) and will cause much trouble before it is widely appreciated as such.”
(Edwards, 1972; p.180.) p
“...the mathematical rules of probability theory are not merely rules for calculating frequencies of
random variables; they are also the unique consistent rules for conducting inference (i.e., plausible
reasoning)” *Correspondence:
Denes Szucs
ds377@cam.ac.uk *Correspondence:
Denes Szucs
ds377@cam.ac.uk (Jaynes, 2003; p. xxii). (Jaynes, 2003; p. xxii). When Null Hypothesis Significance
Testing Is Unsuitable for Research:
A Reassessment 1 Department of Psychology, University of Cambridge, Cambridge, United Kingdom, 2 Meta-Research Innovation Center at
Stanford and Department of Medicine, Department of Health Research and Policy, and Department of Statistics, Stanford
University, Stanford, CA, United States Null hypothesis significance testing (NHST) has several shortcomings that are
likely contributing factors behind the widely debated replication crisis of (cognitive)
neuroscience, psychology, and biomedical science in general. We review these
shortcomings and suggest that, after sustained negative experience, NHST should no
longer be the default, dominant statistical practice of all biomedical and psychological
research. If theoretical predictions are weak we should not rely on all or nothing
hypothesis tests. Different inferential methods may be most suitable for different types
of research questions. Whenever researchers use NHST they should justify its use,
and publish pre-study power calculations and effect sizes, including negative findings. Hypothesis-testing studies should be pre-registered and optimally raw data published. The current statistics lite educational approach for students that has sustained the
widespread, spurious use of NHST should be phased out. THE REPLICATION CRISIS AND NULL HYPOTHESIS
SIGNIFICANCE TESTING (NHST) Received: 03 February 2017
Accepted: 13 July 2017
Published: 03 August 2017 There is increasing discontent that many areas of psychological science, cognitive neuroscience,
and biomedical research (Ioannidis, 2005; Ioannidis et al., 2014) are in a crisis of producing
too many false positive non-replicable results (Begley and Ellis, 2012; Aarts et al., 2015). This
wastes research funding, erodes credibility and slows down scientific progress. Since more
than half a century many methodologists have claimed repeatedly that this crisis may at least
in part be related to problems with Null Hypothesis Significance Testing (NHST; Rozeboom,
1960; Bakan, 1966; Meehl, 1978; Gigerenzer, 1998; Nickerson, 2000). However, most scientists REVIEW
published: 03 August 2017
doi: 10.3389/fnhum.2017.00390 REVIEW published: 03 August 2017
doi: 10.3389/fnhum.2017.00390 Citation: Szucs D and Ioannidis JPA (2017)
When Null Hypothesis Significance
Testing Is Unsuitable for Research:
A Reassessment. Front. Hum. Neurosci. 11:390. doi: 10.3389/fnhum.2017.00390 August 2017 | Volume 11 | Article 390 Frontiers in Human Neuroscience | www.frontiersin.org 1 Null Hypothesis Significance Testing: A Reassessment Szucs and Ioannidis (and in particular psychologists, biomedical scientists, social
scientists, cognitive scientists, and neuroscientists) are still near
exclusively educated in NHST, they tend to misunderstand and
abuse NHST and the method is near fully dominant in scientific
papers (Chavalarias et al., 1990-2015). Here we provide an
accessible critical reassessment of NHST and suggest that while
it may have legitimate uses when there are precise quantitative
predictions and/or as a heuristic, it should be abandoned as the
cornerstone of research. (and in particular psychologists, biomedical scientists, social
scientists, cognitive scientists, and neuroscientists) are still near
exclusively educated in NHST, they tend to misunderstand and
abuse NHST and the method is near fully dominant in scientific
papers (Chavalarias et al., 1990-2015). Here we provide an
accessible critical reassessment of NHST and suggest that while
it may have legitimate uses when there are precise quantitative
predictions and/or as a heuristic, it should be abandoned as the
cornerstone of research. Hence, a single significant result should not represent a “scientific
fact” but should merely draw attention to a phenomenon which
seems worthy of further investigation including replication
(Goodman, 2008). In contrast to the above, until recently
replication studies have been very rare in many scientific fields;
lack of replication efforts has been a particular problem in the
psychological sciences (Makel et al., 2012), but this may hopefully
change now with the wide attention that replication has received
(Aarts et al., 2015). Our paper does not concern specifically the details of
neuro-imaging methodology, many papers dealt with such
details recently (Pernet and Poline, 2015; Nichols et al., 2016,
2017). Rather, we take a more general view in discussing
fundamental problems that can affect any scientific field,
including neuroscience and neuro-imaging. In relation to this it
is important to see that non-invasive neuroscience data related
to behavioral tasks cannot be interpreted if task manipulations
did not work and/or behavior is unclear. This is because most
measured brain activity changes can be interpreted in many
different ways on their own (Poldrack, 2006; see Section 2.7 in
Nichols et al., 2016). NHST as a Weak Heuristic Based on the
p-Value: Fisher p-values were popularized by Fisher (1925). In the context of the
current NHST approach Fisher only relied on the concepts of
the null hypothesis (H0) and the exact p-value (hereafter p will
refer to the p-value and “pr” to probability; see Appendix 1 in
Supplementary Material for terms). He thought that experiments
should aim to reject (or “nullify”; henceforth the name “null
hypothesis”) H0 which assumes that the data demonstrates
random variability according to some distribution around a
certain value. Discrepancy from H0 is measured by a test statistic
whose values can be paired with one or two-tailed p-values which
tell us how likely it is that we would have found our data or more
extreme data if H0 was really correct. Formally we will refer to the
p-value as: pr(data or more extreme data|H0). It is important to
realize that the p-value represents the “extremeness” of the data
according to an imaginary data distribution assuming there is no
bias in data sampling. Crucially, the Neyman–Pearson approach is designed to
work efficiently (Neyman and Pearson, 1933) in the context
of long-run repeated testing (exact replication). Hence, there
is a major difference between the p-value which is computed
for a single data set and α, β, power, Type I, and Type
II error which are so called “frequentist” concepts and they
make sense in the context of a long-run of many repeated
experiments. If we only run a single experiment all we can claim
is that if we had run a long series of experiments we would
have had 100α% false positives (Type I error) had H0 been
true and 100β% false negatives (Type II error) had H1 been
true provided we got the power calculations right. Note the
conditionals. The late Fisher viewed the exact p-value as a heuristic piece of
inductive evidence which gives an indication of the plausibility
of H0 together with other available evidence, like effect sizes
(see Hubbard and Bayarri, 2003; Gigerenzer et al., 2004). Fisher
recommended that H0 can usually be rejected if p ≤0.05 but
in his system there is no mathematical justification for selecting
a particular p-value for the rejection of H0. Rather, this is up
to the substantively informed judgment of the experimenter. Fisher thought that a hypothesis is demonstrable only when
properly designed experiments “rarely fail” to give us statistically
significant results (Gigerenzer et al., 1989, p. 96; Goodman, 2008). Citation: So, as most behavioral data are analyzed by
NHST statistics NHST based inference from behavioral data also
plays a crucial role in interpreting brain data. Neyman and Pearson: A Decision
Mechanism Optimized for the Long-Run
The concepts of the alternative hypothesis (H1), α, power, β,
Type I, and Type II errors were introduced by Neyman and
Pearson (Neyman and Pearson, 1933; Neyman, 1950) who set
up a formal decision procedure motivated by industrial quality
control problems (Gigerenzer et al., 1989). Their approach
aimed to minimize the false negative (Type II) error rate to
an acceptable level (β) and consequently to maximize power
(1-β) subject to a bound (α) on false positive (Type I) errors
(Hubbard and Bayarri, 2003). α can be set by the experimenter
to an arbitrary value and Type-II error can be controlled by
setting the sample size so that the required effect size can be
detected (see Figure 1 for illustration). In contrast to Fisher, this
framework does not use the p-value as a measure of evidence. We merely determine the critical value of the test statistic
associated with α and reject H0 whenever the test statistic is
larger than the critical value. The exact p-value is irrelevant
because the sole objective of the decision framework is long-
run error minimization and only the critical threshold but not
the exact p-value plays any role in achieving this goal (Hubbard
and Bayarri, 2003). Neyman and Pearson rejected the idea
of inductive reasoning and offered a reasoning-free inductive
behavioral rule to choose between two behaviors, accepting
or rejecting H0, irrespective of the researcher’s belief about
whether H0 and H1 are true or not (Neyman and Pearson,
1933). Frontiers in Human Neuroscience | www.frontiersin.org NHST as a Weak Heuristic Based on the
p-Value: Fisher In the Neyman–Pearson framework optimally setting α and
β assures long-term decision-making efficiency in light of our
costs and benefits by committing Type I and Type II errors. However, optimizing α and β is much easier in industrial quality
control than in research where often there is no reason to expect
a specific effect size associated with H1 (Gigerenzer et al., 1989). August 2017 | Volume 11 | Article 390 2 Null Hypothesis Significance Testing: A Reassessment Szucs and Ioannidis FIGURE 1 | NHST concepts make sense in the context of a long run of studies. 3 × 10,000 studies with normally distributed data were simulated for 3 situations (A:
True H0 situation: Mean = 0; SD = 1; n = 16. B: Mean = 0.5; SD = 1; n = 16; Power = 0.46. C: Mean = 0.5; SD = 1; n = 32; Power = 0.78.). One sample
two-tailed t-tests determined whether the sample means were zero. The red dots in the top panels show t scores for 3 × 1,000 studies (not all studies are shown for
better visibility). The vertical dashed lines mark the critical rejection thresholds for H0, t(α/2) for the two-tailed test. The studies producing a t statistic more extreme
than these thresholds are declared statistically significant. The middle panels show the distribution of t scores for all 3 × 10,000 studies (bins = 0.1). The bottom
panels show the distribution of p-values for all 3 × 10,000 studies (bins = 0.01) and state the proportion of significant studies. The inset in the bottom right panel
shows the mean absolute effect sizes in standard deviation units for situations A-C from all significant (Sig.) and non-significant (n.s.) studies with 95% bias corrected
and accelerated bootstrap confidence intervals (10,000 permutations). The real effect size was 0 in situation (A) and 0.5 in situations (B,C). Note that the less is the
power the more statistically significant studies overstate the effect size. Also note that p-values are randomly distributed and the larger is power the more right skewed
is the distribution of p-values. In the true H0 situation the distribution of p-values is uniform between 0 and 1. See further explanation of this figure in Appendix 2 in
Supplementary Material. FIGURE 1 | NHST concepts make sense in the context of a long run of studies. NHST as a Weak Heuristic Based on the
p-Value: Fisher 3 × 10,000 studies with normally distributed data were simulated for 3 situations (A:
True H0 situation: Mean = 0; SD = 1; n = 16. B: Mean = 0.5; SD = 1; n = 16; Power = 0.46. C: Mean = 0.5; SD = 1; n = 32; Power = 0.78.). One sample
two-tailed t-tests determined whether the sample means were zero. The red dots in the top panels show t scores for 3 × 1,000 studies (not all studies are shown for
better visibility). The vertical dashed lines mark the critical rejection thresholds for H0, t(α/2) for the two-tailed test. The studies producing a t statistic more extreme
than these thresholds are declared statistically significant. The middle panels show the distribution of t scores for all 3 × 10,000 studies (bins = 0.1). The bottom
panels show the distribution of p-values for all 3 × 10,000 studies (bins = 0.01) and state the proportion of significant studies. The inset in the bottom right panel
shows the mean absolute effect sizes in standard deviation units for situations A-C from all significant (Sig.) and non-significant (n.s.) studies with 95% bias corrected
and accelerated bootstrap confidence intervals (10,000 permutations). The real effect size was 0 in situation (A) and 0.5 in situations (B,C). Note that the less is the
power the more statistically significant studies overstate the effect size. Also note that p-values are randomly distributed and the larger is power the more right skewed
is the distribution of p-values. In the true H0 situation the distribution of p-values is uniform between 0 and 1. See further explanation of this figure in Appendix 2 in
Supplementary Material. For example, if a factory has to produce screw heads with a
diameter of 1 ± 0.01 cm than we know that we have to be able
to detect a deviation of 0.01 cm to produce acceptable quality
output. In this setting we know exactly the smallest effect size
we are interested in (0.01 cm) and we can also control the
sample size very efficiently because we can easily take a sample
of a large number of screws from a factory producing them by
the million assuring ample power. Frontiers in Human Neuroscience | www.frontiersin.org NHST as a Weak Heuristic Based on the
p-Value: Fisher On the one hand, failing to
detect too large or too small screws (Type II error) will result
in our customers canceling their orders (or, in other industrial
settings companies may deliver faulty cars or exploding laptops to customers exposing themselves to substantial litigation and
compensation costs). On the other hand, throwing away false
positives (Type I error), i.e., completely good batches of screws
which we think are too small or too large, will also cost us a
certain amount of money. Hence, we have a very clear scale
(monetary value) to weigh the costs and benefits of both types
of errors and we can settle on some rationally justified values
of α and β so as to minimize our expenses and maximize our
profit. In contrast to such industrial settings, controlling the sample
size and effect size and setting rational α and β levels is not August 2017 | Volume 11 | Article 390 Frontiers in Human Neuroscience | www.frontiersin.org 3 Null Hypothesis Significance Testing: A Reassessment Szucs and Ioannidis that straightforward in most research settings where the true
effect sizes being pursued are largely unknown and deciding
about the requested size of a good enough effect can be very
subjective. For example, what is the smallest difference of interest
between two participant groups in a measure of “fMRI activity”? Or, what is the smallest difference of interest between two
groups of participants when we measure their IQ or reaction
time? And, even if we have some expectations about the “true
effect size,” can we test enough participants to ensure a small
enough β? Further, what is the cost of falsely claiming that a
vaccine causes autism thereby generating press coverage that
grossly misleads the public (Deer, 2011; Godlee, 2011)? What
is the cost of running too many underpowered studies thereby
wasting perhaps most research funding, boosting the number of
false positive papers and complicating interpretation (Schmidt,
1992; Ioannidis, 2005; Button et al., 2013)? More often than not
researchers do not know the “true” size of an effect they are
interested in, so they cannot assure adequate sample size and
it is also hard to estimate general costs and benefits of having
particular α and β values. NHST as a Weak Heuristic Based on the
p-Value: Fisher While some “rules of thumb” exist
about what are small, modest, and large effects (e.g., Cohen,
1962, 1988; Jaeschke et al., 1989; Sedlmeier and Gigerenzer,
1989), some large effects may not be actionable (e.g., a change
in some biomarker that is a poor surrogate and thus bears little
relationship to major, clinical outcomes), while some small effects
may be important and may change our decision (e.g., most
survival benefits with effective drugs are likely to be small, but
still actionable). FIGURE 2 | The distribution of p-values if the alternative hypothesis (H1) is
true. Each line depicts the distribution of p-values resulting from one-sample
two-tailed t-tests testing whether the sample mean was zero. Effect sizes (ES)
indicate the true sample means for normally distributed data with standard
deviation 1. For each effect size one million simulations were run with 16 cases
in each simulation. The distribution of the p-value is becoming increasingly
right skewed with increasing effect size and power. Note that α, the Type I
error rate, is fix irrespective of what p-value is found in an experiment. FIGURE 2 | The distribution of p-values if the alternative hypothesis (H1) is
true. Each line depicts the distribution of p-values resulting from one-sample
two-tailed t-tests testing whether the sample mean was zero. Effect sizes (ES)
indicate the true sample means for normally distributed data with standard
deviation 1. For each effect size one million simulations were run with 16 cases
in each simulation. The distribution of the p-value is becoming increasingly
right skewed with increasing effect size and power. Note that α, the Type I
error rate, is fix irrespective of what p-value is found in an experiment. Given the above ambiguity, researchers fall back to the default
α = 0.05 level with usually undefined power. So, the unjustified
α and β levels completely discredit the originally intended
“efficiency” rationale of the creators of the Neyman–Pearson
decision mechanism (Neyman and Pearson, 1933). (“positive” and “negative”) is again crucial if we rely on NHST
because only then can they inform us about the distribution of
p-values. Another point to notice is that both p-values and usual
standardized effect size measures (Cohen’s D, correlation values,
etc.) are direct functions of NHST test statistics. Hence, for given
degrees of freedom NHST test statistics, effect size measures and
p-values will have non-linear correspondence as illustrated in
Figure 3. Measures Contrary to the fact that in Figure 1 all 10,000 true H0 and
10,000 true H1 samples were simulated from identical H0 and
H1 distributions, the t scores and the associated p-values reflect
a dramatic spread. That is, p-values are best viewed as random
variables which can take on a range of values depending on the
actual data (Sterling, 1959; Murdoch et al., 2008). Consequently,
it is impossible to tell from the outcome of a single (published)
experiment delivering a statistically significant result whether a
true effect exist. The only difference between the true H0 and
true H1 situations is that when H0 is true in all experiments,
the distribution of p-values is uniform between 0 and 1 whereas
when H1 is true in all experiments p-values are more likely to
fall on the left of the 0–1 interval, that is, their distribution
becomes right skewed. The larger is the effect size and power
the stronger is this right skew (Figure 2). This fact led to the
suggestion that comparing this skew allows us to determine the
robustness of findings in some fields by studying “p curves”
(Hung et al., 1997; Simonsohn et al., 2014a,b). Hence, from this
perspective, replication, and unbiased publication of all results Frontiers in Human Neuroscience | www.frontiersin.org NEGLECTING THE FULL CONTEXT OF
NHST LEADS TO CONFUSIONS ABOUT
THE P-VALUE NHST completely neglects the above mentioned pre-study
information and exclusively deals with rows 2–4 of Table 1. NHST computes the one or two-tailed p-value for a particular
data set assuming that H0 is true. Additionally, NHST logic takes
long-run error probabilities (α and β) into account conditional
on H0 and H1. These long-run probabilities are represented in
typical 2 × 2 NHST contingency tables but note that β is usually
unknown in real studies. Most textbooks illustrate NHST by partial 2 × 2 tables
(see Table 1) which fail to contextualize long-run conditional
probabilities and fail to clearly distinguish between long-run
probabilities and the p-value which is computed for a single
data set (Pollard and Richardson, 1987). This leads to major
confusions about the meaning of the p-value (see Appendix 2 in
Supplementary Material). As we have seen, NHST never computes the probability
of H0 and H1 being true or false, all we have is a decision
mechanism hoping for the best individual decision in view of
long-run Type I and Type II error expectations. Nevertheless,
following the repeated testing logic of the NHST framework, for
many experiments we can denote the long-run probability of H0
being true given a statistically significant result as False Report
Probability (FRP), and the long-run probability of H1 being true
given a statistically significant result as True Report Probability
(TRP). FRP and TRP are represented in row 5 of Table 1 and it is
important to see that they refer to completely different conditional
probabilities than the p-value. pp
y
First, both H0 and H1 have some usually unknown pre-study
or “prior” probabilities, pr(H0) and pr(H1). Nevertheless, these
probabilities may be approximated through extensive substantive
knowledge. For example, we may know about a single published
study claiming to demonstrate H1 by showing a difference
between appropriate experimental conditions. However, in
conferences we may have also heard about 9 highly powered
but failed replication attempts very similar to the original
study. In this case we may assume that the odds of H0:H1
are 9:1, that is, pr(H1) is 1/10. Of course, these pre-study
odds are usually hard to judge unless we demand to see
our colleagues’ “null results” hidden in their drawers because
of the practice of not publishing negative findings. NHST in Its Current Form The current NHST merged the approaches of Fisher and
Neyman and Pearson and is often applied stereotypically as a
“mindless null ritual” (Gigerenzer, 2004). Researchers set H0
nearly always “predicting” zero effect but do not quantitatively
define H1. Hence, pre-experimental power cannot be calculated
for most tests which is a crucial omission in the Neyman–
Pearson framework. Researchers compute the exact p-value as
Fisher did but also mechanistically reject H0 and accept the
undefined H1 if p ≤(α = 0.05) without flexibility following the
behavioral decision rule of Neyman and Pearson. As soon as p
≤α, findings have the supposed right to become a scientific
fact defying the exact replication demands of Fisher and the
belief neutral approach of Neyman and Pearson. Researchers
also interpret the exact p-value and use it as a relative measure
of evidence against H0, as Fisher did. A “highly significant” August 2017 | Volume 11 | Article 390 4 Null Hypothesis Significance Testing: A Reassessment Szucs and Ioannidis FIGURE 3 | The relationship of the p-value, test statistic and effect sizes. (A) The relationship of t values, degrees of freedoms and p-values for Pearson correlation
studies (df = n–2). (B) The relationship of Pearson correlation (r) values, degrees of freedoms, and p-values [r = t/sqrt(df + t2)]. (C) The relationship of r- and t-value
pairs for each degree of freedom at various p-values. The bold black lines mark the usual significance level of α = 0.05. Note that typically only results which exceed
the α = 0.05 threshold are reported in papers. Hence, papers mostly report exaggerated effect sizes. FIGURE 3 | The relationship of the p-value, test statistic and effect sizes. (A) The relationship of t values, degrees of freedoms and p-values for Pearson correlation
studies (df = n–2). (B) The relationship of Pearson correlation (r) values, degrees of freedoms, and p-values [r = t/sqrt(df + t2)]. (C) The relationship of r- and t-value
pairs for each degree of freedom at various p-values. The bold black lines mark the usual significance level of α = 0.05. Note that typically only results which exceed
the α = 0.05 threshold are reported in papers. Hence, papers mostly report exaggerated effect sizes. NHST in Its Current Form not allow for accepting it and because researchers erroneously
often think that the single published positive study has a very
small, acceptable error rate of providing false positive statistically
significant results which equals α, or the p-value. So, they
often spuriously assume that the negative studies somehow
lacked the sensitivity to show an effect while the single positive
study is perceived as a well-executed sensitive experiment
delivering a “conclusive” verdict rather than being a “lucky” false
positive (Bakan, 1966). (See a note on pilot studies in Serious
Underestimation of the Proportion of False Positive Findings in
NHST). result with a small p-value is perceived as much stronger
evidence than a weakly significant one. However, while Fisher
was conscious of the weak nature of the evidence provided
by the p-value (Wasserstein and Lazar, 2016), generations
of scientists encouraged by incorrect editorial interpretations
(Bakan, 1966) started to exclusively rely on the p-value in
their decisions even if this meant neglecting their substantive
knowledge: scientific conclusions merged with reading the p-
value (Goodman, 1999). Frontiers in Human Neuroscience | www.frontiersin.org NEGLECTING THE FULL CONTEXT OF
NHST LEADS TO CONFUSIONS ABOUT
THE P-VALUE FRP =
False positives
All statistically significant results
=
5% of 100 studies
5% of 100 studies + 60% of 100 studies
=
5
5 + 60 =
5
65 = 0.0769 FRP =
False positives
All statistically significant results
=
5% of 100 studies
5% of 100 studies + 60% of 100 studies
=
5
5 + 60 =
5
65 = 0.0769 That is, we will have 5 false positives out of a total of
65 statistically significant outcomes which means that the
proportion of false positive studies amongst all statistically
significant results is 7.69%, higher than the usually assumed 5%. However, this example still assumes that we get every second
hypothesis right. If we are not as lucky and only get every sixth
hypothesis right then if we run 600 studies, 500 of them will
have true H0 true situations and 100 of them will have true H1
situations. Hence, the computation will look like: NEGLECTING THE FULL CONTEXT OF
NHST LEADS TO CONFUSIONS ABOUT
THE P-VALUE Note that the p value does not fit this view as it does not have any long run interpretation besides that it is a random variable (Murdoch et al., 2008). The most important variables
are bolded, familiar signal detection categories are also provided. NHST does not deal with the concepts in italics. NHST textbooks typically only present rows 3 and 4 of this table (Alpha level, Power, Confidence level and Type II error). We follow the NHST view and deal with long run probabilities
only. Note that the p value does not fit this view as it does not have any long run interpretation besides that it is a random variable (Murdoch et al., 2008). The most important variables
are bolded, familiar signal detection categories are also provided. NHST does not deal with the concepts in italics. and in the other 100 studies (true H1) 60% of studies will come up
significant. FRP is the ratio of false positive studies to all studies
which come up significant: and the only parameter NHST computes is the p-value it is well-
documented (Oakes, 1986; Gliner et al., 2002; Castro Sotos et al.,
2007, 2009; Wilkerson and Olson, 2010; Hoekstra et al., 2014)
that many, if not most researchers confuse FRP with the p-value
or α and they also confuse the complement of p-value (1-p) or
α (1-α) with TRP (Pollard and Richardson, 1987; Cohen, 1994). These confusions are of major portend because the difference
between these completely different parameters is not minor, they
can differ by orders of magnitude, the long-run FRP being much
larger than the p-value under realistic conditions (Sellke et al.,
2001; Ioannidis, 2005). The complete misunderstanding of the
probability of producing false positive findings is most probably
a key factor behind vastly inflated confidence in research findings
and we suggest that this inflated confidence is an important
contributor to the current replication crisis in biomedical science
and psychology. Frontiers in Human Neuroscience | www.frontiersin.org NEGLECTING THE FULL CONTEXT OF
NHST LEADS TO CONFUSIONS ABOUT
THE P-VALUE Current
scientific practices appreciate the single published “positive”
study more than the 9 unpublished negative ones perhaps
because NHST logic only allows for rejecting H0 but does Simply put, the p-value is pretty much the only thing that
NHST computes but scientists usually would like to know the
probability of their theory being true or false in light of their data
(Pollard and Richardson, 1987; Goodman, 1993; Jaynes, 2003;
Wagenmakers, 2007). That is, researchers are interested in the
post-experimental probability of H0 and H1. Most probably, for
the reason that researchers do not get what they really want to see August 2017 | Volume 11 | Article 390 5 Null Hypothesis Significance Testing: A Reassessment Szucs and Ioannidis TABLE 1 | “pr” stands for probability. True null effect (H0)
True positive effect (H1)
Pre-experiment probability of H0 and H1
Long run of experiments
pr(H0)
pr(H1)
The conditional probability of having this data or more extreme data
given that H0 is true
Single experiment
p-value
—
The conditional probability of having a significant test result given that
H0 or H1 are true
Long run of experiments
Alpha level (α)
Type I error
False Positive
False Alarm
Power = 1 −β
True Positive Hit
The conditional probability of not having a significant test result given
that H0 or H1 are true
Long run of experiments
1 – α = Confidence level
True Negative Correct Rejection
β = 1 – Power
Type II error
False Negative Miss
Post-experiment probability of H0 and H1 given a significant test result
Long run of experiments
FRP
pr(H0|significant result)
TRP
pr(H1|significant result)
NHST textbooks typically only present rows 3 and 4 of this table (Alpha level, Power, Confidence level and Type II error). We follow the NHST view and deal with long run probabilities
only. Note that the p value does not fit this view as it does not have any long run interpretation besides that it is a random variable (Murdoch et al., 2008). The most important variables
are bolded, familiar signal detection categories are also provided. NHST does not deal with the concepts in italics. TABLE 1 | “pr” stands for probability. NHST textbooks typically only present rows 3 and 4 of this table (Alpha level, Power, Confidence level and Type II error). We follow the NHST view and deal with long run probabilities
only. Serious Underestimation of the Proportion
of False Positive Findings in NHST For example,
if α = 0.05, power = 0.6, and H0:H1 odds = 1 then a 10%
bias (u = 0.1) will raise FRP to 18.47%. A 20% bias will raise
FRP to 26.09%. If H0:H1 odds = 6 then FRP will be 67.92%. Looking at these numbers the replication crisis does not seem
surprising: using NHST very high FRP can be expected even
with modestly high H0:H1 odds and moderate bias (Etz and
Vandekerckhove, 2016). Hence, under realistic conditions FRP
not only extremely rarely equals α or the p-value (and TRP
extremely rarely equals 1-α and/or 1-p-value) but also, FRP is
much larger than the generally assumed 5% and TRP is much
lower than the generally assumed 95%. Overall, α or the p-value
practically says nothing about the likelihood of our research
findings being true or false. experiment is run: We do not know what percent of the published
statistically significant findings are lucky false positives explained
post-hoc (Kerr, 1998) when in fact researchers could not detect
the originally hypothesized effect and/or worked out analyses
depending on the data (Gelman and Loken, 2014). However, it
is reasonable to assume that only the most risk avoidant studies
have lower H0:H1 odds than 1, relatively conservative studies
have low to moderate H0:H1 odds (1–10) while H0:H1 odds can
be much higher in explorative research (50–100 or even higher;
Ioannidis, 2005). The above H0:H1 assumptions are reasonable, as they are
supported by empirical data in many different fields. For
example, half or more of the drugs tested in large, late phase
III trials show higher effectiveness against older comparators
(H0:H1 = <1; Soares et al., 2005). Conversely, the vast majority
of tested hypotheses in large-scale exploratory research reflect
null effects, e.g., in the search of genetic variants associated with
various diseases in the candidate gene era where investigators
were asking hypotheses one or a few at a time (the same
way that investigators continue to test hypotheses in most
other biomedical and social science fields) yielded thousands of
putative discovered associations, but only 1.2% of them were
subsequently validated to be non-null when large-scale consortia
with accurate measurements and rigorous analyses plans assessed
them (Chanock et al., 2007; Ioannidis et al., 2011). Of the
hundreds of thousands to many millions of variables assessed
in current agnostic–omics testing, much less than 1% are likely
to reflect non-null effects (H0:H1>>100). Serious Underestimation of the Proportion
of False Positive Findings in NHST TRP is the ratio of truly positive (H1 is true) statistically significant studies
to all statistically significant studies: 60/65 = 0.9231 = 1 −FRP = 1 −0.0769. FIGURE 4 | Illustration of long run False Positive Probability (FRP) and True Positive Probability (TRP) of studies. Let’s assume that we run 2 × 100 studies, H0 is true
in 100 studies and H1 is true in 100 studies with α = 0.05 and Power = 1−β = 0.6. (A) Shows the outcome of true H0 studies, 5 of the 100 studies coming up
statistically significant. (B) Shows the outcome of true H1 studies, 60 of the 100 studies coming up statistically significant [note that realistically the 60 studies would
be scattered around just as in panel (A) but for better visibility they are represented in a block]. (C) Illustrates that true H0 and true H1 studies would be
indistinguishable. That is, researchers do not know which study tested a true H0 or true H1 situation (i.e., they could not distinguish studies represented by black and
gray squares). All they know is whether the outcome of a particular study out of the 200 studies run was statistically significant or not. FRP is the ratio of false positive
(H0 is true) statistically significant studies to all statistically significant studies: 5/65 = 0.0769. TRP is the ratio of truly positive (H1 is true) statistically significant studies
to all statistically significant studies: 60/65 = 0.9231 = 1 −FRP = 1 −0.0769. findings have far more difficulty to be published and the
researcher may feel that she wasted her efforts. Further, positive
findings are more likely to get cited than negative findings
(Kjaergard and Gluud, 2002; Jannot et al., 2013; Kivimäki et al.,
2014). Consequently, researchers may often be highly biased to
reject H0 and publish positive findings. Researcher bias affects
FRP even if our NHST decision criteria, α and β, are formally
unchanged. Ioannidis (2005) introduced the u bias parameter. The impact of u is that after some data tweaking and selective
reporting (see Section NHST May Foster Selective Reporting
and Subjectivity) u fraction of otherwise non-significant true H0
results will be reported as significant and u faction of otherwise
non-significant true H1 results will be reported as significant. If u increases, FRP increases and TRP decreases. Serious Underestimation of the Proportion
of False Positive Findings in NHST g
Ioannidis (2005) has shown that most published research findings
relying on NHST are likely to be false. The modeling supporting
this claim refers to the long-run FRP and TRP which we
can compute by applying Bayes’ theorem (see Figure 4 for
illustration, see computational details and further illustration in
Appendix 3 in Supplementary Material). The calculations must
consider α, the power (1-β) of the statistical test used, the pre-
study probabilities of H0 and H1, and it is also insightful to
consider bias (Berger, 1985; Berger and Delampady, 1987; Berger
and Sellke, 1987; Pollard and Richardson, 1987; Lindley, 1993;
Sellke et al., 2001; Sterne and Smith, 2001; Ioannidis, 2005). FRP =
False positives
All statistically significant results
=
5% of 500 studies
5% of 500 studies + 60% of 100 studies
=
25
25 + 60 = 25
85 = 0.2941 FRP =
False positives
All statistically significant results
=
5% of 500 studies
5% of 500 studies + 60% of 100 studies
=
25
25 + 60 = 25
85 = 0.2941 Hence, nearly 1/3 of all statistically significant findings will be
false positives irrespective of the p-value. Note that this issue
is basically the consequence of running multiple NHST tests
throughout the whole literature and FRP can be considered the
uncontrolled false discovery rate (FDR) across all studies run (see
Section Family-Wise Error Rate (FWER) and FDR Correction in
NHST). While NHST neglects the pre-study odds of H0 and H1, these
are crucial to take into account when calculating FRP and TRP. For example, let’s assume that we run 200 experiments and in 100
studies our experimental ideas are wrong (that is, we test true H0
situations) while in 100 studies our ideas are correct (that is, we
test true H1 situations). Let’s also assume that the power (1-β) of
our statistical test is 0.6 and α = 0.05. In this case in 100 studies
(true H0) we will have 5% of results significant by chance alone Crucially, estimating pre-study odds is difficult, primarily
due to the lack of publishing negative findings and to the lack
of proper documentation of experimenter intentions before an August 2017 | Volume 11 | Article 390 6 Null Hypothesis Significance Testing: A Reassessment Szucs and Ioannidis FIGURE 4 | Illustration of long run False Positive Probability (FRP) and True Positive Probability (TRP) of studies. Serious Underestimation of the Proportion
of False Positive Findings in NHST Let’s assume that we run 2 × 100 studies, H0 is true
in 100 studies and H1 is true in 100 studies with α = 0.05 and Power = 1−β = 0.6. (A) Shows the outcome of true H0 studies, 5 of the 100 studies coming up
statistically significant. (B) Shows the outcome of true H1 studies, 60 of the 100 studies coming up statistically significant [note that realistically the 60 studies would
be scattered around just as in panel (A) but for better visibility they are represented in a block]. (C) Illustrates that true H0 and true H1 studies would be
indistinguishable. That is, researchers do not know which study tested a true H0 or true H1 situation (i.e., they could not distinguish studies represented by black and
gray squares). All they know is whether the outcome of a particular study out of the 200 studies run was statistically significant or not. FRP is the ratio of false positive
(H0 is true) statistically significant studies to all statistically significant studies: 5/65 = 0.0769. TRP is the ratio of truly positive (H1 is true) statistically significant studies FIGURE 4 | Illustration of long run False Positive Probability (FRP) and True Positive Probability (TRP) of studies. Let’s assume that we run 2 × 100 studies, H0 is true
in 100 studies and H1 is true in 100 studies with α = 0.05 and Power = 1−β = 0.6. (A) Shows the outcome of true H0 studies, 5 of the 100 studies coming up
statistically significant. (B) Shows the outcome of true H1 studies, 60 of the 100 studies coming up statistically significant [note that realistically the 60 studies would
be scattered around just as in panel (A) but for better visibility they are represented in a block]. (C) Illustrates that true H0 and true H1 studies would be
indistinguishable. That is, researchers do not know which study tested a true H0 or true H1 situation (i.e., they could not distinguish studies represented by black and
gray squares). All they know is whether the outcome of a particular study out of the 200 studies run was statistically significant or not. FRP is the ratio of false positive
(H0 is true) statistically significant studies to all statistically significant studies: 5/65 = 0.0769. Frontiers in Human Neuroscience | www.frontiersin.org Serious Underestimation of the Proportion
of False Positive Findings in NHST Lower rates of H0:H1
would be incompatible with logical considerations of how many
variables are needed to explain all the variance of a disease or
outcome risk. At this point it is worth noting that it could be argued
that unpublished pilot experiments may prompt us to run
studies and hence, often H0:H1 odds would be lower than
1. However, unpublished pilot data often comes from small
scale underpowered studies with high FRP, undocumented
initial hypotheses and analysis paths. Hence, we doubt that Besides H0:H1 odds bias is another important determinant of
FRP and TRP (Ioannidis, 2005). Whenever, H0 is not rejected Frontiers in Human Neuroscience | www.frontiersin.org August 2017 | Volume 11 | Article 390 7 Null Hypothesis Significance Testing: A Reassessment Szucs and Ioannidis TABLE 2 | Potential NHST style argument (based on Pollard and Richardson,
1987). H0
Harold is American
H1
Harold is not American
Model for H0
If Harold is American (H0), than he is most probably
not a member of congress. data
Harold is a member of congress. pr(data or more extreme
data|H0)
Very low
Inference
Because pr(data or more extreme data|H0) is very
low, we reject H0 and accept H1 and conclude:
Harold is most probably not American. TABLE 2 | Potential NHST style argument (based on Pollard and Richardson,
1987) statistically
significant
pilot
results
inevitably
mean
low
H0:H1 odds. NHST Misleads Because It Neglects
Pre-data Probabilities We can
assign arbitrary but plausible probabilities: pr(data|H0) = pr(Harold is member of congress | American)
= 10−7
pr(data|H1) = pr(Harold is member of congress | not American)
= 0 pr(data|H0) = pr(Harold is member of congress | American)
= 10−7 pr(data|H1) = pr(Harold is member of congress | not American)
= 0 pr(data|H1) = pr(Harold is member of congress | not American)
= 0 That is, while the data is indeed rare under H0, its probability is
actually zero under H1 (in other words, the data is very unlikely
under both the null and the alternative models). So, even if p ≈
0.0000001, it does not make sense to reject H0 and accept H1
because this data just cannot happen if H1 is true. If we only have
these two hypotheses to choose from then it only makes sense
to accept H0 because the data is still possible under H0 (Jaynes,
2003). In fact, using Bayes’ theorem we can formally show that The Neglect of Power Reinterpreted g
p
In contrast to the importance of power in determining FRP and
TRP, NHST studies tend to ignore power and β and emphasize
α and low p-values. Often, finding a statistically significant
effect erroneously seems to override the importance of power. However, statistical significance does not protect us from false
positives. FRP can only be minimized by keeping H0:H1 odds
and bias low and power high (Pollard and Richardson, 1987;
Button et al., 2013; Bayarri et al., 2016). Hence, power is not
only important so that we increase our chances to detect true
effects but it is also crucial in keeping FRP low. While power
in principle can be adjusted easily by increasing sample size,
power in many/most fields of biomedical science and psychology
has been notoriously low and the situation has not improved
much during the past 50 years (Cohen, 1962; Sedlmeier and
Gigerenzer, 1989; Rossi, 1990; Hallahan and Rosenthal, 1996;
Button et al., 2013; Szucs and Ioannidis, 2017). Clearly, besides
making sure that research funding is not wasted, minimizing FRP
also provides very strong rationale for increasing the typically
used sample sizes in studies. the NHST machinery only computes the p-value conditioned on
H0 and it is able to provide this result even if H1 is not specified
at all. A widespread misconception flowing from the fuzzy attitude
of NHST to H1 is that rejecting H0 allows for accepting a specific
H1 (Nickerson, 2000). This is what most practicing researchers
do in practice when they reject H0 and argue for their specific H1
in turn. However, NHST only computes probabilities conditional
on H0 and it does not allow for the acceptance of either H0, a
specific H1 or a generic H1. Rather, it only allows for the rejection
of H0. Hence, if we reject H0 we will have no idea about how
well our data fits a specific H1. This cavalier attitude to H1 can
easily lead us astray even when contrasting H0 just with a single
alternative hypothesis as illustrated by the invalid inference based
on NHST logic in Table 2 (Pollard and Richardson, 1987). NHST Misleads Because It Neglects
Pre-data Probabilities Besides often being subject to conceptual confusion and
generating misleading inferences especially in the setting of
weak power, NHST has further serious problems. NHST logic is
based on the so-called modus tollens (denying the consequent)
argumentation (see footnote in Appendix 4 in Supplementary
Material): It sets up a H0 model and assumes that if the data
fits this model than the test statistic associated with the data
should not take more extreme values than a certain threshold
(Meehl, 1967; Pollard and Richardson, 1987). If the test statistic
contradicts this expectation then NHST assumes that H0 can be
rejected and consequently its complement, H1 can be accepted. While this logic may be able to minimize Type I error in well-
powered high-quality well-controlled tests (Section Neyman and
Pearson: A decision Mechanism Optimized for the Long-Run),
it is inadequate if we use it to decide about the truth of H1 in a
single experiment, because there is always space for Type I and
Type II error (Falk and Greenbaum, 1995). So, our conclusion
is never certain and the only way to see how much error we
have is to calculate the long-run FRP and TRP using appropriate
α and power levels and prior H0:H1 odds. The outcome of
the calculation can easily conflict with NHST decisions (see
Appendix 4 in Supplementary Material). Our model says that if H0 is true, it is a very rare event
that Harold is a member of congress. This rare event then
happens which is equivalent to finding a small p-value. Hence, we
conclude that H0 can be rejected and H1 is accepted (i.e., Harold
is a member of congress and therefore he is not American.). However, if we carefully explicate all probabilities it is easy to
see that we are being mislead by NHST logic. First, because we
have absolutely no idea about Harold’s nationality we can set
pre-data probabilities of both H1 and H0 to 1/2, which means
that H0:H1 odds are uninformative, 1:1. Then we can explicate
the important conditional probabilities of the data (Harold is
a member of congress) given the possible hypotheses. NHST Neglects Predictions under H1
Facilitating Sloppy Research NHST does not require us to specify exactly what data H1
would predict. Whereas, the Neyman–Pearson approach requires
researchers to specify an effect size associated with H1 and
compute power (1-β), in practice this is easy to neglect because August 2017 | Volume 11 | Article 390 Frontiers in Human Neuroscience | www.frontiersin.org 8 Null Hypothesis Significance Testing: A Reassessment Szucs and Ioannidis the probability of H0 is actually 1 (Appendix 5 in Supplementary
Material). the probability of H0 is actually 1 (Appendix 5 in Supplementary
Material). because H1 is formally undefined and NHST never tells us
anything about it. In light of these problems Jaynes (2003)
suggested that the NHST framework addresses an ill-posed
problem and provides invalid responses to questions of statistical
inference. In most real world problems multiple alternative hypotheses
compete to explain the data. However, by using NHST we can
only reject H0 and argue for some H1 without any formal
justification of why we prefer a particular hypothesis whereas it
can be argued that it only makes sense to reject any hypothesis
if another one better fits the data (Jaynes, 2003). We only have
qualitative arguments to accept a specific H1 and the exclusive
focus on H0 makes unjustified inference too easy. For example,
if we assume that H0 predicts normally distributed data with
mean 0 and standard deviation 1 then we have endless options
to pick H1 (Hubbard and Bayarri, 2003): Does H1 imply that
the data have a mean other than zero, the standard deviation
other than 1 and/or does it represent non-normally distributed
data? NHST allows us to consider any of these options implicitly
and then accept one of them post-hoc without any quantitative
justification of why we chose that particular option. Further,
merging all alternative hypotheses into a single H1 is not only
too simplistic for most real world problems but it also poses an
“inferential double standard” (Rozeboom, 1960): The procedure
pits the well-defined H0 against a potentially infinite number of
alternatives. It is noteworthy that some may argue that Jaynes’s argument
is formally invalid as the NHST approach can be used to
reject a low probability H0 in theory. NHST Neglects Predictions under H1
Facilitating Sloppy Research However, recall that (1)
NHST does not deliver final objective theoretical decisions,
there is no theoretical justification for any α thresholds marking
a boundary of informal surprise and NHST merely aims to
minimize Type I error on the long run (and in fact, Neyman
and Pearson (1933) considered their procedure a theory-free
decision mechanism and Fisher considered it a heuristic). (2)
NHST can only reject H0 (heuristically or in a theory-free
manner) and (3) cannot provide support for any H1. We could
also add that many practicing biomedical and social scientists
may not have clear quantitative predictions under H0 besides
expecting to reject a vague null effect (see Section NHST in
Sciences with and without Exact Quantitative Predictions for the
difference between sciences with and without exact predictions). Hence, their main (ultimate) objective of using NHST is often
actually not the falsification of the exact theoretical predictions
of a well-defined theory (H0). Rather, they are more interested
in arguing in favor of an alternative theory. For example,
with a bit of creativity fMRI “activation” in many different
(perhaps post-hoc defined) ROIs can easily be “explained” by
some theory when H0 (“no activation”) is rejected in any of
the ROIs. However, supporting a specific alternative theory is
just not possible in the NHST framework and in this context
Jaynes’ comment is perfectly valid: NHST provides an ill-defined
framework, after rejecting H0 real-world researchers have no
formal hypothesis test outcomes to support their “positive”
arguments. Vague H1 definitions (the lack of quantitative predictions)
enable researchers to avoid the falsification of their favorite
hypotheses by intricately redefining them (especially in fields
such as psychology and cognitive neuroscience where theoretical
constructs are often vaguely defined) and never providing any
definitive assessment of the plausibility of a favorite hypothesis
in light of credible alternatives (Meehl, 1967). This problem
is reflected in papers aiming at the mere demonstration of
often little motivated significant differences between conditions
(Giere, 1972) and post-hoc explanations of likely unexpected
but statistically significant findings. For example, neuroimaging
studies often attempt to explain why an fMRI BOLD signal
“deactivation” happened instead of a potentially more reasonable
looking “activation” (or, vice versa). Most such findings may
be the consequence of the data randomly deviating into the
wrong direction relative to zero between-condition difference. Even multiple testing correction will not help such studies as
they still rely on standard NHST just with adjusted α thresholds. Frontiers in Human Neuroscience | www.frontiersin.org NHST Neglects Predictions under H1
Facilitating Sloppy Research Similarly, patient studies often try to explain an unexpected
difference between patient and control groups (e.g., the patient
group is “better” on a measure or shows “more” or “less” brain
activation) by some kind of “compensatory mechanism.” In such
cases what happens is that “the burden of inference has been
delegated to the statistical test,” indeed, and simply because p ≤α
odd looking observations and claims are to be trusted as scientific
facts (Bakan, 1966, p. 423; Lykken, 1968). NHST Is Unsuitable for Large Datasets NHST Is Unsuitable for Large Datasets
In consequence of the recent ‘big data’ revolution access to large
databases has increased dramatically potentially increasing power
tremendously (though, large data sets with many variables are
still relatively rare in neuroscience research). However, NHST
leads to worse inference with large databases than with smaller
ones (Meehl, 1967; Khoury and Ioannidis, 2014). This is due to
how NHST tests statistics are computed, the properties of real
data and to the lack of specifying data predicted by H1 (Bruns
and Ioannidis, 2016). Most NHST studies rely on nil null hypothesis testing
(Nickerson, 2000) which means that H0 expects a true mean
difference of exactly zero between conditions with some
variation around this true zero mean. Further, NHST machinery
guarantees that we can detect any tiny irrelevant effect sizes if
sample size is large enough. This is because test statistics are
typically computed as the ratio of the relevant between condition
differences and associated variability of the data weighted by
some function of the sample size [difference/variability ×
f(sample size)]. The p-value is smaller if the test statistic is larger. Thus, the larger is the difference between conditions and/or the
smaller is variability and/or the larger is the sample size the larger Finally, paradoxically, when real life practicing researchers
achieve their “goal” and successfully reject H0 they may be left
in complete existential vacuum because during the rejection of
H0 NHST “saws offits own limb” (Jaynes, 2003; p. 524): If
we manage to reject H0 then it follows that pr(data or more
extreme data|H0) is useless because H0 is not true. Thus, we
are left with nothing to characterize the probability of our data
in the real world; we will not know pr(data|H1) for example, August 2017 | Volume 11 | Article 390 9 Null Hypothesis Significance Testing: A Reassessment Szucs and Ioannidis is the test statistic and the smaller is the p-value (see Figure 3
for examples). Consequently, by increasing sample size enough it
is guaranteed that H0 can be rejected even with miniature effect
sizes (Ziliak and McCloskey, 2008). measurement
precision
and
increased
amounts
of
data
increase the difficulties a theory must pass before it is
accepted. This is because theoretical predictions are well-
defined, numerically precise and it is also easier to control
measurements (Lykken, 1968). Hence, NHST may be used
to aim to falsify exact theoretical predictions. NHST Is Unsuitable for Large Datasets However,
as noted, groups are actually likely to differ and if sample size
increases and variability in data decreases it will become easier
and easier to reject any kind of H0 when following the NHST
approach. In fact, with precise enough measurements, large
enough sample size and repeated “falsification” attempts H0 is
guaranteed to be rejected on the long run (see Section The
Rejection of H0 is Guaranteed on the Long-Run) even if the
underlying processes generating the data in two experimental
conditions are exactly the same. Hence, ultimately any H1 can be
accepted, claiming support for any kind of theory. For example,
in an amusing demonstration Carver (1993) used Analysis of
Variance to re-analyze the data of Michaelson and Morley
(1887) who came up with a “dreaded” null finding and based
on this they suggested that the speed of light was constant
(H0) thereby providing empirical support for Einstein’s theory
of relativity. Carver (1993) found that that the speed of light
was actually not constant at p < 0.001. The catch? The effect
size as measured by Eta2 was 0.005. While some may feel
that Einstein’s theory has now been falsified, perhaps it is also
worth considering that here the statistically significant result is
essentially insignificant. This example also highlights the fact we
are not arguing against the Popperian view of scientific progress
by falsifying theories. Rather, we discuss why NHST is a very
imperfect method for this falsification (see further arguments as
well). The above issues have been demonstrated empirically and
by simulations. For example, Bakan (1966; see also Berkson,
1938; Nunnally, 1960; Meehl, 1967) subdivided the data of
60,000 persons according to completely arbitrary criteria, like
living east or west of the Mississippi river, living in the north
or south of the USA, etc. and found all tests coming up
statistically significant. Waller (2004) examined the personality
questionnaire data of 81,000 individuals to see how many
randomly chosen directional null hypotheses can be rejected. If
sample size is large enough, 50% of directional hypothesis tests
should be significant irrespective of the hypothesis. As expected,
nearly half (46%) of Waller’s (2004) results were significant. Simulations suggest that in the presence of even tiny residual
confounding (e.g., some omitted variable bias) or other bias, large
observational studies of null effects will generate results that may
be mistaken as revealing thousands of true relationships (Bruns
and Ioannidis, 2016). NHST Is Unsuitable for Large Datasets For example,
a theory may predict that a quantity should be let’s say 8
and the experimental setup can assure that really only very
few factors influence measurements—these factors can then
be taken into account during analysis. Hence, increased
measurement precision will make it easier to demonstrate
a departure from numerically exact predictions. So, a “five
sigma” deviation rule may make good sense in physics
where precise models are giving precise predictions about
variables. Parameters of many real data sets are much more likely to
differ than to be the same for reasons completely unrelated
to our hypotheses (Meehl, 1967, 1990; Edwards, 1972). First,
many psychological, social and biomedical phenomena are
extremely complex reflecting the contribution of very large
numbers of interacting (latent) factors, let it be at the level of
society, personality or heavily networked brain function or other
biological networks (Lykken, 1968; Gelman, 2015). Hence, if
we select any two variables related to these complex networks
most probably there will be some kind of at least remote
connection between them. This phenomenon is called “crud
factor” Meehl (1990) or “ambient correlational noise” (Lykken,
1968) and it is unlikely to reflect a causal relationship. In fact
some types of variables, such as intake of various nutrients and
other environmental exposures are very frequently correlated
among themselves and with various disease outcomes without
this meaning that they have anything to do with causing
disease outcomes (Patel and Ioannidis, 2014a,b). Second, unlike
in physical sciences it is near impossible to control for the
relationship of all irrelevant variables which are correlated with
the variable(s) of interest (Rozeboom, 1960; Lykken, 1968). Consequently, there can easily be a small effect linking two
randomly picked variables even if their statistical connection
merely communicates that they are part of a vast complex
interconnected network of variables. Only a few of these tiny
effects are likely to be causal and of any portend (Siontis and
Ioannidis, 2011). In sciences using NHST without clear numerical predictions
the situation is the opposite of the above, because NHST
does not demand the exact specification of H1, so theories
typically only predict a fairly vague “difference” between groups
or experimental conditions rather than an exact numerical
discrepancy between measures of groups or conditions. NHST Is Unsuitable for Large Datasets Experimental studies may also suffer the
same problem, if they have even minimal biases. A typical defense of NHST may be that we actually may
not want to increase power endlessly, just as much as we
still think that it allows us to detect reasonable effect sizes
(Giere, 1972). For example, equivalence testing may be used
to reject the hypothesis that a meaningfully large effect exist
(e.g., Wellek, 2010) or researchers may check for the sign
of expected effects (Gelman and Tuerlinckx, 2000). However,
because typically only statistically significant data is published,
published studies most probably exaggerate effect sizes. So,
estimating true (expected) effect sizes is very difficult. A more
reasoned approach may be to consider explicitly what the NHST May Foster Selective Reporting and
Subjectivity Because NHST never evaluates H1 formally and it is fairly
biased toward the rejection of H0, reporting bias against
H0 can easily infiltrate the literature even if formal NHST
parameters are fixed (see Section Serious Underestimation of
the Proportion of False Positive Findings in NHST about the
“u” bias parameter). Overall, a long series of exploratory tools
and questionable research practices are utilized in search for
statistical significance (Ioannidis and Trikalinos, 2007; John
et al., 2012). Researchers can influence their data during
undocumented analysis and pre-processing steps and by the
mere choice of structuring the data (constituting researcher
degrees of freedom; Simmons et al., 2011). This is particularly a
problem in neuroimaging where the complexity and idiosyncrasy
of analyses is such that it is usually impossible to replicate exactly
what happened and why during data analysis (Kriegeskorte
et al., 2009; Vul et al., 2009; Carp, 2012). Another term that
has been used to describe the impact of diverse analytical
choices is “vibration of effects” (Ioannidis, 2008). Different
analytical options, e.g., choice of adjusting covariates in a
regression model can result in a cloud of results, instead of
a single result, and this may entice investigators to select a
specific result that is formally significant, while most analytical
options would give non-significant results or even results
with effects in the opposite direction (“Janus effect”; Patel
et al., 2015). Another common mechanism that may generate
biased results with NHST is when investigators continue data
collection and re-analyse the accumulated data sequentially
without accounting for the penalty induced by this repeated
testing (DeMets and Lan, 1994; Goodman, 1999; Szucs, 2016). The unplanned testing is usually undocumented and researchers
may not even be conscious that it exposes them to Type
I error accumulation. Bias may be the key explanation why
in most biomedical and social science disciplines, the vast
majority of published papers with empirical data report
statistically significant results (Kavvoura et al., 2007; Fanelli,
2010; Chavalarias et al., 1990-2015). Overall, it is important to
see that NHST can easily be infiltrated by several undocumented
subjective decisions. NHST in Sciences with and without Exact
Quantitative Predictions Due to the combination of the above properties of real-world
data sets and statistical machinery theory testing radically
differs in sciences with exact and non-exact quantitative
predictions (Meehl, 1967). In physical sciences increased August 2017 | Volume 11 | Article 390 Frontiers in Human Neuroscience | www.frontiersin.org 10 Null Hypothesis Significance Testing: A Reassessment Szucs and Ioannidis the larger are H0:H1 odds and bias and the larger is the long-
run FRP. For example, in a large laboratory with 20 post-docs
and PhD students, each person running 5 experiments a year
implementing 10 significance tests in each experiment we can
expect 20 × 5 × 10 × 0.05 = 50 [usually publishable] false
results a year at α = 0.05 if H0 is true. Coupled with the fact
that a large number of unplanned tests may be run in each
study (Simmons et al., 2011; Gelman and Loken, 2014) and that
negative results and failed replications are often not published,
this leads to “unchallenged fallacies” clogging up the research
literature (Ioannidis, 2012; p1; Sterling, 1959; Bakan, 1966;
Sterling et al., 1995). Moreover, such published false positive true
H0 studies will also inevitably overestimate the effect size of the
non-existent effects or of existent, but unimportantly tiny, effects
(Schmidt, 1992, 1996; Sterling et al., 1995; Ioannidis, 2008). These effects may even be confirmed by meta-analyses, because
meta-analyses typically are not able to incorporate unpublished
negative results (Sterling et al., 1995) and they cannot correct
many of the biases that have infiltrated the primary studies. For example, such biases may result in substantial exaggeration
of measured effect sizes in meta-analyses (see e.g., Szucs and
Ioannidis, 2017). consequences (“costs”) are of a false-positive, true-positive, false-
negative, and true-negative result. Explicit modeling can suggest
that the optimal combination of Type 1 error and power may
need to be different depending on what these assumed costs are
(Djulbegovic et al., 2014). Different fields may need to operate
at different optimal ratios of false-positives to false-negatives
(Ioannidis et al., 2011). NHST May Foster Selective Reporting and
Subjectivity (Bayesian methods are often blamed
such subjectivity, see Section Teach Alternative Approaches
Seriously.) Given that the predictions of H1 are rarely precise and
that theoretical constructs in many scientific fields (including
psychology and cognitive neuroscience) are often poorly defined
(Pashler and Harris, 2012), it is easy to claim support for a
popular theory with many kinds of data falsifying H0 even if the
constructs measured in many papers are just very weakly linked
to the original paper, or not linked at all. Overall, the literature
may soon give the impression of a steady stream of replications
throughout many years. Even when “negative” results appear,
citation bias may still continue to distort the literature and the
prevailing theory may continue to be based on the “positive”
results. Hence, citation bias may maintain prevailing theories
even when they are clearly false and unfounded (Greenberg,
2009). NHST Does Not Facilitate Systematic
Knowledge Integration Due to high FRP the contemporary research literature provides
statistically
significant
“evidence”
for
nearly
everything
(Schoenfeld and Ioannidis, 2012). Because NHST emphasizes
all or none p-value based decisions rather than the magnitude
of effects, often only p-values are reported for critical tests,
effect size reports are often missing and interval estimates
and confidence intervals are not reported. In an assessment
of the entire biomedical literature in 1990–2015, 96% of the
papers that used abstracts reported at least some p-value below
0.05, while only 4% of a random sample of papers presented
consistently effect sizes with confidence intervals (Chavalarias
et al., 1990-2015). However, oddly enough, the main NHST
“measure of evidence,” the p-value cannot be compared across
studies. It is a frequent misconception that a lower p-value
always means stronger evidence irrespective of the sample size
and effect size (Oakes, 1986; Schmidt, 1996; Nickerson, 2000). Besides the non-comparable p-values, NHST does not offer any The Rejection of H0 Is Guaranteed on the
Long-Run If H0 is true, with α = 0.05, 5% of our tests will be statistically
significant on the long-run. The riskier experiments we run, August 2017 | Volume 11 | Article 390 Frontiers in Human Neuroscience | www.frontiersin.org 11 Null Hypothesis Significance Testing: A Reassessment Szucs and Ioannidis formal mechanism for systematic knowledge accumulation and
integration (Schmidt, 1996) unlike Bayesian methods which can
take such pre-study information into account. Hence, we end
up with many fragmented studies which are most often unable
to say anything formal about their favorite H1s (accepted in
a qualitative manner). Methods do exist for the meta-analysis
of p-values (see e.g., Cooper et al., 2009) and these are still
used in some fields. However, practically such meta-analyses
still say nothing about the magnitude of the effect size of the
phenomenon being addressed. These methods are potentially
acceptable when the question is whether there is any non-null
signal among multiple studies that have been performed, e.g., in
some types of genetic associations where it is taken for granted
that the effect sizes are likely to be small anyhow (Evangelou and
Ioannidis, 2013). FWER control is a conservative procedure in keeping Type
I error rate low but it also sacrifices power increasing Type II
error. An alternative to FWER control is False Discovery Rate
(FDR) control which allows more Type I errors but assures higher
power. Using the same logic as the computation of FRP discussed
before, FDR control considers the estimated proportion of false
positive statistically significant findings amongst all statistically
significant findings (i.e., the proportion of erroneously rejected
null hypotheses out of all rejected null hypotheses; Benjamini and
Hochberg, 1995). FDR computation is illustrated by Table 3. If
we run M hypothesis tests then a certain number of them are
likely to test true null effects (M0 in Table 3) and some other
number of them are likely to test non-null effects with true
alternative hypotheses (M1 in Table 3). Depending on our α level
and power (1-β), a certain number of the M0 and M1 tests will
reject the null hypothesis (FP and TN, respectively, see Table 3
for abbreviations) while some other number of them will not
reject the null hypothesis (TN and FN, respectively). Family-Wise Error Rate (FWER) and FDR
Correction in NHST An increasingly important problem is that with the advent of
large data sets researchers can use NHST to test multiple, related
hypotheses. For example, this problem routinely appears in
neuro-imaging where a large amount of non-independent data
points are collected and then the same hypothesis test may be
run on tens of thousands of observations, for example, from
a brain volume, or from 256 electrodes placed on the scalp,
each electrode recording voltage 500 times a second. Analysis
procedures that generate different views of data (e.g., time-
frequency or independent component analyses) may further
boost the amount of tests to be run. Of course, in real research settings we do not know how
many of our tests test true null effects and we only know how
many tests we run and how many of them return statistically
significant and non-significant results. So Q can be considered
a random variable. However, as Q cannot be controlled directly
FDR is defined as the expected value of the proportion of false
positive errors: FDR = E[FP/R|R > 0] · pr(R > 0), a variable
which can be controlled (see Benjamini and Hochberg, 1995;
Curran-Everett, 2000; Nichols and Hayasaka, 2003; Bennett et al.,
2009; Benjamini, 2010; Goeman and Solari, 2014). Some FDR
estimation procedures can also factor in dependency between
tests (Benjamini and Yekutieli, 2001). Regarding these multiple testing situations, a group of
statistical tests which are somehow related to each other can
be defined as a “family of comparisons.” The probability that a
family of comparisons contains at least one false positive error
is called the family wise error rate (FWER). If the repeated
tests concern independent data sets where H0 is true than the
probability of having at least one Type I error in k independent
tests, each with significance level α, is αTOTAL = 1 - (1 - α)k. For example if k = 1, 2, 3, 4, 5, and 10 than αTOTAL is 5, 9.75,
14.26, 18.55, 22.62, and 40.13%, respectively (see Curran-Everett,
2000; Szucs, 2016 for graphical illustrations and simulations for
non-independent data). j
In contrast to FDR, using the notation in Table 3, FWER
can be expressed as FWER = pr(FP≥1) = 1 −pr(FP=0) that
is, the probability that there is at least one false positive Type
I error in a family of observations. The Rejection of H0 Is Guaranteed on the
Long-Run If we know
the exact numbers in Table 3 then the proportion of false positive
statistically significant findings can be computed as the ratio of
false positive results to all statistically significant results: Q =
FP/(FP + TP) = FP/R (assuming that R̸= 0). Frontiers in Human Neuroscience | www.frontiersin.org Family-Wise Error Rate (FWER) and FDR
Correction in NHST That is, 42.86% of statistically significant results will be false positives. FDR = Q · pr(R > 0) = Q · [α · (M0/M) + Power · (M1/M)] = 0.045. If we only test true null effects then Q
= false positives/all significant results = α·M / α·M = 1; and FDR = Q · pr(R>0) = Q · α = α = FWER [pr(R>0) = α because all significant results are coming from true null situations]. Note that in real research only R, M, and M-R are known whereas M0, M1, and Q are not known. H0 (leftmost column) stands for the null hypothesis. The proportion of false positive statistically significant test outcomes to all statistically significant test outcomes is Q = FP/(FP + TP)
= FP/R (R̸= 0). The numbers give an example for the case when α = 0.05 (so, FWER = α = 0.05) and β =0.4, so Power = 1 −β = 0.6. In the example we run 1,000 null hypothesis
tests. We test 9 times as many true null situations than situations with true alternative hypotheses (that is, every 10th of our experimental ideas are correct). In this case Q = 45/105 =
0.4286. That is, 42.86% of statistically significant results will be false positives. FDR = Q · pr(R > 0) = Q · [α · (M0/M) + Power · (M1/M)] = 0.045. If we only test true null effects then Q
= false positives/all significant results = α·M / α·M = 1; and FDR = Q · pr(R>0) = Q · α = α = FWER [pr(R>0) = α because all significant results are coming from true null situations]. Note that in real research only R, M, and M-R are known whereas M0, M1, and Q are not known. findings when a single hypothesis is tested simultaneously in
many data points (e.g., voxels) but do nothing to protect
against many of the other problems discussed in this paper
(e.g., generating a high amount of false positives across the
literature; being sensitive to undocumented biasing procedures;
neglecting predictions under H1; not providing probability
statements for H1; neglecting pre-data probabilities; being unable
to effectively integrate study results). These problems are valid
even when just one single NHST test is run. Family-Wise Error Rate (FWER) and FDR
Correction in NHST If the null hypothesis is
true in all tests we run then FDR = FWER while if there are
situations with true alternative hypotheses then FDR < FWER
(see Table 3 for example). Also, various other FDR and FWER
measures can be derived (see the above cited reviews). It can be
argued that controlling FDR is more useful in research where a
very large number of tests are carried out routinely, like neuro-
imaging or genetics but less useful in behavioral psychological
and social science research where fewer hypotheses may be
tested at any one time and accepting any single hypothesis
as statistically significant may have large impact on inferences
(Gelman et al., 2012). This last statement is also true for
behavioral data used to support the interpretation of neuro-
imaging findings. There are numerous procedures which can take multiple
testing into account by correcting p-values. The simplest of these
procedures is Bonferroni correction which computes an adjusted
p-value threshold as α/n where α is the statistical significance
threshold for a single test and n is the number of tests run. Hence,
if we run 5 tests which can be defined as a family of tests and our
original α is 0.05 then the Bonferroni corrected adjusted α level
is 0.05/5 = 0.01. Any p-values above this threshold should not be
considered to demonstrate statistically significant effects. Besides
the Bonferroni correction there are other alternative methods of
FWER correction, like the Tukey Honestly Significant Difference
test, the Scheffe test, Holm’s method, Sidak’s method; Hochberg’s
method, etc. Some of these corrections also take the dependency
(non-independence) of tests into account (see e.g., Shaffer, 1995;
Nichols and Hayasaka, 2003 for review). Most relevant to our paper, both FWER and FDR error
rate corrections are based on the same NHST procedure. That is, they do not modify the procedure in any ways
other than aiming to decrease Type I error toward initially
expected levels when multiple NHST tests are run. That is,
these methods can help in constraining the number of random Frontiers in Human Neuroscience | www.frontiersin.org August 2017 | Volume 11 | Article 390 12 Null Hypothesis Significance Testing: A Reassessment Szucs and Ioannidis TABLE 3 | Illustrating the logic behind FDR computation. Family-Wise Error Rate (FWER) and FDR
Correction in NHST In addition,
empirical analyses of large fMRI data sets found that the most
popular fMRI analysis software packages implemented erroneous
multiple testing corrections and hence, generate much higher
levels of false positive results than expected (Eklund et al.,
2012, 2016). This casts doubts on a substantial part of the
published fMRI literature. Further, Carp (2012) reported that
about 40% of 241 relatively recent fMRI papers actually did
not report having used multiple testing correction. So, a very
high percentage of fMRI literature may have been exposed to
high false positive rates either multiple correction was used
or not (see also (Szucs and Ioannidis, 2017) on statistical
power). to study model response behavior. This may offer a way to
judge the reasonableness of analyses offering richer information
than NHST accept/reject decisions (Gelman et al., 2014). The
challenge of course is the development of models. However,
below we argue that efficient model development can only
happen if we refocus our efforts on understanding data patters
from the testing of often very vaguely defined hypotheses. In addition, Bayesian methods are also able to formally
aggregate data from many experiments (e.g., adding data serially
and by hierarchical models; Gelman et al., 2014). This can
further maximize large-scale joint efforts for better model
specifications. Family-Wise Error Rate (FWER) and FDR
Correction in NHST Null hypothesis is true
Alternative hypothesis is true
Sums
H0 is rejected
(statistically significant outcome)
FP = False Positives
45 (if α = 0.05; 900·0.05 = 45)
TP = True Positives
60 (if power = 0.6; 100·0.6 = 60)
R
105
H0 is not rejected
(statistically non-significant outcome)
TN = True Negatives
855 (if α = 0.05; 900·0.95 = 855)
FN = False Negatives
40 (if β = 0.4; 100·0.4 = 40)
M – R
895
Sums
M0
900
M1
100
M
1000
H0 (leftmost column) stands for the null hypothesis. The proportion of false positive statistically significant test outcomes to all statistically significant test outcomes is Q = FP/(FP + TP)
= FP/R (R̸= 0). The numbers give an example for the case when α = 0.05 (so, FWER = α = 0.05) and β =0.4, so Power = 1 −β = 0.6. In the example we run 1,000 null hypothesis
tests. We test 9 times as many true null situations than situations with true alternative hypotheses (that is, every 10th of our experimental ideas are correct). In this case Q = 45/105 =
0.4286. That is, 42.86% of statistically significant results will be false positives. FDR = Q · pr(R > 0) = Q · [α · (M0/M) + Power · (M1/M)] = 0.045. If we only test true null effects then Q
= false positives/all significant results = α·M / α·M = 1; and FDR = Q · pr(R>0) = Q · α = α = FWER [pr(R>0) = α because all significant results are coming from true null situations]. Note that in real research only R, M, and M-R are known whereas M0, M1, and Q are not known. TABLE 3 | Illustrating the logic behind FDR computation. H0 (leftmost column) stands for the null hypothesis. The proportion of false positive statistically significant test outcomes to all statistically significant test outcomes is Q = FP/(FP + TP)
= FP/R (R̸= 0). The numbers give an example for the case when α = 0.05 (so, FWER = α = 0.05) and β =0.4, so Power = 1 −β = 0.6. In the example we run 1,000 null hypothesis
tests. We test 9 times as many true null situations than situations with true alternative hypotheses (that is, every 10th of our experimental ideas are correct). In this case Q = 45/105 =
0.4286. THE STATE OF THE ART MUST CHANGE NHST Is Unsuitable as the Cornerstone of
Scientific Inquiry in Most Fields Frontiers in Human Neuroscience | www.frontiersin.org When and How to Use NHST The currently dominant, NHST influenced approach is that
instead of understanding raw data researchers often just focus
on the all or nothing rejection of a vaguely defined H0 and shift
their attention to interpreting brain “activations” revealed by
potentially highly misleading statistical parameter maps. Based
on these maps then strong (qualitative) claims may be made
about alternative theories whose support may in fact never be
tested. So, current approaches seem to reward exuberant theory
building based on small and underpowered studies (Szucs and
Ioannidis, 2017) much more than meticulous data collection and
understanding and modeling extensive raw data patterns. For
analogy, in astronomy theories typically built on thousands of
years of sky observation data open to everyone. For example,
Kepler could identify the correct laws of planetary motion
because he had access to the large volume of observational
data accumulated by Tycho Brache who devoted decades of
his life to much more precise data collection than previously
done. Similarly, the crucial tests of Einstein’s theories were
precise predictions about data which could be verified or falsified
(Smolin, 2006; Chaisson and McMillan, 2017). Importantly, we do not want to ban NHST (Hunter, 1997),
we realize that it may be reasonable to use it in some well-
justified cases. In all cases when NHST is used its use must be
justified clearly rather than used as an automatic default and
single cornerstone procedure. On the one hand, NHST can be
used when very precise quantitative theoretical predictions can
be tested, hence, both power and effect size can be estimated
well as intended by Neyman and Pearson (1933). On the other
hand, when theoretical predictions are not precise, reasonably
powered NHST tests may be used as an initial heuristic look at
the data as Fisher (1925) intended. However, in these cases (when
well-justified theoretical predictions are lacking) if studies are not
pre-registered (see below) NHST tests can only be considered
preliminary (exploratory) heuristics. Hence, their findings should
only be taken seriously if they are replicated, optimally within the
same paper (Nosek et al., 2013). These replications must be well
powered to keep FRP low. As discussed, NHST can only reject
H0 and can accept neither a generic or specific H1. So, on its own
NHST cannot provide evidence “for” something even if findings
are replicated. NHST Is Unsuitable as the Cornerstone of
Scientific Inquiry in Most Fields q
y
In summary, NHST provides the illusion of certainty through
supposedly ‘objective’ binary accept/reject decisions (Cohen,
1994; Ioannidis, 2012) based on practically not very useful p-
values (Bakan, 1966). However, researchers usually never give any
formal assessment of how well their theory (a specific H1) fits the
facts and, instead of gradual model building (Gigerenzer, 1998)
and comparing the plausibility of theories, they can get away
with destroying a strawman: they disprove an H0 (which happens
inevitably sooner or later) with a machinery biased to disproving
it without ever going into much detail about the exact behavior of
variables under exactly specified hypotheses (Kranz, 1999; Jaynes,
2003). NHST also does not allow for systematic knowledge
accumulation. In addition, both because of its shortcomings and
because it is subject to major misunderstandings it facilitates
the production of non-replicable false positive reports. Such
reports ultimately erode scientific credibility and result in wasting
perhaps most of the research funding in some areas (Ioannidis,
2005; Macleod et al., 2014; Kaplan and Irvin, 2015; Nosek et al.,
2015). p
In the NHST framework the multiple comparison problem
is exacerbated by the fact that we may test a very large
number of precise null hypotheses (Neath and Cavanaugh,
2006), often without much theoretical justification (e.g., in many
explorative whole brain analyses). However, as the H0:H1 odds
may be high the NHST mechanism may produce a very large
number of falsely significant results. Similarly high numbers
of false alarms are produced under realistic conditions even if
some rudimentary model is used for the data (e.g., expecting
positive or negative difference between conditions; Gelman
and Tuerlinckx, 2000). In contrast, while currently there is
no standard way to correct for multiple comparisons with
Bayesian methods, Bayesian methods have been shown to be
more conservative than NHST in some situations (Gelman
and Tuerlinckx, 2000) and they offer various methods for
correcting for multiple comparisons (e.g., Westfall et al., 1997;
Gelman et al., 2014). In addition, Bayesian methods are
strongly intertwined with explicit model specifications. These
models can then be used to generate simulated data and NHST seems to dominate biomedical research for various
reasons. First, it allows for the easy production of a large
number of publishable papers (irrespective of their truth value)
providing a response to publication pressure. NHST Is Unsuitable as the Cornerstone of
Scientific Inquiry in Most Fields Second, NHST August 2017 | Volume 11 | Article 390 Frontiers in Human Neuroscience | www.frontiersin.org 13 Null Hypothesis Significance Testing: A Reassessment Szucs and Ioannidis seems deceptively simple: because the burden of inference
(Bakan, 1966) has been delegated to the significance test all too
often researchers’ statistical world view is narrowed to checking
an inequality: is p ≤0.05 (Cohen, 1994)? After passing this test,
an observation can become a “scientific fact” contradicting the
random nature of statistical inference (Gelman, 2015). Third, in
biomedical and social science NHST is often falsely perceived as
the single objective approach to scientific inference (Gigerenzer
et al., 1989) and alternatives are simply not taught and/or
understood. their variables, make model predictions and then confirm these
with likelihood-based and/or Bayesian methods. Making such
predictions would probably require intimate familiarity with a lot
of raw data. seems deceptively simple: because the burden of inference
(Bakan, 1966) has been delegated to the significance test all too
often researchers’ statistical world view is narrowed to checking
an inequality: is p ≤0.05 (Cohen, 1994)? After passing this test,
an observation can become a “scientific fact” contradicting the
random nature of statistical inference (Gelman, 2015). Third, in
biomedical and social science NHST is often falsely perceived as
the single objective approach to scientific inference (Gigerenzer
et al., 1989) and alternatives are simply not taught and/or
understood. Ways to Change In most biomedical, neuroscience, psychology, and social science
fields currently popular analysis methods are based on NHST. It is clear that analysis software and researcher knowledge
cannot be changed overnight. Below we summarize some further
recommendations which we think can minimize the negative
features of NHST even if it continues to be dominant for a while. A very important practical goal would be to change the incentive
structure of biomedical and social science to bring it in line
with these and similar other recommendations (Wagenmakers
et al., 2011; Begley and Ellis, 2012; Nosek et al., 2013; Stodden
et al., 2016). Also note that we are not arguing against statistical
inference which we consider the “logic of science” (Jaynes, 2003;
p. xxii.), quantitative and well justified statistical inference should
be at the core of the scientific enterprise. We have now decades of negative experience with NHST
which gradually achieved dominance in biomedical and social
science since the 1930s (Gigerenzer et al., 1989). Critique of
NHST started not much later (Jeffreys, 1939, 1948, 1961) and
has been forcefully present since then (Jeffreys, 1939, 1948, 1961;
Eysenck, 1960; Nunnally, 1960; Rozeboom, 1960; Clark, 1963;
Bakan, 1966; Meehl, 1967; Lykken, 1968) and continues to-date
(Wasserstein and Lazar, 2016). The problems are numerous, and
as Edwards (1972, p. 179) concluded 44 years ago: “any method
which invites the contemplation of a null hypothesis is open to grave
misuse, or even abuse.” Time has proven this statement and that
problems are unlikely to go away. We suggest that that it is really
time for change now. Frontiers in Human Neuroscience | www.frontiersin.org When and How to Use NHST Overall, if we just consider competing “theories” without ever
deeply considering extensive raw data patterns it is unlikely that
major robust scientific breakthroughs will be done whereas many
different plausible looking theories can be promoted. Imagine,
for example, a situation where astronomers would have only
published the outcomes of their NHST tests, some rejecting that
the sun is in the middle of the universe while others rejecting
that the earth is in the middle of the universe while publishing
no actual raw data. Meta-analyses of published effect sizes would
have confirmed both positions as both camps would have only
published test statistics which passed the statistical significance
threshold. Luckily, real astronomers recorded a lot of data and
derived testable theories with precise predictions. For example, if initially researches do not know where to
expect experimental effects in a particular experimental task, they
could run a whole brain, multiple-testing corrected search for
statistical significance in a group of participants. Such a search
would provide heuristic evidence if they identify some brain areas
reacting to manipulations. In order to confirm these effects they
would need to carefully study for example the BOLD signal or
EEG amplitude changes in areas or over electrodes of interest,
make predictions about the behavior of these variables, replicate
measurements and minimally confirm the previous NHST results
before the findings can be taken seriously. Much better, if
researchers can also provide some model for the behavior of August 2017 | Volume 11 | Article 390 14 Null Hypothesis Significance Testing: A Reassessment Szucs and Ioannidis In basic biomedical and psychology research we often cannot
provide very well worked out hypotheses and even a simple
directional hypothesis may seem particularly enlightening. Such
rudimentary state of knowledge can be respected. However,
in such pre-hypothesis stage substantively blind all or nothing
accept/reject decisions may be unhelpful and may maintain
our ignorance rather than facilitate organizing new information
into proper quantitative scientific models. It is much more
meaningful to focus on assessing the magnitude of effects along
with estimates of uncertainty, let these be error terms, confidence
intervals or Bayesian credible intervals (Edwards, 1972; Luce,
1988; Schmidt, 1996; Jaynes, 2003; Gelman, 2013a,b; see Morey
et al., 2016 on the difference between classical confidence
intervals and Bayesian credible intervals). These provide more
direct information on the actual “empirical” behavior of our
variables and/or the precision of interval estimation. Publish All Analysis Scripts with Analysis Settings Publish All Analysis Scripts with Analysis Settings
Another cost-free improvement is to publish all analysis scripts
with the ability to regenerate all figures and tables (Laine
et al., 2007; Peng, 2009, 2011; Diggle and Zeger, 2010; Keiding,
2010; Doshi et al., 2013). This does not require large storage
space and can also be done in Supplementary Material. If
researchers keep this expectation in mind from the start of a
project then implementing it becomes relatively straightforward. Program code will often provide information which is missing
from papers. With regard to missing analysis information it is
important to be conscious of the fact that seemingly innocuous
and irrelevant analysis settings (e.g., slightly changing initial
filtering parameters) can have major impact on final statistical
outcomes at the end of a complicated processing pipeline. For
example, modified initial settings may change the statistically
significant/non-significant status of final important test statistics. This can be an issue if multiple settings can be justified and/or
if some settings leading to significant outcomes are actually less
justified than alternative settings. Publishing scripts will also
provide more information on potential statistical errors (Bakker
and Wicherts, 2001; Nuijten et al., 2016). The change of emphasis suggested here would require that
instead of p-values and reporting the outcomes of all/nothing
hypothesis tests studies should focus on reporting data in
original units of measurement as well as providing derived
effect sizes. It is important to publish data summaries (means,
standard errors, nowadays extremely rarely plotted empirical
data distributions) in original units of measurement as derived
measures may be highly biased by some (undocumented)
analysis techniques. If we have clear and pre-registered
hypotheses then it is relatively straightforward to publish raw
data summaries (e.g., mean BOLD signal or ERP amplitude
change with standard errors) related to those hypotheses. Clear data presentation usually gets difficult when there are
lots of incidental findings. Usually unlimited amount of data
summaries can now be published cost free in Supplementary
Materials. August 2017 | Volume 11 | Article 390 Frontiers in Human Neuroscience | www.frontiersin.org When and How to Use NHST Gaining
enough experience with interval estimates and assuring their
robustness by building replication into design (Nosek et al., 2013)
may then allow us to describe the behavior of variables by more
and more precise scientific models which may provide more clear
predictions (Schmidt, 1996; Jaynes, 2003; Gelman, 2013a,b). by high-dimensional neuroscience data (Kriegeskorte et al., 2009;
Vul et al., 2009; Carp, 2012) pre-registration seems a necessary
pre-condition of robust hypothesis driven neuroscience research. Pre-registration would likely help to cleanse non-replicable
“unchallenged fallacies” (Ioannidis, 2012) from the literature. For
example, Kaplan and Irvin (2015) found that pre-registering the
primary hypotheses of clinical studies decreased the proportion
of positive findings from 57% (17 of 30 studies) to 8% (2 out of 25
studies). Hence, another benefit of pre-registration would be to
decrease publication volumes. This would require changing the
incentive system motivating scientists (Nosek et al., 2013). It is to note that honesty regarding pre-registration and
challenging questionable research practices (Simmons et al.,
2011) is the shared responsibility of all co-authors. Some fields
in medical research have already over 10 years of experience
with pre-registration. This experience shows that pre-registration
needs to be thorough to be reliable. For example, many
observational studies claimed to have been registered but closer
scrutiny shows that registration has actually happened after the
study/analysis was done (Boccia et al., 2016). In other cases,
clinical trials may be seemingly properly pre-registered before
they start recruiting patients, but analyses and outcomes were
still manipulated after registration (Ioannidis et al., 2017). Hence,
proper safeguards should be put in place to ensure that scientists
are accountable for any misconduct regarding breaching pre-
registration rules. The above problem does not only concern perceived “soft
areas” of science where measurement, predictions, control and
quantification are thought to be less rigorous than in “hard”
areas (Meehl, 1978). In many fields, for example, in cognitive
neuroscience, the measurement methods may be “hard” but
theoretical predictions and analysis often may be just as “soft”
as in any area of “soft” psychology: Using a state of the art fMRI
scanner for data collection and novel but extremely complicated
and often not well understood analysis paths will not make a
badly defined theory well-defined. Publish Data (Summaries) Irrespective of Statistical
Significance, Promote Building Good Quality
Datasets Including Large Replication Studies It is important to publish data summaries and/or data sets,
including the ones not resulting in statistically significant
findings. Without these datasets true effect sizes simply cannot
be determined. This will require that these datasets become
citeable so that their authors can be rewarded if data is used
for secondary analyses. Considering for example the above
mentioned case of Tycho Brache it is clear that his data
collection exercise was a necessary precondition of crowning
the Copernican/Newtonian revolution of astronomy (Chaisson
and McMillan, 2017). Hence, we should be able to reward
the mere collection of large volumes of good quality data:
such activity can prove to be an immense service to the
whole profession. Initiatives, like registered multi-lab replication
studies should also be prioritized when the validity of important
proposals is at stake. Funders are currently often reluctant
to fund such studies. However, they should realize that the
continuous seeking of new results and theories may just waste
most of their resources (Ioannidis et al., 2014; Kaplan and Irvin,
2015). All too often statistical understanding is perceived as
something external to the subject matter of substantive research. However, it is important to see that statistical understanding
influences most decisions about substantive questions, because
it underlies the thinking of researchers even if this remains
implicit. While common sense “statistics” may be able to
cope with simple situations, common sense is not enough to
decipher scientific puzzles involving dozens, hundreds, or even
thousands of interrelated variables. In such cases well justified
applications of probability theory are necessary (Jaynes, 2003). Hence, instead of delegating their judgment to “automatized”
but ultimately spurious decision mechanisms, researchers should
have confidence in their own informed judgment when they make
an inference. Such confidence requires deep study. y
Understanding probability is difficult. Common sense is
notoriously weak in understanding phenomena based on
probabilities (Gigerenzer et al., 2005). We cannot assume that
without proper training biomedical and social science graduates
would get miraculously enlightened about probability. Pre-registration It is to note that in-house analysis scripts may provide
substantial competitive advantage to researchers who are able
to programme these. Hence, the unconditional release of these
scripts may deprive researchers from an important competitive
asset. In such cases in house scripts could be documented in
brief methods papers which could be cited when the relevant
scripts are used so that researchers benefit from citations. Perhaps
specialized methods repository journals could be set up for
this purpose. For some recent recommendations for improving
computational reproducibility practices see Stodden et al. (2016). In our view one of the most important and virtually cost-
free (to researchers) improvement would be to pre-register
hypotheses and analysis parameters and approaches (in line with
Section 5.2 in Nichols et al., 2016; p11; Gelman and Loken,
2014). Pre-registration can easily be done for example, at the
website of the Open Science Foundation (osf.org), also in a
manner that it does not immediately become public. Hence,
competitors will not be able to scoop good ideas before the study
is published. Considering the extreme analysis flexibility offered August 2017 | Volume 11 | Article 390 August 2017 | Volume 11 | Article 390 Frontiers in Human Neuroscience | www.frontiersin.org 15 Null Hypothesis Significance Testing: A Reassessment Szucs and Ioannidis Publish Data (Summaries) Irrespective of Statistical
Significance, Promote Building Good Quality
Datasets Including Large Replication Studies Some
of the best symbolic thinking minds of humanity devoted
hundreds of years to the proper understanding of probability
and statisticians still do not agree on how best to draw statistical
inference (Stigler, 1986; Gigerenzer et al., 1989), e.g., the
recent American Statistical Association statement on p-values
(Wasserstein and Lazar, 2016) was accompanied by 21 editorials
from the statisticians and methodologists who participated
in crafting it and who disagreed in different aspects among
themselves. Publish Raw Data In an ideal world researchers should publish all raw data. This
is easy with small volumes of behavioral data but it has serious
monetary and time investment costs with large neural data
volumes (see also Nichols et al., 2017). Some repositories have
already been set up and it is important that funders cover
these costs and optimally provide infrastructure (see Pernet
and Poline, 2015; Nichols et al., 2017). Incentives such as a
badge system may help promote availability of more raw data
(Nosek et al., 2015). In our opinion it is important to publish
unprocessed raw data because processed data may already have
been distorted/biased in undocumented ways. In general, it is
more and more usual to reanalyse data from large repositories,
so much further development can be expected in this area (e.g.,
Eklund et al., 2012). A core problem seems to be that the statistical subject knowledge
of many researchers in biomedical and social science has been
shown to be poor (Oakes, 1986; Gliner et al., 2002; Castro Sotos
et al., 2007, 2009; Wilkerson and Olson, 2010; Hoekstra et al.,
2014). NHST perfectly fits with poor understanding because of
the perceived simplicity of interpreting its outcome: is p ≤0.05
(Cohen, 1994)? We suggest that the weak statistical understanding is probably
due to inadequate “statistics lite” education. This approach
does not build up appropriate mathematical fundamentals
and does not provide scientifically rigorous introduction into
statistics. Hence, students’ knowledge may remain imprecise,
patchy, and prone to serious misunderstandings. What this
approach achieves, however, is providing students with false
confidence of being able to use inferential tools whereas
they usually only interpret the p-value provided by black box
statistical software. While this educational problem remains
unaddressed, poor statistical practices will prevail regardless of
what procedures and measures may be favored and/or banned by
editorials. Frontiers in Human Neuroscience | www.frontiersin.org August 2017 | Volume 11 | Article 390 Increase Statistical Power and Publish Pre-study
Power Calculations In real world research it is usually impossible to determine
the statistical power of NHST tests exactly. However, if raw
data summaries and/or raw data is published irrespective of
statistical significance (Section Pre-registration, Publish Raw
Data, and Publish Data (Summaries) Irrespective of Statistical
Significance, Promote Building Good Quality Datasets Including
Large Replication Studies) then we have a much better chance of
trying to determine power. Another option is to determine power
to detect pre-defined standardized effect sizes. In any case, power
in psychology and neuroscience should be much higher than
what it is nowadays (Szucs and Ioannidis, 2017). We hypothesize
that pre-registration would facilitate increasing power because
researchers could less expect to rely on incidentally finding
something statistically significant to report from their studies. Hence, they would have more interest in assuring that they are
able to respond their primary, registered hypotheses. There is no reason to assume that understanding twenty
first and twenty second century science will require less
mathematical and statistical understanding than before. Such
as there is no royal road to mathematics, there is no royal
road to statistics which is heavily based on mathematics. If
statistical understanding does not improve it will not matter
whether editorials enforce bootstrapping, likelihood estimation
or Bayesian approaches, they will all remain opaque to the August 2017 | Volume 11 | Article 390 16 Null Hypothesis Significance Testing: A Reassessment Szucs and Ioannidis untrained mind and open to abuse such as the NHST of the
twentieth century. as they allow for formal knowledge integration from previous
studies. One approach would be to phase out the ‘statistics lite’
education approach for all research stream students and teach
statistics rigorously. A typical research stream undergraduate
training could include, for example, 3–4 semesters of calculus,
one semester of introductory statistics, three more semesters
of calculus based statistics, and then finally two semesters of
more specialized statistics. An alternative and/or complementary
approach would be to enhance the training of professional
applied statisticians and to ensure that all research involves
knowledgeable statisticians or equivalent methodologists. At a
minimum, all scientists should be well trained in understanding
evidence and statistics and being in a position to recognize that
they may need help from a methodologist expert (Marusic and
Marusic, 2003; Moharari et al., 2009; Vujaklija et al., 2010). AUTHOR NOTE A previous version of this manuscript was available as a pre-
print (http://biorxiv.org/content/early/2016/12/20/095570). The
copyright holder for this preprint is the author/funder. It is made
available under a CC-BY-NC-ND 4.0 International license. ACKNOWLEDGMENTS It is important to note that Bayesian methods are often
accused of subjectivity because they can take prior information
into account. However, Bayesian methods are able to consider
prior expectations formally and explicitly in their models
provided that necessary information (e.g., raw data and/or
extensive reporting of data parameters) from previous studies is
available. In contrast, as we have discussed NHST can be latently
biased by subjectivity at many points without ever revealing any
of the biases. In contrast, different reasonable Bayesian priors can
be implemented and their impact on outcomes can be debated
explicitly and ultimately, the goodness of model predictions can
be tested. Hence, we do not see the use of Bayesian priors
as a drawback. Rather, explicit priors can represent a strength DS is supported by a twenty-first Century Science Initiative in
Understanding Human Cognition Scholar Award (220020370)
from the James S. McDonnell Foundation. METRICS is
supported from a grant from the Laura and John Arnold
Foundation. AUTHOR CONTRIBUTIONS DS wrote the first draft of the manuscript. DS and JI revised
successive drafts. Teach Alternative Approaches Seriously Teach Alternative Approaches Seriously
It is important that researchers are conscious that NHST only
represents a small segment of available statistical techniques. Besides NHST, Bayesian and likelihood based approaches should
also be taught, with explanation of the strengths and weaknesses
of each inferential method. Hypotheses could be tested by
either likelihood ratio testing, and/or Bayesian methods which
usually view probability as characterizing the state of our
beliefs about the world (Pearl, 1988; Jaynes, 2003; MacKay,
2003; Sivia and Skilling, 2006; Gelman et al., 2014; for
neuroscience data see e.g., Lorenz et al., 2017). The above
alternative approaches typically require model specifications
about alternative hypotheses, they can give probability statements
about H0 and alternative hypotheses, they allow for clear
model comparison, are insensitive to data collection procedures
and do not suffer from problems with large samples. In
addition, Bayesian methods can also factor in pre-study (prior)
information into model evaluations which may be important
for integrating current and previous research findings. Hence,
the above alternative approaches seem more suitable for the
purpose of scientific inquiry than NHST and ample literature
is available on both. The problem is that usually none of these
alternative approaches are taught properly in statistics courses
for students in psychology, neuroscience, biomedical science
and social science. For example, across 1,000 abstracts randomly
selected from the biomedical literature of 1990–2015, none
reported results in a Bayesian framework (Chavalarias et al.,
1990-2015). Frontiers in Human Neuroscience | www.frontiersin.org There Is No Automatic Inference: New-Old
Dangers Ahead? g
Perhaps the most worrisome false belief about statistics is the
belief in automatic statistical inference (Bakan, 1966; Gigerenzer
and Marewski, 1998), the illusion that plugging in some numbers
into some black box algorithm will give a number (perhaps
the p-value or some other metric) that conclusively proves or
disproves hypotheses (Bakan, 1966). There is no reason to assume
that any kind of “new statistics” (Cumming, 2014) will not
suffer the fate of NHST if statistical understanding is inadequate. For example, it has been shown that confidence intervals are
misinterpreted just as badly as p-values by undergraduates,
graduates, and researchers alike and self-declared statistical
experience even slightly positively correlates with the number
of errors (Hoekstra et al., 2014). Or, many times black box
machine learning algorithms may be run uncritically and/or
on relatively small data volumes. However, the more complex
is a dataset the more chance such substantively blind search
algorithms have to find some relationships where nothing worthy
of mention exist. So, uncritical applications are likely to further
boost the proportion of false positive findings irrespective of the
sophistication of the algorithms (Skokic et al., 2016). Similarly,
the proper use of Bayesian methods may require use of advanced
simulation methods and a clear understanding and justification
of probability distribution models. In contrast to this, it is
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The authors declare that the research was
conducted in the absence of any commercial or financial relationships that could
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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. Waller, N. G. (2004). The fallacy of the null hypothesis in soft psychology. Appl. Prevent. Psychol. 11, 83–86. doi: 10.1016/j.appsy.2004.02.015 Wasserstein,
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Video observations of treatment administration to children on antiretroviral therapy in rural KwaZulu-Natal
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AIDS CARE, 2016
VOL. 28, NO. S2, 34–41
http://dx.doi.org/10.1080/09540121.2016.1176674 AIDS CARE, 2016
VOL. 28, NO. S2, 34–41
http://dx.doi.org/10.1080/09540121.2016.1176674 AIDS CARE, 2016
VOL. 28, NO. S2, 34–41
http://dx.doi.org/10.1080/09540121.2016.1176674 AIDS CARE, 2016
VOL. 28, NO. S2, 34–41
http://dx.doi.org/10.1080/09540121.2016.1176674 ABSTRACT For children younger than five years, caregivers are responsible for the measurement and
administration of antiretroviral medication doses to children. Failure to adhere to the regimen as
prescribed may lead to high viral loads (VLs), immune suppression and ultimately drug
resistance. In the content of this study, adherence refers to adequate dosing of the medication
by a caregiver. Acquired drug resistance to antiretroviral therapy (ART) is prevalent amongst
children in South Africa, and poor adherence to the dosing regimen by caregivers may be
associated with this problem. In this qualitative study, we purposively recruited 33 caregiver–
child dyads from the Hlabisa HIV Treatment and Care Programme database. Children were
divided into three groups based on their VL at the time of recruitment. Children with a VL ≥400
cps/ml were grouped as unsuppressed (n = 11); children with a VL ≤400 cps/ml were grouped as
suppressed (n = 12); and children with no VL data were grouped as newly initiated (n = 10). Caregiver–child dyads were visited at their households twice to document, by means of video
recording, how treatment was administered to the child. Observational notes and video
recordings were entered into ATLAS.ti v 7 and analysed thematically. Results were interpreted
through the lens of Ecological Systems Theory and the information–motivation–behavioural skills
model was used to understand and reflect on several of the factors influencing adherence within
the child’s immediate environment as identified in this study. Thematic video analysis indicated
context- and medication-related factors influencing ART adherence. Although the majority of
children in this sample took their medicine successfully, caregivers experienced several
challenges with the preparation and administration of the medications. In the context of
emerging drug resistance, efforts are needed to carefully monitor caregiver knowledge of
treatment administration by healthcare workers during monthly clinic visits. KEYWORDS
HIV; adherence; barriers;
facilitators; paediatric; anti-
retroviral treatment (Davies et al., 2011; Fox, Ive, Long, Maskew, & Sanne,
2010; Lessells et al., 2014). Video observations of treatment administration to children on antiretroviral
therapy in rural KwaZulu-Natal Bronwyne Coetzeea, Ashraf Kageea and Ruth Blandb,c,d aDepartment of Psychology, Stellenbosch University, Stellenbosch, South Africa; bAfrica Centre for Population Health, KwaZulu-Natal, South
Africa; cInstitute of Health and Wellbeing, and Royal Hospital for Sick Children, University of Glasgow, Glasgow, UK; dSchool of Public Health,
Faculty of Health Sciences, University of Witwatersrand, Johannsburg, South Africa ARTICLE HISTORY
Received 29 January 2016
Accepted 23 March 2016 © 2016 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group
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.
CONTACT Bronwyne Coetzee
bronwyne@sun.ac.za KEYWORDS
HIV; adherence; barriers;
facilitators; paediatric; anti-
retroviral treatment Participants and procedure Purposive sampling was used to select and recruit partici-
pants from two primary healthcare clinics in the study
area. VL status at the time of recruitment was used to
differentiate three groups of children invited to take
part in the study. Children on ART for more than a year
and with two consecutive VLs ≥400 copies per millilitre
(cps/ml) were grouped as unsuppressed (n = 11); children
on ART for more than a year with a VL ≤400 cps/ml
were grouped as suppressed (n = 12); and children with
no VL data were grouped as newly initiated (n = 10). To our knowledge, no study has observed and docu-
mented the barriers and facilitators associated with
treatment administration of ART to children younger
than five years by their caregivers in their homes during
medication times in the morning and evening. The aim
of this study was to document, by means of video
recording, the barriers to and facilitators of adherence
to ART among children younger than five years in
rural KwaZulu-Natal. g
p
y
Adherence counsellors at each of the clinics invited
caregivers to meet with the primary investigator (PI)
(BC) and research assistant (RA) in a private room
after their clinic visit to inform them of the study and
to obtain written informed consent for participation. Caregivers were informed that the PI and an isiZulu-
speaking RA (to act as an interpreter) were going to
visit each of the recruited caregiver–child dyads at their
homes twice (one morning visit and one evening visit)
to document, by means of video recording, how children
received their ARVs. Participants were not excluded if
they did not consent to video recording. All participants
gave written informed consent, and none objected to
video recording. Furthermore, all participants had indi-
cated that household members were aware that the
child was taking medications. Following each home
visit, participants were handed a food parcel to the
value of R150.00 (£6.26) as a token of our appreciation
for their willingness to participate and their time in
this research. This study was conceptualised, interpreted and under-
stood through the lens of Ecological Systems Theory
(EST) (Bronfenbrenner, 1979). Ecological theories of
health, like EST, consider individual (situated at the
micro level) as well as environmental (contextual) factors
(meso-, exo- and macro levels) when attempting to under-
stand, or when examining, a target behaviour (see Figure
1). Ethics The study received ethical approval from Stellenbosch
University (S12/05/135), permission from the local com-
munity advisory board and the hospital management
before entering into the field. Multiple visits to partici-
pants’ households were used to minimise reactivity bias
during observations. Further, both the PI and the RA
documented the visits by means of field notes and
semi-structured observation schedules, separately. A
debriefing session was held after each visit to compare
and discuss observations. Introduction In the context of limited antiretroviral (ARV) drug options
available for adults and children in South Africa (Davies
et al., 2011), adherence to a first-line regimen is key to
ensure optimal and prolonged benefits of treatment
(Bangsberg et al., 2000; Bangsberg et al., 2003). According
to the latest guidelines on ART initiation among infants,
children and adolescents in South Africa, all children
younger than five years old should be initiated on ART
and are usually initiated on a first-line ART regimen
(Department of Health South Africa, 2014). Second-line
regimens, although available, remain costly and often dif-
ficult to access (Davies et al., 2011). Particular concern has
been raised regarding developing resistance to second-line
regimens, especially as monitoring of patient viral loads
(VLs) is unavailable in many resource-limited settings For children younger than five years who rely on par-
ental or non-parental caregivers to administer their
medication to them daily, adherence becomes an even
greater task to manage. In the context of this study,
adherence refers to adequate dosing of the medication
by a caregiver. In addition to having to provide for chil-
dren’s most basic needs, caregivers have to integrate
and manage complicated regimens into their daily
lives (Coetzee, Kagee, & Bland, 2015). For example, in
the absence of fixed-dose combinations available to
children younger than five years, caregivers of children
in this age group are required to carefully and accu-
rately measure and administer volumes of liquid drug
formulations to children on ART twice a day (World
Health Organisation [WHO], 2013). Moreover, some CONTACT Bronwyne Coetzee © 2016 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group
This is an Open Access article distributed under the terms of the Creative Commons Attribu
distribution, and reproduction in any medium, provided the original work is properly cited AIDS CARE
35 35 Trust-funded Africa Centre for Population Health
(www.africacentre.ac.za). of the medications have special storage requirements
and some are unpalatable and require creative strategies
to mask their taste in order to aid administration
(Department of Health South Africa, 2014). These fac-
tors highlight several characteristics of paediatric ART
that complicates adherence. Setting The research was conducted in Northern KwaZulu-Natal
(South Africa) under the auspices of the Wellcome Figure 1. The four systems of Bronfenbrenner’s EST. Participants and procedure In addition to EST, we used concepts associated with
the information–motivation–behavioural skills (IMB)
model to discuss and explain specific observed behaviours
in this study (Fisher, Fisher, Amico, & Harman, 2006). Results The median number of adults and children per house-
hold was three. Households had up to six occupants in
a dwelling consisting of one bedroom and one living
area (approximately 30 m2 in total (Hunter & Posel,
2012)). Thus, overcrowded households were common
amongst the participants in this study and inhibited
the ease with which medication was administered to
the child. Medication storage Nearly all of the caregivers stored the medications in the
bedroom. Abacavir (ABC) and Lamivudine (3TC) (both
liquid ARV formulations) and tablets (such as Efavirenz
(EFV), Zidovudine and Stavudine (d4 T)) do not require
refrigeration and it was therefore appropriate to store
these medications at room temperature. The directions
for one of the medications, Lopinavir/ritonavir (LPV/r),
state that it should be kept refrigerated (Department of
Health South Africa, 2013). Although most caregivers
(30 out of 33) had access to a refrigerator, not all caregivers
stored LPV/r in the refrigerator. Compared to children
with suppressed VLs, fewer caregivers of children newly
initiated and with unsuppressed VLs kept LPV/r refriger-
ated (Table 1). Caregiver characteristics The median age of the caregivers was 35 years (IQR:
27.5–48), 29 years (IQR: 24–40) and 28.5 (IQR: 25.5–
51) for caregivers of children with suppressed VLs,
unsuppressed VLs and newly initiated on ART, respect-
ively. A greater proportion of caregivers to children with
suppressed VLs (7 out of 12) were married and/or living
with a significant other in a permanent union compared
to the other two groups (2 out of 11 – unsuppressed, and
2 out of 10 – new enrollers). Furthermore, a greater pro-
portion of caregivers to children with suppressed VLs
had completed high school (5 out of 12), compared to
the other two groups (2 out of 11 – unsuppressed, and
1 out of 10 – new enrollers). Most of the caregivers (27
out of 33) were unemployed and were receiving either
a child support grant or disability grant from the South
African government. More than half of the caregivers
(19 out of 33) were also ART users. Only 24 (73%) had
received the pre-ART HIV education sessions before
the child was enrolled on treatment. Most caregivers
(25 out of 33) indicated that they were not solely respon-
sible for treatment administration or for attending clinic
visits (11 out of 33), but shared this responsibility with
another member of the household, usually an older sib-
ling. Caregivers reported that all members of the Medications stored in the bedroom were either hid-
den (when kept in plastic bags and stored in a cupboard
in the bedroom), or not hidden (when kept visible on a
table in the bedroom). Compared to children with
supressed and unsuppressed VLs, more of the caregivers
of children who were newly initiated (see Table 1) on
ART did not keep LPV/r in the fridge and preferred to
store medications out of sight. Based on this observation,
it is possible that they were hiding the medications from
younger children, other household members or visitors. Child characteristics The median age of the children at recruitment was 3.5
years (interquartile range (IQR), 2–4.75) for those
with suppressed VLs, 4 years (IQR: 3–5) for those
with unsuppressed VLs and 2 years (IQR: 2–5) for
those newly initiated on ART. Twelve children were
between 0 and 2 years old, 5 children were 3 years
old, 4 were 4 years old, and 12 were aged 5 years. The
majority of children were female (19 out of 33) and
most of them (18 out of 33) had their biological mother
as primary caregiver. Other primary caregivers were
grandmothers (9 out of 33), fathers (1 out of 33) and
aunts (5 out of 33). The average time on ART was 2.3
years, 2.8 years and 0.4 years amongst children with
suppressed VLs, unsuppressed VLs and those newly
initiated, respectively. Data analysis The sources of data were the video recordings, field notes
and data from the semi-structured observation schedules Figure 1. The four systems of Bronfenbrenner’s EST. B. COETZEE ET AL. 36 used during home visits. Demographic data of the chil-
dren (such as age, gender, etc.) as well as their caregivers
(such as age, marital status, level of education, etc.) were
entered and analysed using SPSS v 22. Field notes and
video data were analysed thematically using ATLAS.ti
v 7 (www.atlasti.com). used during home visits. Demographic data of the chil-
dren (such as age, gender, etc.) as well as their caregivers
(such as age, marital status, level of education, etc.) were
entered and analysed using SPSS v 22. Field notes and
video data were analysed thematically using ATLAS.ti
v 7 (www.atlasti.com). household were aware that the child was on treatment. However, it was not fully ascertained whether the house-
hold members knew this treatment was for HIV,
especially in the case of other siblings in the household. Food given before treatment administration During the first visit to the homes, all of the caregivers
reported that they usually gave food to the child before
treatment administration in the mornings and evenings. However, there were only four morning (three newly
initiated, and one suppressed VL) and two evening visits
(both newly initiated) in which we had seen a child eat-
ing before treatment was administered or had seen vis-
ible signs that food had been prepared and given. We
did not observe any food given during our visits to the AIDS CARE
37 37 Table 1. Context-related observations. Table 1. Context-related observations. Newly initiated
Suppressed VL
Unsuppressed VL
Morning visit
(n = 10)
Evening visit
(n = 6)
Morning visit (n = 12)
Evening visit (n = 8)
Morning visit (n = 11)
Evening visit (n = 9)
Medication workspace
Lounge
5
4
9
6
2
4
Bedroom
3
2
2
1
7
3
Kitchen
2
0
1
1
2
2
Medication storage
In bedroom (hidden)
5
3
5
3
3
5
In bedroom (not hidden)
2
1
6
5
7
4
In lounge (hidden)
3
2
0
0
1
0
In lounge (not hidden)
0
0
1
0
0
LPV/r refrigerateda
4 out of 8
2 out of 8
9 out of 10
7 out of 10
5 out of 9
4 out of 9
Food givenb
3
2
1
0
0
0
a27 (10 out of 12 suppressed; 9 out of 11 unsuppressed; 8 out of 10 newly initiated) children were receiving LPV/r as part of their regimen. bFood given before treatment administration. a27 (10 out of 12 suppressed; 9 out of 11 unsuppressed; 8 out of 10 newly initiated) children were receiving LPV/r as part of th
bFood given before treatment administration. households of children with unsuppressed VLs. There
was therefore a mismatch between what caregivers had
stated during the initial home visit and what we observed
during the morning and evening home visits. (usually ranging between 1 and 5 ml) and administer it
to the child. Of children newly initiated on ART, 9
received ABC, 10 received 3TC and 8 received LPV/r
as part of their regimen. Of children with suppressed
VLs, 10 received ABC, 9 received 3TC and 10 received
LPV/r as part of their regimen. Of children with unsup-
pressed VLs, 7 received ABC, 7 received 3TC and 9
received LPV/r as part of their regimen. Liquid formulations Apart from one child with a suppressed VL, all of the
children received liquid ARV formulations as part of
their regimen. For children under three years (<10
kgs), a first-line regimen is a combination of ABC, Lami-
vudine (3TC) and LPV/r. All three of these medications
are available as liquid formulations, and dose changes
vary depending on the weight of the child. For children
between three and 10 years old (>10 kg), a combination
of ABC, 3TC and EFV is recommended (Department of
Health South Africa, 2014). Caregivers are required to
measure the millilitres prescribed for each medication The majority of caregivers (observed 20 times during
morning administration, and 13 times during evening
administration) used a separate syringe to measure and
administer each dose (Table 2). One way to aid accurate
measurements of liquid formulations using syringes is
through the use of a syringe nozzle. Syringe nozzles are
small plastic attachments that fit into the opening of
medication bottles and are made especially for syringes. These devices are typically packaged with the medi-
cations ABC and 3TC. The medication LPV/r does not
come
with
such
a
device. Syringe
nozzles
were Table 2. Medication-related observations. Newly initiated
Suppressed VL
Unsuppressed VL
Morning visit
(n = 10)
Evening visit
(n = 6)
Morning visit
(n = 12)
Evening visit
(n = 8)
Morning visit
(n = 11)
Evening visit
(n = 9)
Medication measurement tools
Syringe
6
3
7
5
7
5
Syringe + nozzle
4
2
4
3
1
1
Measuring cup
0
1
1
0
3
3
Measurement issues
Bubble in syringe
1
0
2
1
1
1
Dose checkinga
ABC
2
3
2
0
4
3
3TC
2
3
1
1
4
3
LPV/r
3
3
8
6
6
7
Medication administration (directly or indirectly)c
Syringe (directly)
6
3
9
7
4
4
Measuring cup (indirectly)
4
3
2
1
7
5
Missed dosesb
1
1
0
0
0
0
aDose checking: caregiver checks the measurement dose amount in the syringe for accuracy. bMissed doses: medication is not taken/given on the day. cMedication administration: directly – medication injected into child’s mouth using syringe. Indirectly – medication transferred into a cup. Table 2. Medication-related observations. Table 2. Medication-related observations. aDose checking: caregiver checks the measurement dose amount in the syringe for accuracy. bMissed doses: medication is not taken/given on the day. Palatability y
Most of the children (no problems with palatability in 38
out of 56 observations) ingested their medications with-
out much resistance (Table 3). However, we observed
four instances (one newly initiated, one suppressed,
two unsuppressed) of children spitting medications
out; two instances of vomiting (one newly initiated,
one unsuppressed); seven instances of coughing (two
newly initiated, four suppressed, one unsuppressed)
during medication administration or moaning by the
child; five instances of child refusal (one instance in
which the child ran away at the time of medication
administration (newly initiated); and four instances
(two newly initiated and two suppressed) where the
child pushed the caregiver’s hand away (Table 3). In all
instances of palatability difficulty, the caregiver com-
pleted dose administration. In both instances of vomiting
in particular, the caregivers only administered a partial
dose. As such, children who vomited their medications
did not receive their full dosage. Figure 2. Dose measurement of 3TC taken at an angle. The
image illustrates the caregiver of a suppressed child on ART
taking a measurement of 3TC at a horizontal angle. underutilised amongst the caregivers (only used 15 times
across 56 observations) (Table 2). Observation revealed
that syringe nozzles were used more frequently by care-
givers of children newly initiated (observed six times)
and suppressed on ART (observed seven times) com-
pared to unsuppressed children on ART (observed
twice). Fewer instances were observed among those
with unsuppressed VLs as a large proportion was receiv-
ing LPV/r via syringe only. However, even with a syringe nozzle intact, caregivers
who chose to use them did so erroneously. In at least two
instances where measurements were taken at a horizon-
tal angle, these measurements led to bubble formations
in the syringe. Figure 2 shows a caregiver (biological
mother) taking a measurement of 3TC with a syringe
nozzle intact. The caregiver can be seen holding the bot-
tle horizontally while the measurement is taken. The
angle at which the doses were measured resulted in
bubbles forming within the syringe, suggesting possible
dose inaccuracy. The dose was not checked (for accu-
racy) by the caregiver after the measurement was
made. In four other instances, it was clear from the
video data that a large bubble had formed in the syringe
(Table 2). In neither of these instances was the bubble
removed, indicating possible under-dosing of that
medication. Tablet formulations Tablets and capsules were either swallowed whole or dis-
solved in water or mixed with a taste distractor (e.g. drinking yoghurt). All the children with suppressed
VLs received their tablets/capsules directly and they
were subsequently swallowed whole. Discussion To our knowledge, this is the first study to directly
observe factors influencing adherence to ART amongst
children younger than five years living in a rural area
in
a
resource-constrained
environment. Ecological
models, like EST, favour behaviour-specific approaches
to interventions, such as improving adherence to ART
amongst children younger than five years. Thus, by
using EST in this study, we were able to identify contex-
tual and medication-related factors influencing adher-
ence to ART among children in a rural area of South
Africa. Liquid formulations cMedication administration: directly – medication injected into child’s mouth using syringe. Indirectly – medication transferred into a cup. 38
B. COETZEE ET AL. B. COETZEE ET AL. B. COETZEE ET AL. 38 Figure 2. Dose measurement of 3TC taken at an angle. The
image illustrates the caregiver of a suppressed child on ART
taking a measurement of 3TC at a horizontal angle. The tablets of four out of five children in the unsup-
pressed group were dissolved in water before being
administered. Dissolving tablets into solutions was pro-
blematic in cases where the residue of the tablets
remained behind in the container (usually a cup) and
was discarded by the caregiver when she rinsed out the
medication cup. In all four instances where either a tablet
or capsule had been dissolved in water, residue of the
medications remained behind in glass/cup from which
it was administered. Once administered, the caregiver
rinsed the administration tool and discarded the residue
that remained left over, indicating a possible under-dos-
ing of that medication. Context-related factors Caregivers in this study showed a preference for keeping
medications hidden. Although caregivers were not AIDS CARE
39 AIDS CARE 39 Table 3. Child-related observations. Table 3. Child-related observations. Newly initiated
Suppressed VL
Unsuppressed VL
Morning visit
(n = 10)
Evening visit
(n = 6)
Morning visit
(n = 12)
Evening visit
(n = 8)
Morning visit
(n = 11)
Evening visit
(n = 9)
Palatability
No palatability issues
5
4
10
3
8
8
Spitting
1
0
1
0
2
0
Vomiting
1
0
0
0
0
1
Cough/moans
2
0
0
4
1
0
Refusal
1
2
1
1
0
0
Taste distracter used (yes)
0
1
2
2
1
1 medication, but lacked the necessary skills with which
to perform accurate measurements of the doses. In sev-
eral
instances,
erroneous
medication
preparation
appeared to lead to under-dosing. According to the
IMB model (Fisher et al., 2006), information is a necess-
ary condition for behavioural change, but it is not suffi-
cient to bring about that change in itself. Adequate
motivation to adhere to the regimen as well as behav-
ioural skills to administer the medication effectively are
required for behavioural change to occur. While care-
givers used medication tools such as syringes and medi-
cation cups to measure doses of the medications, they
lacked the necessary skills required for performing accu-
rate measurements, such as dose checking (especially
checking that all of the contents of dissolved tablets
and capsules were administered) and removing bubbles
in syringes. These findings concur with other studies
assessing dosing accuracy among caregivers on ART
(Howard et al., 2014; Yin et al., 2008). explicitly asked why they chose to store medications in
this way, one might presume that caregivers were hiding
the medications from young children or from visitors. Keeping medications hidden adds to the large body of lit-
erature on HIV-related stigma (Bogart, Cowgill, & Ken-
nedy, 2008; Brown, Macintyre, & Trujillo, 2003; Katz
et al., 2013; Rintamaki, Davis, Skripkauskas, Bennett, &
Wolf, 2006; Ware, Wyatt, & Tugenberg, 2006). In this
instance, perceived stigma prevented caregivers from
accessing cue-based strategies, such as visibility of the
medications in their homes, for reminder purposes
(Marhefka et al., 2008). We also observed little to no food administered
prior to treatment administration. However, the lack
of food and the subsequent administration of medi-
cation suggest that caregivers were willing to adminis-
ter treatment without food. Context-related factors Caregivers’ willingness to
adhere to ART in the absence of available resources
has often been associated with what Olds, Kiwanuka,
Ware, Tsai, and Haberer (2015) have referred to as
the “Lazarus effect” (p. 586). In their study, based on
the observable improvements in children’s health due
to ART use, caregivers of children in rural Uganda
were less inclined to miss treatment out of fear that
the child may become ill again (Olds et al., 2015). Fur-
thermore, willingness to provide treatment in the
absence of food also contradicts evidence from care-
givers and healthcare workers. For example, in our pre-
vious work, caregivers to children in the same study
area as well as doctors, nurses and counsellors reported
that lack of food was one of the most important barriers
to adherence to ART(Coetzee et al., 2015). While this
barrier has been shown to be true in other contexts
as well (Haberer & Mellins, 2009; Simoni et al., 2007;
Vreeman, Wiehe, Pearce, & Nyandiko, 2008), our
data suggest that this does not always mean that
doses are missed. Caregivers to children living in rural areas have low
levels of health literacy (Howard et al., 2014), which
may contribute to misunderstandings of how best to
administer the medication to children. In other research,
we showed that adherence counsellors provided little
information to caregivers during monthly clinic sessions,
which we attributed to the rushed training that adher-
ence counsellors received and the lack of follow-up and
debriefing (Coetzee et al., 2015). However, caregivers to
children with suppressed and unsuppressed VLs tended
to use devices such as syringe nozzles less frequently than
those newly initiated on ART. Thus, carelessness may
also be a consequence of treatment fatigue (Bagenda
et al., 2011). While most of the children took their medicines suc-
cessfully, several still struggled with the palatability of the
formulations. In the few instances where vomiting was
observed, this may have been attributable to the lack of
food in household. Other studies have shown that lack
of food before ART is often accompanied by noxious
side effects (Biadgilign, Deribew, Amberbir, & Deribe,
2009; Fetzer et al., 2011). Taste-masking and pill-swal-
lowing interventions have contributed suggestions for References Bagenda, A., Barlow-Mosha, L., Bagenda, D., Sakwa, R.,
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shown to improve adherence to medication in children. Grant 88903 is gratefully acknowledged. Opinions expressed
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necessarily to be attributed to the NRF. The Africa Centre is
funded by the Wellcome Trust, UK, under Grant #097410/Z/ These findings need to be interpreted against several
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Medicine, 162(9), 814–822. doi:10.1001/archpedi.162.9.814 Lessells, R. J., Stott, K. E., Manasa, J., Naidu, K. K., Skingsley,
A., Rossouw, T., & de Oliveira, T. (2014). Implementing
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Africa: Early experiences, achievements and challenges.
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Increased structural connectivity in high schizotypy
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a n o p e n a c c e s s
j o u r n a l Corresponding Author:
Eirini Messaritaki
messaritakie2@cardiff.ac.uk
Handling Editor:
Alex Fornito
Copyright: © 2022
Massachusetts Institute of Technology
Published under a Creative Commons
Attribution 4.0 International
(CC BY 4.0) license
The MIT Press Keywords: Schizophrenia, Schizotypy, Brain networks, Structural connectivity, Tractography Keywords: Schizophrenia, Schizotypy, Brain networks, Structural connectivity, Tractography RESEARCH a n o p e n a c c e s s
j o u r n a l The link between brain structural connectivity and schizotypy was explored in two healthy
participant cohorts, collected at two different neuroimaging centres, comprising 140 and
115 participants, respectively. The participants completed the Schizotypal Personality
Questionnaire (SPQ), through which their schizotypy scores were calculated. Diffusion-MRI
data were used to perform tractography and to generate the structural brain networks of the
participants. The edges of the networks were weighted with the inverse radial diffusivity. Graph theoretical metrics of the default mode, sensorimotor, visual, and auditory subnetworks
were derived and their correlation coefficients with the schizotypy scores were calculated. To
the best of our knowledge, this is the first time that graph theoretical measures of structural
brain networks are investigated in relation to schizotypy. A positive correlation was found
between the schizotypy score and the mean node degree and mean clustering coefficient of
the sensorimotor and the default mode subnetworks. The nodes driving these correlations were
the right postcentral gyrus, the left paracentral lobule, the right superior frontal gyrus, the left
parahippocampal gyrus, and the bilateral precuneus, that is, nodes that exhibit compromised
functional connectivity in schizophrenia. Implications for schizophrenia and schizotypy are
discussed. Citation: Messaritaki, E., Foley, S.,
Barawi, K., Ettinger, U., & Jones, D. K. (2023). Increased structural
connectivity in high schizotypy. Network Neuroscience, 7(1), 213–233. https://doi.org/10.1162/netn_a_00279
DOI:
https://doi.org/10.1162/netn_a_00279
Supporting Information:
https://doi.org/10.1162/netn_a_00279
Received: 8 July 2022
Accepted: 14 September 2022
Competing Interests: The authors have
declared that no competing interests
exist. Corresponding Author:
Eirini Messaritaki
messaritakie2@cardiff.ac.uk
Handling Editor:
Alex Fornito
Copyright: © 2022
Massachusetts Institute of Technology
Published under a Creative Commons
Attribution 4.0 International
(CC BY 4.0) license
The MIT Press Citation: Messaritaki, E., Foley, S.,
Barawi, K., Ettinger, U., & Jones, D. K. (2023). Increased structural
connectivity in high schizotypy. Network Neuroscience, 7(1), 213–233. https://doi.org/10.1162/netn_a_00279
DOI:
https://doi.org/10.1162/netn_a_00279
Supporting Information:
https://doi.org/10.1162/netn_a_00279
Received: 8 July 2022
Accepted: 14 September 2022
Competing Interests: The authors have
declared that no competing interests
exist. Corresponding Author:
Eirini Messaritaki
messaritakie2@cardiff.ac.uk
Handling Editor:
Alex Fornito
Copyright: © 2022
Massachusetts Institute of Technology
Published under a Creative Commons
Attribution 4.0 International
(CC BY 4.0) license
The MIT Press Citation: Messaritaki, E., Foley, S.,
Barawi, K., Ettinger, U., & Jones, D. K. (2023). Increased structural
connectivity in high schizotypy. Network Neuroscience, 7(1), 213–233. https://doi.org/10.1162/netn_a_00279
DOI:
https://doi.org/10.1162/netn_a_00279
Supporting Information:
https://doi.org/10.1162/netn_a_00279
Received: 8 July 2022
Accepted: 14 September 2022
Competing Interests: The authors have
declared that no competing interests
exist. Increased structural connectivity in
high schizotypy Eirini Messaritaki1
, Sonya Foley1
, Kali Barawi2, Ulrich Ettinger3
, and Derek K. Jones1
1Cardiff University Brain Research Imaging Centre (CUBRIC), School of Psychology, Cardiff University, Cardiff, UK
2School of Medicine, Cardiff University, Cardiff, UK
3Department of Psychology, University of Bonn, Bonn, Germany Eirini Messaritaki1
, Sonya Foley1
, Kali Barawi2, Ulrich Ettinger3
, and Derek K. Jones1 1Cardiff University Brain Research Imaging Centre (CUBRIC), School of Psychology, Cardiff University, Cardiff, UK
2School of Medicine, Cardiff University, Cardiff, UK
3Department of Psychology, University of Bonn, Bonn, Germany INTRODUCTION Schizophrenia is a serious psychiatric disorder of unknown aetiology, while schizotypy
encompasses a set of stable personality traits that are thought to reflect the subclinical expres-
sion of schizophrenia. Specifically, people with high levels of schizotypy show cognitive and
neural patterns resembling the deficits observed in schizophrenia (Ettinger et al., 2014, 2015). Studying schizotypy is, therefore, important for a number of reasons. Firstly, given that partly
overlapping factors underlie both conditions (Fanous et al., 2007), studying schizotypy can
shed light on the aetiology of schizophrenia. In addition, people with high schizotypy suffer
from lower social, educational, and professional levels of functioning, and high levels of dis-
tress (Barrantes-Vidal et al., 2010; Cohen & Davis, 2009); therefore, studying schizotypy can
help us better understand these problems and develop interventions to ameliorate them. More-
over, studying schizotypy can reveal characteristics (in the brains of healthy high-schizotypy
participants) that protect or compensate against schizophrenia; those characteristics may be
used to inform the development of novel treatments (Ettinger et al., 2014, 2015). Finally,
the study of schizotypy is free from any confounds that stem from pharmacological interven-
tions, and as such it is of value to both clinical and nonclinical research. Given this similarity of schizophrenia and schizotypy, in building our aims for this study, we
looked to the topological properties of the structural and functional brain networks of schizo-
phrenia patients. The functional brain networks of schizophrenia patients are compromised and generally
exhibit lower connectivity and less efficient organisation, with few exceptions, compared to
healthy individuals. For example, a meta-analysis of 13 studies of functional whole-brain net-
works, in which the functional connectivity was measured via functional MRI (fMRI), magne-
toencephalography (MEG), or electroencephalography (EEG), showed significantly lower
small-worldness and measures of local organisation (Kambeitz et al., 2016). Additional studies
that were not included in this meta-analysis corroborate the findings. For example, fMRI-
measured functional connectivity was lower within the default mode network in schizophre-
nia patients compared to healthy controls (Pankow et al., 2015). Schizophrenia patients
showed lower activation compared to healthy controls in the lateral frontal cortex bilaterally,
in the left basal ganglia and the cerebellum (Landin-Romero et al., 2014). The lateral default
mode network exhibited lower functional connectivity with the sensorimotor cortex, but
higher connectivity with association areas in schizophrenia patients (Wang et al., 2015). Clustering coefficient:
A measure of how interconnected
the neighbours of that node are. AUTHOR SUMMARY We investigated the topological organisation of the structural brain networks, derived via
tractography, of healthy participants with varying schizotypy scores, in two independent
cohorts comprising 140 and 115 participants, respectively. In order to capture the possible
impact of myelination or axonal density on the structural networks, we used the inverse radial
diffusivity as the edge weight. We observed that participants with the higher schizotypy scores
exhibited higher node degree and clustering coefficient in the sensorimotor and default mode
networks. This provides evidence of stronger structural connectivity in participants with higher
schizotypy, which could indicate a possible protective mechanism against schizophrenia, or
imply that structural alterations observed in schizophrenia are a correlate or consequence of
the disease rather than its cause. Copyright: © 2022
Massachusetts Institute of Technology
Published under a Creative Commons
Attribution 4.0 International
(CC BY 4.0) license Copyright: © 2022
Massachusetts Institute of Technology
Published under a Creative Commons
Attribution 4.0 International
(CC BY 4.0) license The MIT Press Increased structural connectivity in high schizotypy Tractography:
A computational method that uses
diffusion-MRI data to reconstruct and
visualize the white matter tracts of
the brain. INTRODUCTION The anterior and posterior default mode networks exhibited higher and lower connectivity
with the right control and lateral visual networks, respectively, in schizophrenia patients
(Wang et al., 2015). Analysis of dynamic functional connectivity showed that schizophrenia
patients activated the default mode network less frequently than healthy controls, but each
activation lasted 4–5 s longer than in the healthy controls (Kottaram et al., 2019). Altered func-
tional connectivity was observed in sensorimotor, visual, auditory, social, high level cognitive,
and motor processing nodes in schizophrenia patients compared to controls (Berman et al.,
2016; Jimenez et al., 2019; Kaufmann et al., 2015), with most of the alterations indicating
lower functional connectivity. Schizophrenia patients exhibited lower clustering coefficient,
global efficiency and local efficiency, and higher characteristic path length of functional net-
works (Zhu et al., 2016). Finally, compared to healthy controls, significant hypoconnectivities
were observed between seed regions and the areas in the auditory, default mode, self-
referential, and somatomotor network in schizophrenia patients (Li et al., 2019). The structural brain networks of schizophrenia patients have been shown predominantly to
have a less central and less efficient organisation compared to those of healthy controls, but Network Neuroscience Network Neuroscience Network Neuroscience 214 Increased structural connectivity in high schizotypy there are some exceptions. For example, the global efficiency, local efficiency, clustering coef-
ficient, and mean connectivity strength of structural networks weighted by the connectivity
probability were significantly lower in schizophrenia patients compared to controls, while
the mean betweenness centrality was significantly higher (Shon et al., 2018). Significantly lon-
ger path lengths of frontal and temporal regions were observed in structural networks (with
edges weighted by the magnetisation-transfer ratio) of schizophrenia patients compared to
controls, and frontal hubs of patients showed a significant reduction of betweenness centrality
(van den Heuvel et al., 2010). Structural networks of schizophrenia patients with auditory
visual hallucinations showed higher characteristic path length compared with those of healthy
controls (Zhu et al., 2016). Additionally, studies have shown that schizophrenia patients have higher radial diffusivity
and mean diffusivity in some white matter tracts (left thalamo-occipital tracts, right uncinate
fascicle, right middle longitudinal fascicle) compared to healthy controls (Abdul-Rahman
et al., 2011; Joo et al., 2018). METHODS Two datasets were collected at the Cardiff University Brain Research Imaging Centre (CUBRIC)
between 2014 and 2016 (Cardiff data) and at the University of Munich between 2009 and
2011 (Munich data). The scanners and sequences used were different, and the sensitivity of
the data to microstructural parameters is, therefore, different. For that reason, the two datasets
were analysed separately. INTRODUCTION Another study showed that schizophrenia patients have higher
mean diffusivity in the right anterior thalamic radiations, the forceps minor, the bilateral infe-
rior fronto-occipital fasciculus, the left superior longitudinal fasciculus, and the bilateral unci-
nate (Clark et al., 2011). Radial diffusivity, although nonspecific in principle, can be influenced
by myelination and/or axonal density (Chen et al., 2009; Cykowski et al., 2010; Jones et al.,
2013), with higher values potentially indicating lower myelination or axonal density. In this study we used tractography-derived structural brain networks to investigate possible
differences in the organisation of the brain networks of healthy participants with varying schi-
zotypy scores. Various microstructural metrics, such as the number of reconstructed stream-
lines or the fractional anisotropy, have been used in past studies to assign connectivity strength
to structural network edges (Caeyenberghs et al., 2016; Messaritaki et al., 2019, 2021). In this
study, in order to capture the possible impact of altered myelination or axonal density on the
strength of the connections, we used the inverse radial diffusivity to assign edge weights. The
aims of the study were to explore whether previously reported alterations of white matter con-
nectivity in schizophrenia could also be observed in relation to high-schizotypy scores,
thereby providing a test of the (dis)continuity between high schizotypy and schizophrenia. 88/netn_a_00279.pdf by guest on 13 April 2023 Downloaded from http://direct.mit.edu/netn/article-pdf/7/1/213/2072088/netn_a_00279.pdf by guest on 13 April 2023 Munich data. One hundred fifteen healthy participants (55 female, aged 18–50 years, mean
age = 27.6 years, SD = 8.0 years) were recruited through advertisements in and around
Munich, Germany. All experimental procedures were approved by the research ethics com-
mittee of the Faculty of Medicine at the University of Munich, in accordance with the current
division of the Declaration of Helsinki. Participants were included after thorough clinical
screening and only if they did not meet any of the exclusion criteria: any DSM-IV Axis I dis-
orders, first-grade relatives with psychotic disorders, former or current neurological disorders,
current physical conditions, current medication except for contraceptives, uncorrected visual
impairments. Further inclusion criteria were age between 18 and 55 and German as a first
language. All subjects volunteered to take part in the study, gave written informed consent,
and received financial compensation for their participation. All participants completed the German version (Klein et al., 1997) of the SPQ (Raine, 1991)
to yield their total schizotypy score. In the Munich data, the SPQ subscales showed acceptable
to good reliability; the Cronbach’s alpha for the total score was 0.882. High-resolution T1-weighted structural images were acquired on a 3T MAGNETOM Verio
scanner (Siemens, Erlangen, Germany) using a 12-channel head matrix Rx-coil. A three-
dimensional MPRAGE sequence was used, with repetition time TR = 2,400 ms, echo time
TE = 3.06 ms, flip angle = 9° with 160 slices, slice thickness = 1.0 mm, voxel size = 1.0 ×
1.0 × 1.0 mm, FOV = 256 mm. Diffusion-weighted data were acquired using a diffusion-
weighted interleaved spin-echo, echo planar imaging sequence with the following parameters:
25 axial slices; slice thickness = 5.2 mm; TE = 107 ms; TR = 5 s; gap between slices = 5.2 mm;
FOV = 230 mm; matrix = 128 × 128; flip angle = 90; bandwidth = 1,395 Hz/Px; voxel size = 1.8 ×
1.8 × 5.2 mm. Diffusion gradients were applied in 20 directions with b = 1,000 s/mm2, with each
image acquired three times. Three images with no diffusion weighting were also acquired. Data Collection Cardiff data. One hundred forty healthy participants (92 female, 19–55 years old, mean =
24.7 years, SD = 5.0 years) were recruited in and around Cardiff, UK. All procedures were
given ethical approval by the Cardiff University School of Psychology Ethics Committee. All
participants gave written informed consent before taking part and received financial compen-
sation for their participation. All participants had normal or corrected-to-normal vision and
had no history of neurological or neuropsychiatric disorders. All participants completed the English version of the Schizotypal Personality Questionnaire
(SPQ) (Raine, 1991), through which their total schizotypy score was calculated. The schizo-
typy score is the sum score of nine subscales: ideas of reference, odd beliefs or magical Network Neuroscience 215 Increased structural connectivity in high schizotypy thinking, unusual perceptual experiences, suspiciousness, excessive social anxiety, no close
friends, constricted affect, odd or eccentric behaviour, odd speech. The higher the schizotypy
score, the more pronounced the schizotypal traits in a participant. For the questionnaire, ade-
quate internal consistency and criterion validity have been demonstrated (Fonseca-Pedrero
et al., 2018; Klein et al., 2001). In the Cardiff data, the SPQ subscales showed acceptable
to good reliability; the Cronbach’s alpha for the total score was 0.815. MRI was carried out on a GE Signa HDx 3T scanner (GE Healthcare, Milwaukee, WI). T1-
weighted structural data were acquired using an axial three-dimensional fast spoiled gradient
recalled sequence with the following parameters: TR = 8 ms, TE = 3 ms, TI = 450 ms; flip
angle = 20°; voxel size = 1 mm; field of view (FOV) ranging from 256 × 192 × 160 mm3 to
256 × 256 × 256 mm3 (anterior-posterior/left-right/superior-inferior). The T1 images were
downsampled to 1.5-mm isotropic resolution. Diffusion-weighted MRI data were acquired
using a peripherally cardiac-gated sequence with b = 1,200 s/mm2, TR = 20 s, TE = 90 ms,
voxel size 2.4 × 2.4 × 2.4 mm3, zero slice gap, FOV = 230 mm. Data were acquired along
30 unique and isotropically distributed gradient orientations. Three images with no diffusion
weighting were also acquired. The diffusion images were coregistered to the T1-weighted
images and corrected for head movement and eddy current distortions. Free-water correction
was also performed (Metzler-Baddeley et al. 2012; Pasternak et al., 2009). Parcellation and Network Construction To validate our analysis against the use of different cortical/subcortical parcellations (Parker
et al., 2014), two atlases were employed to identify the areas of the cerebrum that formed
the nodes of the structural brain areas: (i) the AAL atlas (Tzourio-Mazoyer et al., 2002), which
comprises 90 areas, and (ii) the Desikan–Killiany atlas (Desikan et al., 2006), which comprises
84 areas. FreeSurfer (https://surfer.nmr.mgh.harvard.edu) was used to parcellate the T1-
weighted anatomical scans (Fischl et al., 2002, 2004) and the Desikan–Killiany atlas was used
to identify the 84 areas of the cerebrum that formed the nodes of the structural brain networks. The AAL parcellation was calculated using ExploreDTI 4.8.6 (Leemans et al., 2009). Each
dataset was analysed with both atlas parcellations. However, ExploreDTI failed to give a reli-
able parcellation for the Munich data, possibly due to the lower resolution of the MRI images
in that dataset. For that reason, the subsequent analysis for the Munich data was done only
using the Desikan–Killiany atlas. In order to exclude tracts that are not reliable due to having been recovered with a small
number of streamlines, we applied a threshold NSthr and excluded edges with fewer than NSthr
streamlines. Because that threshold is arbitrary (Buchanan et al., 2020), we repeated the anal-
ysis for thresholds that correspond to mean network sparsity over participants in the range of
0.85 to 0.25. Edges that survived thresholding were weighted with the inverse mean radial diffusivity
calculated along the voxels spanning the corresponding white matter tracts. Networks were
constructed for the auditory, sensorimotor, visual, and default mode systems, that is, for sub-
networks that have consistently been shown to be altered in schizophrenia patients compared
to controls. Three graph theoretical metrics, namely the mean node degree, mean clustering
coefficient and mean nodal strength, were calculated for all networks using the Brain Connec-
tivity Toolbox (Rubinov & Sporns, 2010). These metrics provide insight about different aspects
of the topological organisation of the brain networks. Specifically, the node degree and the
nodal strength measure the connectivity of a node within the network, while the clustering
coefficient measures the connectivity within the neighbours of each node. We also note that
the node degree is independent of the edge weighting, while the clustering coefficient and the
nodal strength do depend on it. Sparsity:
The number of edges in the network
divided by the total possible edges
in it. Parcellation and Network Construction In order to identify any graph theoretical metrics that were
correlated with each other, correlation coefficients were calculated between graph theoretical
metrics for each subnetwork. The mean nodal strength exhibited correlation coefficients with
absolute values higher than 0.85 with the mean node degree, and for that reason it was
removed from the analysis. Node degree:
The number of edges that connect
the node to the rest of the network. Nodal strength:
The sum of the weights of the edges
connected to the node. Tractography Probabilistic, anatomically constrained streamline tractography was performed using MRtrix
(Tournier et al., 2019), employing the iFOD2 algorithm (Smith et al., 2012, 2015; Tournier Network Neuroscience Network Neuroscience 216 Increased structural connectivity in high schizotypy et al., 2010). The maximum angle between successive steps was 50°, the minimum and max-
imum streamline lengths were 30 mm and 25 0mm, respectively, and the FOD amplitude cut-
off was 0.06. Two million streamlines were generated for each participant, with the seed points
on the interface between grey matter and white matter. Visual inspection of the tractograms
was performed to verify that the white matter was adequately covered and that streamlines did
not extend outside of the white matter. Nodal strength:
The sum of the weights of the edges
connected to the node. Demographic Information The schizotypy scores (i.e., SPQ total scores) of the participants in the Cardiff data ranged
between 0 and 43 (mean: 12.4, SD: 10.1), while for the Munich data they ranged between
0 and 35 (mean: 8.2, SD: 7.0). No statistically significant correlations were found between
schizotypy score and age or sex, or between age and sex of the participants, for the Cardiff
or the Munich data. The correlation coefficients and p values between these variables are
given in Table 1 for the two datasets. Statistical Analysis Correlation coefficients and the corresponding p values were calculated between the demo-
graphic characteristics of the participants, that is, schizotypy score, age, and sex, to verify that
those were not correlated with each other. Network Neuroscience Network Neuroscience 217 Increased structural connectivity in high schizotypy Partial correlation coefficients and the corresponding p values were calculated between the
schizotypy score and the graph theoretical metrics of the participants, correcting for age and
sex. The mean Cook’s distance (Cook, 1977) was used to identify outliers, which were
subsequently investigated further. Data points with Cook’s distance greater than four times
the mean Cook’s distance were excluded from further analysis, because they either had
isolated (unconnected) nodes in their structural connectivity matrices, or were outliers in the
degree distribution. The p values that resulted from the correlation analysis were corrected for
multiple comparisons using the false discovery rate algorithm (Benjamini & Yekutieli, 2001),
applied over all graph theoretical measures of all subnetworks. In order to understand whether any correlations between the graph theoretical metrics and
the schizotypy score are due to all the nodes of each subnetwork, or whether they are driven
by a few specific nodes of it, we calculated the correlation coefficients between the schizotypy
score and the node-specific graph theoretical metrics for those graph theoretical metrics that
exhibited statistically significant correlations. We considered that a node is driving the corre-
lations if the related p value was lower than 0.05. The analysis pipeline is shown in Figure 1. Cardiff Data: Desikan–Killiany Atlas Significant positive correlations that survived multiple comparison correction and persisted
across NSthr were identified between schizotypy score and the mean node degree of the sen-
sorimotor network, and between the schizotypy score and the mean clustering coefficient of
the default mode network. The related correlation coefficients and p values are shown in
Figure 5. The corresponding scatter plots are shown in Figure 6. Cardiff Data: AAL Atlas Significant positive correlations that survived multiple comparison correction and persisted
across NSthr were identified between schizotypy score and the mean node degree and mean
clustering coefficient for the sensorimotor and the default mode networks. The correlation
coefficients and p values across NSthr are shown in Figure 3. Scatter plots for these correlations
are shown in Figure 4. Network Analysis The NSthr required to achieve a given mean sparsity for the whole-brain network in the three
cases investigated is shown in Figure 2. In order to achieve sparsities as low as 0.25, we use
NSthr of [0, 160] for the Cardiff/AAL pair, [0, 60] for the Cardiff/Desikan–Killiany pair, and [0,
120] for the Munich/Desikan–Killiany pair. In the following subsections, in addition to show-
ing the correlation coefficients and p values for all these thresholds, we also show scatter plots
for the networks that correspond to a sparsity of 0.3, that is, NSthr = 86 for the Cardiff/AAL case,
NSthr = 36 for the Cardiff/Desikan–Killiany case, and NSthr = 55 for the Munich/Desikan–
Killiany case. Figure 1. Analysis pipeline. Figure 1. Analysis pipeline. 218 Network Neuroscience Increased structural connectivity in high schizotypy Table 1. Correlation coefficients and p values (the latter in parentheses) between the demographic
data in the two datasets Table 1. Correlation coefficients and p values (the latter in parentheses) between the demographic
data in the two datasets
Sz score vs. age
Sz score vs. sex
Age vs. sex
Cardiff data
0.05 (0.59)
−0.10 (0.25)
−0.07 (0.43)
Munich data
−0.14 (0.15)
0.17 (0.06)
−0.10 (0.29) Munich Data: Desikan–Killiany Atlas Significant positive correlations were identified between the schizotypy score and the mean
node degree of the sensorimotor network, and the mean node degree of the visual network. These correlations were nominally significant, that is, did not survive multiple comparison cor-
rection, but persisted across NSthr. The correlation coefficients and p values are shown in
Figure 7. The corresponding scatter plots are shown in Figure 8. Figure 2. NSthr required to achieve a given sparsity in the whole-brain structural networks for the
three dataset/atlas combinations. Figure 2. NSthr required to achieve a given sparsity in the whole-brain structural networks for the
three dataset/atlas combinations. Network Neuroscience Network Neuroscience 219 Increased structural connectivity in high schizotypy Figure 3. Correlation coefficients and p values between graph theoretical metrics and schizotypy
score for the Cardiff data and the AAL atlas. Only the two subnetworks that showed persistent cor-
relations across thresholds are shown. Coloured markers indicate correlation coefficients and p
values that survived multiple comparison correction, while blank (fainter coloured) markers indicate
correlations that were nominally significant ( p < 0.05), but did not survive multiple comparison
correction. Figure 3. Correlation coefficients and p values between graph theoretical metrics and schizotypy
score for the Cardiff data and the AAL atlas. Only the two subnetworks that showed persistent cor-
relations across thresholds are shown. Coloured markers indicate correlation coefficients and p
values that survived multiple comparison correction, while blank (fainter coloured) markers indicate
correlations that were nominally significant ( p < 0.05), but did not survive multiple comparison
correction. Node-Specific Analysis Cardiff data, AAL atlas. The node-specific analysis for the sensorimotor network showed that
the correlations between the schizotypy score and the mean node degree are driven by the
right middle frontal gyrus and the right postcentral gyrus, while the correlations between the Figure 4. Scatter plots for the correlations observed in the Cardiff data for the AAL atlas parcella-
tion for NSthr = 86. The best fit line and its 95% confidence interval are also shown. Figure 4. Scatter plots for the correlations observed in the Cardiff data for the AAL atlas parcella-
tion for NSthr = 86. The best fit line and its 95% confidence interval are also shown. Network Neuroscience Network Neuroscience 220 Increased structural connectivity in high schizotypy Figure 5. Correlation coefficients and p values between graph theoretical metrics and schizotypy
score for the Cardiff data and the Desikan–Killiany atlas. Only the two subnetworks that show per-
sistent correlations across thresholds are shown. Coloured markers indicate correlation coefficients
and p values that survived multiple comparison correction, while blank (fainter coloured) markers
indicate correlations that were nominally significant ( p < 0.05), but did not survive multiple com-
parison correction. Figure 5. Correlation coefficients and p values between graph theoretical metrics and schizotypy
score for the Cardiff data and the Desikan–Killiany atlas. Only the two subnetworks that show per-
sistent correlations across thresholds are shown. Coloured markers indicate correlation coefficients
and p values that survived multiple comparison correction, while blank (fainter coloured) markers
indicate correlations that were nominally significant ( p < 0.05), but did not survive multiple com-
parison correction. schizotypy score and the mean clustering coefficient are driven by the right precentral gyrus
and the right postcentral gyrus. For the default mode network, the correlations between schi-
zotypy score and mean node degree are driven by the left middle frontal gyrus, the right infe-
rior frontal gyrus (opercular part), the left inferior frontal gyrus (orbital and triangular parts), the
left and right middle cingulate, the left hippocampus, and the left thalamus. Also, for the
default mode network, the correlations between schizotypy score and the mean clustering
coefficient were driven by the left and right superior frontal gyrus, the right inferior frontal
gyrus (triangular part), the left anterior cingulate, the left and right middle cingulate, the left
posterior cingulate, the left parahippocampal area, the left middle occipital gyrus, the left Figure 6. Node-Specific Analysis Scatter plots for the correlations observed in the Cardiff data for the Desikan–Killiany
parcellation for NSthr = 36. The best fit line and its 95% confidence interval are also shown. Figure 6. Scatter plots for the correlations observed in the Cardiff data for the Desikan–Killiany
parcellation for NSthr = 36. The best fit line and its 95% confidence interval are also shown. Network Neuroscience Network Neuroscience 221 Increased structural connectivity in high schizotypy Figure 7. Correlation coefficients and p values between graph theoretical metrics and schizotypy
score for the Munich data and the Desikan–Killiany atlas. Only the sensorimotor and the visual
subnetworks showed persistent, nominally significant correlations across thresholds. Figure 7. Correlation coefficients and p values between graph theoretical metrics and schizotypy
score for the Munich data and the Desikan–Killiany atlas. Only the sensorimotor and the visual
subnetworks showed persistent, nominally significant correlations across thresholds. angular gyrus, and the left and right precuneus. These areas are shown in Figure 9. Their
correlation coefficients and respective p values are given in Table S1 in the Supporting Infor-
mation section. Cardiff data, Desikan–Killiany atlas. The node-specific analysis showed that the correlations
between the schizotypy score and the mean node degree of the sensorimotor network were
driven by the left paracentral lobule, the right postcentral gyrus, and the right superior frontal
gyrus. For the default mode network, the correlations between schizotypy score and the mean
clustering coefficient were driven by the left caudal middle frontal gyrus, the left isthmus cin-
gulate, the left lateral orbitofrontal gyrus, the left and right parahippocampal area, the left and Figure 8. Scatter plots for the correlations observed in the Munich data for the Desikan–Killiany
parcellation for NSthr = 55. The best fit line and its 95% confidence interval are also shown. Figure 8. Scatter plots for the correlations observed in the Munich data for the Desikan–Killiany
parcellation for NSthr = 55. The best fit line and its 95% confidence interval are also shown. Network Neuroscience Network Neuroscience 222 222 Increased structural connectivity in high schizotypy Figure 9. Cardiff data: AAL atlas. Nodes that drive the correlations between the schizotypy score
and the sensorimotor mean node degree (top left), sensorimotor mean clustering coefficient (top
right), default mode mean node degree (bottom left), and default mode mean clustering coeffi-
cient (bottom right). Node-Specific Analysis The large markers indicate the nodes of each network that drive the corre-
lations; these nodes are also labelled. The small markers indicate the remaining nodes of each
network. Downloaded from http://direct.mit.edu/netn/article pdf/7/1/213/2072088/netn_a_00279.pdf by guest on 13 April 2023 Downloaded from http://direct.mit.edu/netn/article-pdf/7/1/213/2072088/netn_a_00279.pdf by guest on 13 April 2023 2088/netn_a_00279.pdf by guest on 13 April 2023 Figure 9. Cardiff data: AAL atlas. Nodes that drive the correlations between the schizotypy score
and the sensorimotor mean node degree (top left), sensorimotor mean clustering coefficient (top
right), default mode mean node degree (bottom left), and default mode mean clustering coeffi-
cient (bottom right). The large markers indicate the nodes of each network that drive the corre-
lations; these nodes are also labelled. The small markers indicate the remaining nodes of each
network. right precuneus, the left and right thalamus, the right hippocampus, the right pars triangularis,
and the right rostral anterior cingulate. These areas are shown in Figure 10. Their correlation
coefficients and p values shown in Table S1 in the Supporting Information section. Munich data, Desikan–Killiany atlas. The node-specific analysis showed that the correlations
between schizotypy score and the mean node degree of the sensorimotor network were driven
by the left paracentral lobule, the left and right postcentral gyrus, the left supramarginal gyrus,
the right precentral gyrus, and the right superior frontal gyrus. The correlations between Network Neuroscience 223 Increased structural connectivity in high schizotypy Figure 10. Cardiff data: Desikan–Killiany atlas. Nodes that drive the correlations between the
schizotypy score and the sensorimotor mean node degree (left) and default mode mean clustering
coefficient (right). The large markers indicate the nodes of each network that drive the correla-
tions; these nodes are also labelled. The small markers indicate the remaining nodes of the
network. Downloaded from http://direct.mit.edu/netn/article-pdf/7/1/213/2072088/netn_a_00279.pdf by guest on 13 April 2023 Figure 10. Cardiff data: Desikan–Killiany atlas. Nodes that drive the correlations between the
schizotypy score and the sensorimotor mean node degree (left) and default mode mean clustering
coefficient (right). The large markers indicate the nodes of each network that drive the correla-
tions; these nodes are also labelled. The small markers indicate the remaining nodes of the
network. schizotypy score and the mean node degree of the visual network were driven by the left lin-
gual gyrus, the right cuneus, and the right fusiform area. These areas are shown in Figure 11. DISCUSSION We used graph theoretical metrics to investigate the topology of the structural brain networks
of healthy participants with a range of schizotypy scores. To the best of our knowledge, this is
the first time these properties are investigated in relation to schizotypy. We used two large
datasets, comprising 140 and 115 participants, respectively, collected at different locations,
in different MRI scanners and with different scanning parameters. Our results provide evidence that higher levels of schizotypy in healthy participants relate
to higher structural connectivity in the sensorimotor and default mode networks, with some
increases also possibly present in the visual network. No evidence of any alterations was found
for the auditory network. The edges of the networks were weighted by the inverse radial dif-
fusivity, which partly reflects differences in myelination or axon density (Chen et al., 2009;
Cykowski et al., 2010). Both myelination and axon density support effective communication
in the brain (Drakesmith et al., 2019; Lebel & Deoni, 2018). The edge weighting chosen
impacts our results because the clustering coefficient (which exhibited statistically significant
correlations with the schizotypy score for the sensorimotor and default mode network)
depends on the values of the edges. On the other hand, the node degree (which also exhibited
statistically significant correlations with the schizotypy score) is independent of the edge
weights and is only impacted by the number of edges. This means that had we used a different
measure to weight the edges (such as, for example, the number of streamlines or the fractional
anisotropy of the diffusion tensor, which are sometimes used in network studies), the results
pertaining to the node degree would have been the same. The positive correlations observed between the schizotypy score and the graph theoretical
metrics of the sensorimotor network were replicated across the two datasets and atlases. Even
though they only reached nominal statistical significance in the Munich data, they were per-
sistent across a large range of values of NSthr. On the other hand, the positive correlations
observed for the default mode network were not replicated in the Munich data. This could
be due to the slightly smaller sample or the lower resolution of the Munich data, or the fact
that there were fewer diffusion directions acquired in the diffusion data, as well as a lower
b-value—the latter two facts can impact the calculation of the radial diffusivity. Node-Specific Analysis Their correlation coefficients and p values shown in Table S1 in the Supporting Information
section. The visualizations of the nodes were done using BrainNet Viewer (Xia et al., 2013). 2088/netn_a_00279.pdf by guest on 13 April 2023 Figure 11. Munich data: Desikan–Killiany atlas. Nodes that drive the correlations between the
schizotypy score and the sensorimotor mean node degree (left) and visual mean node degree
(right). The large markers indicate the nodes of each network that drive the correlations; these
nodes are also labelled. The small markers indicate the remaining nodes of the network. Figure 11. Munich data: Desikan–Killiany atlas. Nodes that drive the correlations between the
schizotypy score and the sensorimotor mean node degree (left) and visual mean node degree
(right). The large markers indicate the nodes of each network that drive the correlations; these
nodes are also labelled. The small markers indicate the remaining nodes of the network. Network Neuroscience 224 Increased structural connectivity in high schizotypy DISCUSSION As mentioned in the Introduction, a pattern of lower functional connectivity has been
reported in the literature for schizophrenia patients compared to healthy controls, except for
some evidence of higher connectivity in the default mode network. This higher connectivity,
however, can be responsible for a failure to completely deactivate the default mode network
during tasks (as happens in healthy participants; Raichle et al., 2001), which can lead to com-
promised task performance in schizophrenia. Structural connectivity has also been observed
to be lower in schizophrenia, although the findings are less coherent, most likely due to dif-
ferences in the analysis pipelines used. In this context, there are two possible interpretations for
our results. The first possible interpretation is that our results support the notion that higher structural
connectivity (in the presence of higher schizotypy scores) is a protective characteristic against
schizophrenia. Specifically, the higher structural connectivity in the sensorimotor network of
healthy high-schizotypy participants could be enhancing communication among sensorimotor
regions, that is, regions implicated in action observation and imitation (Caspers et al., 2010),
which are functions crucial for successful social interactions. Additionally, the higher structural
connectivity in the default mode network could be leading to more effective communication
among regions that are responsible for the emotional engagement during social interactions
and the understanding others’ mental states (Li et al., 2014), or enhancing self-referential acti-
vation (Davey et al., 2016). This more effective communication could be protecting these
participants from the development of schizophrenia. This interpretation also indicates that
structural deficiencies in the sensorimotor and default mode networks should be further inves-
tigated as potential causes of schizophrenia. We note that such potential compensatory effects
have been previously reported. For example, increased frontal grey matter and higher frontal
activity in schizotypal personality disorder have been suggested as a possible reason for the
lower magnitude of cognitive and deficit symptoms in the disorder compared to schizophrenia
(Siever et al., 2002; Siever & Davis, 2004). At the same time, we also note that schizophrenia is
normally diagnosed in the late teenage years to the early 20s in men and in the late 20s to
early 30s in women. Some participants in the two samples we used were under 30 years old at
the time that the data were acquired. DISCUSSION A recent study (Wang et al., 2020a, 2020b) showed that high-schizotypy participants exhib-
ited increased white matter connectivity probability between the right middle frontal gyrus and
the right insula, and within the default mode network. Higher strength values were found
between the left superior frontal gyrus and the right rolandic operculum area, and between
the right superior frontal gyrus and the right medial superior frontal gyrus (Wang et al.,
2020a, 2020b). This pattern of higher structural connectivity in high schizotypy agrees with
our results. The same study also showed that this enhanced structural connectivity appears
alongside lower static functional connectivity in the sensorimotor and default mode networks
in the higher schizotypy participants. While the general pattern of higher white matter connec-
tivity in relation to higher schizotypy agrees with our finding, we note that differences between
the studies include that the study of Wang et al. (2020a, 2020b) had participants with higher
schizotypy scores than the ones in our study, they did not use graph theory in their analysis,
and their connectivity measure is more closely related to the number of streamlines rather than
to the myelination or to axonal density in the white matter tracts that we used. Other studies (Hazlett et al., 2012; Nakamura et al., 2005; Pfarr & Nenadic, 2020) have
shown altered microstructural properties in white matter tracts in various schizotypy dimen-
sions. For example, reduced fractional anisotropy (FA) of the cingulum bundle and the unci-
nate fasciculus was related to symptom severity in schizotypal personality disorder (Hazlett Network Neuroscience 225 Increased structural connectivity in high schizotypy et al., 2011, 2012). Also, the FA of the right superior longitudinal fasciculus (SLF) was nega-
tively correlated with disorganised schizotypy (Pfarr & Nenadic, 2020). In the same study (Pfarr
& Nenadic, 2020), the axial diffusivity of the right SLF, right anterior thalamic radiation (ATR),
right uncinate fasciculus (UF), and forceps minor was positively correlated with negative schi-
zotypy. Finally, the mean diffusivity of the right UF, the right ATR, and the forceps minor was
positively correlated with negative schizotypy. It should be pointed out that these results per-
tain to specific white matter tracts, in contrast to our results which pertain to the topological
organisation of the entire brain. Those studies also predominantly refer to specific schizotypy
dimensions rather than overall schizotypy. DISCUSSION Therefore, some of the higher schizotypy participants in
our samples may, at some time after the data were acquired, develop schizophrenia, which
would indicate that the increased structural connectivity in the sensorimotor and default mode
networks could not fully protect them from developing the disease. A second possible interpretation is that the white matter structural alterations that have been
reported in schizophrenia (Abdul-Rahman et al., 2011; Joo et al., 2018; Shon et al., 2018; van
den Heuvel et al., 2010; Zhu et al., 2016) may be the pathophysiological correlate or conse-
quence of worsening schizophrenia symptoms and/or pharmacological treatment, particularly
in chronic schizophrenia, rather than the cause of the disease. This is in agreement with the
dysconnection hypothesis (Friston et al., 2016), indicating that functional deficiencies precede
structural ones in the disease. Network Neuroscience 226 Increased structural connectivity in high schizotypy Our findings relating to the nodes of the subnetworks that drive the higher structural con-
nectivity can also be put in the context of existing findings in the literature. We note that there
were some similarities and some differences in the nodes driving the correlations for the three
dataset/atlas pairs. For example, the right postcentral gyrus drove the correlations between
schizotypy score and mean node degree for the sensorimotor network in all three cases
(Cardiff/AAL, Cardiff/Desikan–Killiany, and Munich/Desikan–Killiany), while the left paracen-
tral lobule and right superior frontal gyrus drove those correlations for the two cases (Cardiff/
Desikan–Killiany and Munich/Desikan–Killiany). Additionally, the left parahippocampal gyrus
and the left and right precuneus drove the correlations between the schizotypy score and the
mean clustering coefficient of the default mode network for both the Cardiff/AAL and
Cardiff/Desikan–Killiany cases. We now discuss the nodes that consistently drove our
observed correlations. s
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Downloaded from http://direct.mit.edu/netn/article-pdf/7/1/213/2072088/netn_a_00279.pdf by guest on 13 April 2023 The right postcentral gyrus has been shown to have lower functional connectivity with its
left counterpart in schizophrenia patients (Kaufmann et al., 2015). It has also been shown to
have lower grey matter density in schizophrenia patients (Glahn et al., 2008; Li et al., 2015). Additionally, the global brain functional connectivity of the postcentral gyrus was lower in
adolescent-onset schizophrenia patients compared to controls (Zhao et al., 2022). DISCUSSION The left paracentral lobule was found to have lower regional homogeneity, a quantity that
reflects local functional connectivity and integration of information processing in adolescent-
onset schizophrenia patients (Wang et al., 2018). It was also shown to have lower cortical
thickness in psychosis patients compared to healthy participants (van Lutterveld et al.,
2014), along with the left pars orbitalis, the right fusiform gyrus, and the right inferior temporal
gyrus. The resting-state global brain functional connectivity (measured via fMRI) of the right supe-
rior frontal gyrus was higher in schizophrenia patients and their siblings compared to controls
(Ding et al., 2019). The right superior frontal gyrus also showed lower grey matter in schizo-
phrenia patients compared to controls (Xiao et al., 2013). In healthy participants, the grey
matter volume of the superior frontal gyrus correlated positively with their SPQ-measured
schizotypy scores (Wang et al., 2020a, 2020b). Differential functional performance of the right
superior frontal gyrus in low versus high-schizotypy individuals (albeit measured via the
Unusual Experiences subscale of the short form of the Oxford and Liverpool Inventory of
Feelings and Experiences, O-LIFE; Mason et al., 2005) has also been reported (Premkumar
et al., 2012), where it was observed that in viewing rejection scenes, low schizotypy partici-
pants activated that gyrus, while high-schizotypy participants deactivated it. The higher node degree of these brain areas (right postcentral gyrus, left paracentral lobule,
and right superior frontal gyrus) within their respective subnetworks (sensorimotor and default
mode) could be responsible for enhanced neural communication in higher schizotypy individ-
uals. This could be protective against some of the functional deficiencies relating to these sub-
networks which have been observed in schizophrenia. Abnormalities in the left parahippocampal gyrus region are a ubiquitous finding in schizo-
phrenia (Friston et al., 1992). In an EEG study (Soni et al., 2020), the left parahippocampal
gyrus was found to have higher power spectral density in patients compared to controls. Sev-
eral structural MRI studies have also reported lower volume in the bilateral parahippocampal
gyrus in schizophrenia (Honea et al., 2005; Lawrie & Abukmeil, 1998; Sim et al., 2006; Soh
et al., 2015; Wright et al., 2000). The precuneus was shown to have higher connectivity with the posterior cingulate cortex in
schizophrenia patients (Sendi et al., 2021). DISCUSSION The resting-state functional connectivity between Network Neuroscience 227 Increased structural connectivity in high schizotypy precuneus and hippocampus was found to have deficits in unmedicated patients with schizo-
phrenia compared to healthy controls (Kraguljac et al., 2014). The functional connectivity
between the precuneus and the bilateral Heschl’s gyri was found to be abnormal in first-
episode schizophrenia (Guo et al., 2014). In healthy participants, the schizotypy score
was positively associated with gyrification in the precuneus in a study that used the Munich
data used in our study (Meller et al., 2019). The precuneus is involved in self-processing,
mental imagery, episodic memory retrieval, and discrimination of self versus others (Cavanna
& Trimble, 2006); all these processes might be related to inferring the intention of others,
which corroborates the possible role of the precuneus for symptoms within the psychosis
spectrum. The higher clustering coefficient of the left parahippocampal gyrus and the precuneus
within the default mode network implies that their neighbourhoods are better connected in
higher schizotypy individuals. This could be compensating for the functional abnormalities
observed in these brain areas in schizophrenia. Specifically, if the nodes connected to the pre-
cuneus can be used to bypass it, then any functional deficiencies of the precuneus would not
affect the brain’s performance in the related functions. The differences in the nodes that drive the correlations for the different atlases used can be
attributed to the fact that the two atlases allocate the cortical and subcortical areas differently,
with the AAL atlas comprising 90 cortical and subcortical areas while the Desikan–Killiany
atlas comprising 82 cortical and subcortical areas with the remaining two areas corresponding
to the two hemispheres of the cerebellum. It has been shown that there are differences in the
network sparsity (and therefore the node degree) and in the clustering coefficient as a result of
the choice of atlas, for example, AAL versus Desikan–Killiany, for the same tractography
methods (Parker et al., 2014). We note, however, that the study presented by Parker et al. (2014) used a different edge weight (i.e., connection density) in contrast to the inverse radial
diffusivity used in our analysis. A strength of our study stems from the fact that we used two large datasets, comprising 140
and 115 participants. CONCLUSIONS We investigated the topological properties of structural brain subnetworks in healthy partici-
pants of varying schizotypy scores. We found evidence of higher structural connectivity of the
sensorimotor and default mode networks in participants with high-schizotypy score. This may
indicate a possible protective mechanism against schizophrenia or could provide further sup-
port for the hypothesis that functional (rather than structural) dysconnection could be the
underlying cause of schizophrenia. ACKNOWLEDGMENTS For the purpose of Open Access, the author has applied a CC BY public copyright license to
any Author Accepted Manuscript version arising from the submission. AUTHOR CONTRIBUTIONS Eirini Messaritaki: Conceptualization; Formal analysis; Funding acquisition; Investigation;
Methodology; Software; Visualization; Writing – original draft; Writing – review & editing. Sonya Foley: Data curation; Formal analysis. Kali Barawi: Data curation. Ulrich Ettinger: Data
curation; Methodology; Writing – review & editing. Derek K. Jones: Funding acquisition;
Methodology; Supervision; Writing – review & editing. FUNDING INFORMATION Eirini Messaritaki, Wellcome Trust (https://dx.doi.org/10.13039/100010269), Award ID:
204824/Z/16/Z. Derek K. Jones, Wellcome Trust (https://dx.doi.org/10.13039/100010269),
Award ID: 096646/Z/11/Z. Derek K. Jones, Wellcome Trust (https://dx.doi.org/10.13039
/100010269), Award ID: 104943/Z/14/Z. Sonya Foley, Wellcome Trust (https://dx.doi.org/10
.13039/100010269), Award ID: 504182. Ulrich Ettinger, Deutsche Forschungsgemeinschaft
(https://dx.doi.org/10.13039/501100001659), Award ID: ET 31/2-1. Eirini Messaritaki, Wellcome Trust (https://dx.doi.org/10.13039/100010269), Award ID:
204824/Z/16/Z. Derek K. Jones, Wellcome Trust (https://dx.doi.org/10.13039/100010269), Eirini Messaritaki, Wellcome Trust (https://dx.doi.org/10.13039/100010269), Award ID:
204824/Z/16/Z. Derek K. Jones, Wellcome Trust (https://dx.doi.org/10.13039/100010269),
Award ID: 096646/Z/11/Z. Derek K. Jones, Wellcome Trust (https://dx.doi.org/10.13039 .13039/100010269), Award ID: 504182. Ulrich Ettinger, Deutsche Forschungsgemeinschaft
(https://dx.doi.org/10.13039/501100001659), Award ID: ET 31/2-1. SUPPORTING INFORMATION Supporting information for this article is available at https://doi.org/10.1162/netn_a_00279. DISCUSSION Despite the differences in scanners, scanning sequences, and the lan-
guage in which the SPQ was administered, the findings of stronger structural connectivity in
the sensorimotor network were replicated in the two datasets, and employing two different
atlases for brain parcellation. Additionally, the finding of stronger structural connectivity in
the default mode network was replicated when using the two different atlases in the Cardiff
dataset. Our tractography generation is state of the art, utilising an algorithm that limits the
presence of false positive tracts. Using the inverse radial diffusivity to assign strength to the
structural connections relates them to the myelination and the axonal density of the white
matter tracts, which are measures that better reflect efficient brain communication than the
more frequently used number of streamlines or FA (Messaritaki et al., 2021). One limitation
of our study is the fact that the schizotypy scores went only up to 43, when the maximum
possible value of the SPQ total score is 74. This is a typical range for studies of healthy vol-
unteers but indicates that our results should be interpreted with regards to that population,
while generalisations to individuals with very high-schizotypy levels need to be drawn with
caution. In the future, MRI studies should include dedicated sequences with differential sen-
sitivity to myelin (e.g., magnetization-transfer imaging (Wolff & Balaban, 1989) or myelin-
water-based imaging (Mackay et al., 1994)) and to axonal properties, including higher b-value
acquisitions, to provide higher sensitivity to the intra-axonal signal fraction and therefore mea-
sures such as apparent axon density (Assaf & Basser, 2005; Zhang et al., 2012) and axon
diameter (Barazany et al., 2009). This will allow us to further investigate structural connectiv-
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The Temporal Relationship Between Terrestrial Gamma‐Ray Flashes and Associated Optical Pulses From Lightning
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Citation (APA):
Skeie, C. A., Østgaard, N., Mezentsev, A., Bjørge‐Engeland, I., Marisaldi, M., Lehtinen, N., Reglero, V., &
Neubert, T. (2022). The temporal relationship between Terrestrial Gamma‐ray flashes and associated optical
pulses from lightning. Journal of Geophysical Research: Atmospheres, 127(17), Article e2022JD037128.
https://doi.org/10.1029/2022JD037128 Downloaded from orbit.dtu.dk on: Oct 24, 2024 Downloaded from orbit.dtu.dk on: Oct 24, 2024 Downloaded from orbit.dtu.dk on: Oct 24, 2024 General rights
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You may not further distribute the material or use it for any profit-making activity or commercial gain 1. Introduction Received 16 MAY 2022
Accepted 18 AUG 2022 Terrestrial Gamma-ray Flashes (TGFs) are bursts of hard X- and gamma-rays produced via bremsstrahlung from
runaway electrons accelerated in the electric fields of thunderstorms (Dwyer, 2012; Gurevich et al., 1992; Moss
et al., 2006; Wilson, 1925). The TGFs are reported to typically last a few tens, up to a few hundred microsec-
onds, and have individual photon energies up to ∼40 MeV (Briggs et al., 2013; Fishman et al., 1994; Marisaldi
et al., 2010, 2014; Smith et al., 2005). Analysis of the energy spectra of TGFs and lightning radio atmospherics
indicate that they are produced below 21 km altitude, most likely between 10 and 15 km (Carlson et al., 2007;
Cummer et al., 2014; Dwyer & Smith, 2005; Lindanger et al., 2021; Mailyan et al., 2016; Pu et al., 2019; Stanley
et al., 2006; Xu et al., 2012). The underlying mechanism for creating the observed TGF photon fluxes is still
unclear. There are two leading models for explaining the observed fluxes based on where and how the electrons
are accelerated. Both theories build on electrons being accelerated in electric fields that are strong enough to
overcome the friction force of the air, in what is called the runaway process (Wilson, 1925), before being multi-
plied in a relativistic runaway electron avalanche process (Gurevich et al., 1992). In the first model, an electron
flux is created in an avalanche process developing in the large-scale electric fields within the thunderclouds. Back-scattered X-rays created by bremsstrahlung, and positrons created by pair-production, seed additional elec-
tron avalanches in what is called a relativistic feedback mechanism (Dwyer, 2008). In the other model the initial
electron flux is created in small, intense, transient overlapping electric fields of streamers, lightning leader and
thundercloud electric field. The overlapping electric fields are strong enough to let electrons runaway, and then
undergo bremsstrahlung and produce X- and gamma-rays (Celestin & Pasko, 2011; Moss et al., 2006). These two
models can also be at play simultaneously, as one does not exclude the other. TGFs were first reported by Fishman et al. (1994), using the Burst and Transient Source Experiment on the
Compton Gamma Ray Observatory. © 2022. The Authors.
This is an open access article under
the terms of the Creative Commons
Attribution License, which permits use,
distribution and reproduction in any
medium, provided the original work is
properly cited. The Temporal Relationship Between Terrestrial Gamma-Ray
Flashes and Associated Optical Pulses From Lightning
C. A. Skeie1
, N. Østgaard1
, A. Mezentsev1
, I. Bjørge-Engeland1
, M. Marisaldi1,2
,
N. Lehtinen1
, V. Reglero3, and T. Neubert4 The Temporal Relationship Between Terrestrial Gamma-Ray
Flashes and Associated Optical Pulses From Lightning The temporal relationship between Terrestrial Gamma‐ray flashes and associated optical
pulses from lightning The temporal relationship between Terrestrial Gamma‐ray flashes and associated optical
pulses from lightning Skeie, C. A.; Østgaard, N.; Mezentsev, A.; Bjørge‐Engeland, I.; Marisaldi, M.; Lehtinen, N.; Reglero, V.; Neubert,
T. Published in:
Journal of Geophysical Research: Atmospheres
Link to article, DOI:
10.1029/2022JD037128 Published in:
Journal of Geophysical Research: Atmospheres Document Version
Publisher's PDF, also known as Version of record Link back to DTU Orbit Citation (APA):
Skeie, C. A., Østgaard, N., Mezentsev, A., Bjørge‐Engeland, I., Marisaldi, M., Lehtinen, N., Reglero, V., &
Neubert, T. (2022). The temporal relationship between Terrestrial Gamma‐ray flashes and associated optical
pulses from lightning. Journal of Geophysical Research: Atmospheres, 127(17), Article e2022JD037128. https://doi.org/10.1029/2022JD037128 Key Points: A., Østgaard, N., Mezentsev,
A., Bjørge-Engeland, I., Marisaldi,
M., Lehtinen, N., et al. (2022). The
temporal relationship between terrestrial
gamma-ray flashes and associated
optical pulses from lightning. Journal
of Geophysical Research: Atmospheres,
127, e2022JD037128. https://doi. org/10.1029/2022JD037128
Received 16 MAY 2022
Accepted 18 AUG 2022 Correspondence to:
C. A. Skeie,
Chris.Skeie@uib.no Skeie, C. A., Østgaard, N., Mezentsev,
A., Bjørge-Engeland, I., Marisaldi,
M., Lehtinen, N., et al. (2022). The
temporal relationship between terrestrial
gamma-ray flashes and associated
optical pulses from lightning. Journal
of Geophysical Research: Atmospheres,
127, e2022JD037128. https://doi. org/10.1029/2022JD037128 Skeie, C. A., Østgaard, N., Mezentsev,
A., Bjørge-Engeland, I., Marisaldi,
M., Lehtinen, N., et al. (2022). The
temporal relationship between terrestrial
gamma-ray flashes and associated
optical pulses from lightning. Journal
of Geophysical Research: Atmospheres,
127, e2022JD037128. https://doi.
org/10.1029/2022JD037128 Received 16 MAY 2022
Accepted 18 AUG 2022 Key Points: C. A. Skeie1
, N. Østgaard1
, A. Mezentsev1
, I. Bjørge-Engeland1
, M. Marisaldi1,2
,
N. Lehtinen1
, V. Reglero3, and T. Neubert4 C. A. Skeie1
, N. Østgaard1
, A. Mezentsev1
, I. Bjørge-Engeland1
, M. Marisald
N. Lehtinen1
, V. Reglero3, and T. Neubert4 • Terrestrial Gamma-ray Flashes
(TGFs) are produced before or
simultaneously with the onset of an
optical pulse 1Birkeland Centre for Space Science, Institute of Physics and Technology, University of Bergen, Bergen, Norway,
2INAF-OAS Bologna, Bologna, Italy, 3Imaging Processing Laboratory, University of Valencia, Valencia, Spain, 4National
Space Institute, Technical University of Denmark, Kongens Lyngby, Denmark • For some events the delay of the
optical pulse is too long to be
explained by the light scattering in
the cloud Abstract We present 221 Terrestrial Gamma-ray Flashes (TGFs) and associated optical pulses observed
by the Atmosphere-Space Interactions Monitor (ASIM) on board the International Space Station. The events
were detected between the end of March 2019 and November 2020 and consist of X- and gamma-ray energy
detections, as well as photometer data (180–230, 337, and 777 nm) and optical camera data (337 and 777 nm). Using the available ASIM data and applying a consistency check based on TGF characteristics and lightning
detections from lightning radio atmospherics close in time, we determine the most likely position of the TGFs
in relation to the photometer field of view (FoV), and the association to the observed optical pulses. Out of the
221 events we find 72 events where the TGF and optical data are determined to be associated and inside the
photometer FoV. Using the measured TGF durations and the time between the onsets of the TGFs and optical
pulses we find: (a) That the TGF onsets are always before or at the same time as the optical pulse onsets (taking
into account cloud scattering). (b) A tendency for longer duration TGFs to have longer delays between onsets. (c) Two groups of events: (a) where there is a possible overlap between the TGFs and the optical emissions,
as the TGFs last longer than the delay between onsets and (b) where the TGFs and optical emissions do not
overlap, as there are long delays between the onsets, which cannot be explained by cloud scattering. • Longer duration TGFs tend to have
longer delays between onsets of the
TGFs and the optical pulses Correspondence to:
C. A. Skeie,
Chris.Skeie@uib.no
Citation:
Skeie, C. 10.1029/2022JD037128
RESEARCH ARTICLE Key Points: Journal of Geophysical Research: Atmospheres Journal of Geophysical Research: Atmospheres 10.1029/2022JD037128 The atmosphere-space interactions monitor (ASIM) is the first instrument specifically designed to observe TGFs,
as well as transient luminous events. ASIM is mounted on the Columbus module of the International Space
Station (ISS), and has been gathering data since April 2018. ASIM has multiple detectors consisting of high and
low energy X- and gamma-ray detectors, photometers, and optical cameras (Neubert et al., 2019). Past studies using radio data have shown that TGFs likely occur during the early phase of intracloud (IC) lightning
(Lu et al., 2010; Pu et al., 2019; Shao et al., 2010; Østgaard et al., 2013, 2021). This is also shown in Lindanger
et al. (2022) who used TGF detections paired with optical measurements of lightning activity to show that TGFs
are produced during the initial phase of a lightning flash. The sequence of the TGF and optical signal of the flash
is still uncertain. Østgaard et al. (2013) were the first to report simultaneous observation of a TGF and optical
light from lightning. Using a TGF detected by the Ramaty High Energy Solar Spectroscopic Imager (RHESSI)
and optical data from the lightning imaging sensor (LIS) on board the tropical rainfall measuring mission satel-
lite, they conclude that the TGF was produced in the initial stage of an IC lightning propagating upwards in the
cloud. Gjesteland et al. (2017) reinvestigated the TGF-optical sequence using two TGFs detected by RHESSI and
optical data from LIS, as well as lightning radio atmospherics from the World Wide Lightning Location Network. However, due to the relative timing uncertainties of the instruments (±1.6 ms), stemming mainly from the time
resolution of LIS, they could not determine the sequence of TGF and optical signal of the flash. Alnussirat
et al. (2019) also investigated the sequence of TGF and optical signals, using optical data from the Geostationary
Lightning Mapper, with a time resolution of 2 ms and TGFs detected by the Fermi Gamma-ray Burst Monitor. Based on their observations, they find that the TGFs are likely produced during the last stages of the development
of the lightning leader channel. However, they could not determine the sequence of TGF-optical emission itself. Journal of Geophysical Research: Atmospheres More recent observations from ASIM have shown that the majority of TGFs occur before or at the onset of the
optical emissions, given the uncertainties in the measurements (Heumesser et al., 2021; Neubert et al., 2020;
Østgaard, Neubert, et al., 2019). A relevant aspect in the determination of the sequence of the TGF and optical
emissions is the cloud scattering of the optical signals. Satellite detection of optical light from lightning including
cloud effects such as scattering, has recently been modeled (Luque et al., 2020; Peterson, 2020). The optical light
emitted from lightning in different wavelength bands is associated with different processes in a lightning flash,
such as the hot channel of a lightning discharge or streamer activity before the discharge (Chanrion et al., 2019). In this work we will investigate the temporal relationship between TGFs and optical emissions from lightning. For this purpose we will use a set of upwards directed TGFs with accompanying optical detections observed
from space by ASIM. We start by investigating the sequence of TGF and the main optical pulse (defined in
Section 3.6), which has been addressed using ASIM before in Østgaard, Neubert, et al. (2019) and Heumesser
et al. (2021). The results of Østgaard, Neubert, et al. (2019) were hampered by the relative timing accuracy
(±80 µs) between the Modular X- and Gamma-ray Sensor (MXGS) and modular multi-spectral imaging array
(MMIA) instruments. In this paper, we will use a larger data sample from a later period, where the relative accu-
racy between the instruments has been improved to ±5 µs, and more sophisticated and accurate methodology. Heumesser et al. (2021) also analyzed TGFs and optical data observed by ASIM, and concluded that the sequence
of TGF-optical cannot be addressed due to the uncertainties in timing and the model they used. For this work we
have carefully inspected each event and applied a consistency check (outlined in Section 3), where we determine
the relationship of each detected TGF and optical pulse. For this purpose we inspected the geolocation source of
the radio atmospherics from the lightning discharges, together with the optical detections of lightning and TGF
characteristics to determine which of the observed optical pulses are most likely associated with the TGFs. Journal of Geophysical Research: Atmospheres The
events where the TGF and optical pulse are found to be associated will then be used to investigate the sequence
of the TGF and optical pulse, as well as the relationship between TGF durations and the time delay between TGF
and the onset of the optical pulse, to help understanding the processes involved and sequence of events. 1. Introduction Since then many observations of TGFs have been made, using mostly
satellite-based (Briggs et al., 2010; Marisaldi et al., 2010, 2014; Østgaard, Neubert, et al., 2019; Smith
et al., 2005), but also aircraft (Bowers et al., 2018; Smith et al., 2011), and ground based instruments (Abbasi
et al., 2018; Dwyer et al., 2004, 2012; Hare et al., 2016; Kereszy et al., 2022; Tran et al., 2015; Wada et al., 2019). 1 of 14 1 of 14 SKEIE ET AL. SKEIE ET AL. 3. Methodology Between end of March 2019 and November 2020 we have observed 221 TGFs, where also optical data from
MMIA are available with a relative timing accuracy of ±5 µs. To determine the association between the TGFs
and the optical pulses we investigated the photometer data in three main steps. (a) We search for an optical pulse
in the 337 and 777 nm optical band within 5 ms of the TGF. If there is no optical pulse the TGF is most likely
outside the MMIA FoV and we exclude the event. (b) For the remaining events the MMIA FoV is determined and
the surrounding lightning activity within 15 min of the TGF is investigated to determine a possible location of the
TGF. (c) A consistency check is performed (as outlined in Section 3.3) using the surrounding lightning activity,
camera images (83.3 ms resolution), and TGF characteristics, such as number of counts and their energies. 2. Instruments and Data The relative timing accuracy between the MXGS and
MMIA instrument was ±80 µs until a software update in April 2019, which
reduced it to its nominal performance of ±5 µs. Due to a non-optimal timing
interface between the ISS and the ASIM instrument, the absolute timing
accuracy is found to be ∼−10 to +40 ms (determined using lightning detec-
tion location from lightning radio atmospherics). This timing accuracy can be
improved for some events by using lightning detections together with optical
data to reduce the absolute timing accuracy to ±1 ms. Figure 1. Illustration of how to determine the corner-point of the modular
multi-spectral imaging array (MMIA) field of view (FoV) projected from the
International Space Station (ISS) to ground level on Earth in three steps. 1)
Rotation due to ISS yaw angle (±Z-direction), 2) move according to roll angle
and the 5° tilt of the MMIA instrument in the roll direction (±Y-direction). 3)
Move according to the pitch angle (±X-direction). Lightning radio atmospherics used in this work is provided by Vaisala's
Global Lightning Detection Network (GLD360) and give us time and loca-
tion data of lightning flashes. GLD360 is a ground-based very-low-frequency
(VLF) and lower part of low-frequency (LF) radio lightning detection network which employs time of arrival
and magnetic direction finding at each sensor to determine the location of individual lightning discharges. The
expected GLD360 detection efficiency is ∼75%–85% for cloud-to-ground flashes, ∼40%–50% for IC pulses, with
∼2–6 km uncertainty in median location accuracy (Demetriades et al., 2010; Said & Murphy, 2016). irection). 2. Instruments and Data The ASIM payload (Neubert et al., 2019) on board the ISS consists of two main instruments: the MMIA and
the MXGS. The MMIA (Chanrion et al., 2019) consists of three photometers and two cameras, which are tilted
5° upwards from nadir (toward starboard of ISS) to avoid potential obstructions from payloads on the bottom of
the mounting platform. The photometers operate in 180–230 nm (UV), 337 nm (blue) with a 4 nm bandwidth,
777.4 nm (red) with a 5 nm bandwidth, and have a sample rate of 100 kHz. The two optical cameras capture up to
12 frames per second, operate in the 337 and 777 nm bands, and have a 400 × 400 m resolution at nadir. Both the 2 of 14 SKEIE ET AL. SKEIE ET AL. Journal of Geophysical Re
Figure 1. Illustration of how to determine the corner-point of the modular
multi-spectral imaging array (MMIA) field of view (FoV) projected from the
International Space Station (ISS) to ground level on Earth in three steps. 1)
Rotation due to ISS yaw angle (±Z-direction), 2) move according to roll angle
and the 5° tilt of the MMIA instrument in the roll direction (±Y-direction). 3)
Move according to the pitch angle (±X-direction). Journal of Geophysical Research: Atmospheres 10.1029/2022JD037128 337 and 777 nm photometers and cameras have a square field of view (FoV)
(80° diagonal), while the UV photometer has a circular FoV (80° diameter). As we are only using the 777 and 337 nm band in this study we will refer to
the square FoV as MMIA FoV throughout this paper. To prevent damage by
sunlight, the MMIA instrument is only active during night time. The MXGS
(Østgaard, Balling, et al., 2019) consists of a high and a low energy detector
(LED). The high-energy detector (HED) is always active while outside the
South Atlantic Anomaly, detects energies between 300 keV and 30 MeV, and
has a time resolution of 28.7 ns. The LED is only active during night, due to
optical photon contamination during day time, detects energies between ∼50
and 400 keV, and has a resolution of 1 µs. The ASIM instrument includes
a cross triggering system between MXGS and MMIA, such that if either
instrument triggers data from both will be kept for a period of ∼2 s, centered
on the trigger time. Journal of Geophysical Research: Atmospheres 10.1029/2022JD037128 3.2. Lightning Detections Using the location data from lightning radio atmospherics (detected by GLD360) in the surrounding area from
within ±1,000 s we get three different scenarios. (a) All lightning activity is outside the MMIA FoV, meaning the
TGF is most likely from outside the FoV. (b) All lightning activity is inside the MMIA FoV, meaning the TGF
is most likely from inside the FoV. (c) There is lightning activity both inside and outside the MMIA FoV. For all
of these groups of events we apply the consistency check (see Section 3.3), where the lightning activity is used
together with (a) TGFs individual photon energies and fluence and (b) camera images. The GLD360 detections are also used to improve the absolute timing accuracy of ASIM, wh The GLD360 detections are also used to improve the absolute timing accuracy of ASIM, which is found to be
−10 to +40 ms before improvement. For many of the events we can improve the absolute timing of ASIM by time
alignment of the measured optical pulses and GLD360 detections. This is done by first finding the ISS time of
the GLD360 detections, by adding the lights' travel time from source to the ISS, to the given GLD360 detection
time. We then use multiple triggers of MMIA data (up to three, consisting of the trigger containing the TGF and
one trigger before and after the trigger containing the TGF) to align as many optical pulse peaks as possible to
the GLD360 detections. A minimum of two alignments is required, with a minimum of one optical pulse aligned
from the MMIA trigger containing the TGF. Aligning the GLD360 detections to the optical pulse peaks is practical, although this approach does not take into
account the time delay due to light scattering through the clouds. However, given the typical rise times of the
optical pulses, this is well within the error of the method. The lightning detection locations and camera images
are then checked for consistency. Using this technique we can get the absolute timing accuracy between MMIA
and the GLD360 detections down to ±1 ms. This method of improving absolute timing accuracy has already
been implemented in Maiorana et al. (2021), Lindanger et al. (2022), and independently developed and applied in
Heumesser et al. (2021). Using the method outlined here we found 95 alignments for the total sample of events. 3.3. Consistency Check A consistency check is performed to determine if the TGFs are likely to be within the MMIA FoV and have an
association with the optical pulses. For this purpose we use the TGF fluence and individual photon energies,
and compare to optical camera and lightning activity. For the consistency check we consider a TGF emission
half-cone of 30–40° without tilt. The TGF fluence is expected to be reduced as the distance between the TGF and
ISS-footpoint increases. This is due to the scattering of photons in the atmosphere and the increasing distance
(1/R 2 effect). Furthermore, we expect TGFs observed within the production cone to have more high energy
(above energy channel 1,000, which is approximately 10 MeV) counts than the TGFs from outside the production
cone. For TGFs observed outside the initial production cone the photons will have undergone Compton scatter-
ing and have reduced energies (Carlson et al., 2007; Gjesteland et al., 2011; Lindanger et al., 2021). This means
the TGFs with no or very few counts with high energies are more likely to be produced outside the MMIA FoV,
because the half-cone angle (30–40°) is similar to the MMIA FoV (diagonal angle of 40°). We also investigate
the lightning activity surrounding the TGF and check for a GLD360 detection associated to the TGF and optical
pulse. If such a pulse is found we compare the location of the GLD360 detection to the optical camera, as well
as the TGF characteristics. We do not use any of these assumptions as hard limitations, but as an indicator to
differentiate TGFs inside the MMIA FoV from those outside the MMIA FoV. 3.1. MMIA Field of View To determine the square MMIA photometer FoV we first interpolate the ISS foot point at the time of the TGF,
using the closest ISS locations before and after the TGF, as well as the ISS velocity and the difference in time
between the TGF detection and the two points. From the ISS foot-point we map out the 337 and 777 nm photom-
eter FoV, which is a square with 80° diagonal for both photometers (Chanrion et al., 2019), with the sides going
along and across the ISS direction of travel (X and Y in Figure 1). This is done by using eight points, namely the
four corners of the square and points at the middle of each side. These eight points are then shifted using yaw,
roll, and pitch angles of the ISS at the time of the TGF, as well as the 5° tilt in the roll direction, as illustrated in
Figure 1. The photometer FoV was compared with coastline camera images, acquired specifically to optimize
the pointing accuracy. The difference in the determined FoV and coastal camera images was found to be at most
∼20 km, which is acceptable for the purposes of this study. 3 of 14 3 of 14 SKEIE ET AL. SKEIE ET AL. Journal of Geophysical Research: Atmospheres Journal of Geophysical Research: Atmospheres Journal of Geophysical Research: Atmospheres 10.1029/2022JD037128 Figure 2. Example of an event where we have determined the Terrestrial Gamma-ray Flash (TGF) to be inside the modular multi-spectral imaging array (MMIA) field
of view (FoV) and associated to the observed optical pulse. (a) Detected counts in high-energy detector (HED). (b–d) Light curves from the photometers, 337, 777, and
180–230 nm. (e) Map of the area around the International Space Station (ISS) footpoint. The blue square shows the MMIA FoV, the black dots are lightning activity
within ±15 min of the TGF, the magenta stars are lightning activity within a second of the TGF, and the green star shows the lightning detection aligned to the optical
pulse. (f, g) Camera 337 and 777 nm show the optical camera images from the frame containing the TGF (Y-axis is along the flight path of the ISS, which is always
eastwards/right in the images). Figure 2. Example of an event where we have determined the Terrestrial Gamma-ray Flash (TGF) to be inside the modular multi-spectral imaging array (MMIA) field
of view (FoV) and associated to the observed optical pulse. (a) Detected counts in high-energy detector (HED). (b–d) Light curves from the photometers, 337, 777, and
180–230 nm. (e) Map of the area around the International Space Station (ISS) footpoint. The blue square shows the MMIA FoV, the black dots are lightning activity
within ±15 min of the TGF, the magenta stars are lightning activity within a second of the TGF, and the green star shows the lightning detection aligned to the optical
pulse. (f, g) Camera 337 and 777 nm show the optical camera images from the frame containing the TGF (Y-axis is along the flight path of the ISS, which is always
eastwards/right in the images). that the optical pulses come from the same location as the GLD360 detections. Panel (a) shows the counts, with
energies from the HED. The TGF has a hard spectrum (five counts above channel 1,000) with many counts (97)
in a short time interval (∼100 µs), which is consistent with a TGF within a small radial distance ∼200–300 km
to the ISS foot-point, that is, within the MMIA FoV. ISS is moving eastward and the camera images have Y-axis
along the path and X-axis toward starboard. This all indicates that the TGF is from within the MMIA FoV and
associated to the observed optical pulse. 3.4. Event Examples Following are examples that show events where the consistency check is used to help determine the TGF-optical
pulse association. Following are examples that show events where the consistency check is used to help determine the TGF-optical
pulse association. Figure 2 shows an example of an event where we have determined that the TGF and optical pulse is associated. For this event enough GLD360 detections could be aligned with optical pulses so that the total timing accuracy
was reduced to ±1 ms. As seen panel (e) of the figure, most lightning activity is inside the MMIA FoV, with
multiple lightning detections within 1 s of the TGF clustered at the same area. The magenta stars show the
GLD360 detections within 1 s of the TGF time, with the green star showing the GLD360 detection found to be
aligned in time to the optical pulse. No other GLD360 detections were found to be within the −10 to +40 ms
window (blue stars), corresponding to the total timing accuracy of ASIM. The camera images (f and g) support 4 of 14 SKEIE ET AL. SKEIE ET AL. Journal of Geophysical Research: Atmospheres Figure 3 shows another example of an event where the TGF is determined to be within the MMIA FoV. For
this event there is only lightning activity within the MMIA FoV. There were not enough GLD360 detections to
improve the timing accuracy for this event. Two GLD360 detections are seen inside the MMIA FoV within the
minimum and maximum of the absolute timing correction (blue stars). One of the detections is most likely an
ionospheric reflection, considering the time, distance, and peak currents (opposite but similar magnitude). This is
also supported by the camera images, where only one active area is seen. An ELVE (Emission of Light and Very
Low Frequency perturbations due to Electromagnetic Pulse Sources) event is observed (UV pulse starting at 0 µs)
at the same time as the TGF. Thirteen such ELVEs were found in this data set, these events are also a part of the
data set analyzed and presented in Bjørge-Engeland et al. (2022). A total of 72 events was found, where we could determine an association between the TGF and the optical pulse
such as the events shown in Figures 2 and 3. Fourty five absolute timing corrections were determined for these
72 events, where 33 ended up with a GLD360 detection aligned with the optical pulse associated to the TGFs. Figure 4 shows an event for which we have determined that the TGF is not from within the MMIA FoV, and
therefore the observed optical pulse is not associated with the TGF. The lightning activity map (e) shows there
are many centers of activity outside the MMIA FoV, but no lightning activity within 1 s of the TGF (magenta,
blue, or green stars). The camera images (f–g) show that the optical pulses come from a location close to the ISS
foot-point. The TGF (a) is found to be long, with relatively few counts in HED (19) and only one count above
energy channel 1,000. The TGF characteristics in this instance do not match our expectations of a TGF found 5 of 14 5 of 14 SKEIE ET AL. SKEIE ET AL. Journal of Geophysical Research: Atmospheres 10.1029/2022JD037128 Figure 3. Example of an event where we have determined the Terrestrial Gamma-ray Flash (TGF) to be inside the Modular multi-spectral imaging array (MMIA) field
of view (FoV) and associated to the observed optical pulse, and there is only lightning activity from within the FoV. Journal of Geophysical Research: Atmospheres (a) Detected counts in high-energy detector (HED)
(b–d) Light curves from the photometers, 337, 777, and 180–230 nm. (e) Map of the area around the International Space Station (ISS) footpoint. The blue square show
the MMIA FoV, the black dots are lightning activity within ±15 min of the TGF, the magenta stars are lightning activity within a second of the TGF, and the green star
shows the lightning detection aligned to the optical pulse. (f, g) Cameras 337 and 777 nm show the optical camera images from the frame containing the TGF (Y-axis i
along the flight path of the ISS, which is always eastwards/right in the images). Figure 3. Example of an event where we have determined the Terrestrial Gamma-ray Flash (TGF) to be inside the Modular multi-spectral imaging array (MMIA) field
of view (FoV) and associated to the observed optical pulse, and there is only lightning activity from within the FoV. (a) Detected counts in high-energy detector (HED). (b–d) Light curves from the photometers, 337, 777, and 180–230 nm. (e) Map of the area around the International Space Station (ISS) footpoint. The blue square shows
the MMIA FoV, the black dots are lightning activity within ±15 min of the TGF, the magenta stars are lightning activity within a second of the TGF, and the green star
shows the lightning detection aligned to the optical pulse. (f, g) Cameras 337 and 777 nm show the optical camera images from the frame containing the TGF (Y-axis is
along the flight path of the ISS, which is always eastwards/right in the images). inside the MMIA FoV and close to the ISS foot-point. In this case it is likely that the TGF is produced in one of
the active areas outside the FoV and is not associated with the optical pulses we observe. We found 57 events of
this type, where the TGF is most likely outside the FoV, and consequently not included in our study. The last group of events is the 88 events excluded in the beginning where we have no association, as there is no
observed optical pulse within ±5 ms of the TGF. Figure 5 shows one of these events, with no lightning activity
within the MMIA FoV as further support. Some lightning activity is observed within 1 s of the TGF just outside
the MMIA FoV, shown as the three magenta stars. Journal of Geophysical Research: Atmospheres The TGF has few counts, with energies below channel 300,
which is consistent with being produced outside the MMIA FoV. For four events there are difficulties in determining optical pulse onset or TGF association to the optical pulse. This is due to there either being multiple TGFs within the 5 ms time window, but only one optical pulse, or
difficulties identifying the pre-activity and determining the onset of the optical pulse. As we do not want to make
an assumption on which TGF is associated to the optical pulse, or what is pre-activity and main optical pulse,
we decided to exclude these events going forward. Table 1 shows a summary of the number of events in each
category. Journal of Geophysical Research: Atmospheres Journal of Geophysical Research: Atmospheres 10.1029/2022JD037128 Figure 4. Example of an event where we have determined the Terrestrial Gamma-ray Flash (TGF) to be outside the modular multi-spectral imaging array (MMIA)
field of view (FoV) and is therefore not associated to the observed optical pulse. (a) Detected counts in high-energy detector (HED). (b–d) Light curves from the
photometers, 337, 777, and 180–230 nm. (e) Map of the area around the International Space Station (ISS) footpoint. The blue square shows the MMIA FoV, the black
dots are lightning activity within ±15 min of the TGF. No lightning detections are within 1 min of the detected TGF (magenta, blue, or green stars). (f, g) Camera 33
and 777 nm show the optical camera images from the frame containing the TGF (up is along the flight path of the ISS, which is always eastwards/right in the images have determined the Terrestrial Gamma-ray Flash (TGF) to be outside the modular multi-spectral imaging array (MMIA) Figure 4. Example of an event where we have determined the Terrestrial Gamma-ray Flash (TGF) to be outside the modular multi-spectral imaging array (MMIA)
field of view (FoV) and is therefore not associated to the observed optical pulse. (a) Detected counts in high-energy detector (HED). (b–d) Light curves from the
photometers, 337, 777, and 180–230 nm. (e) Map of the area around the International Space Station (ISS) footpoint. The blue square shows the MMIA FoV, the black
dots are lightning activity within ±15 min of the TGF. No lightning detections are within 1 min of the detected TGF (magenta, blue, or green stars). (f, g) Camera 337
and 777 nm show the optical camera images from the frame containing the TGF (up is along the flight path of the ISS, which is always eastwards/right in the images). Figure 5. Example of an event where the Terrestrial Gamma-ray Flash (TGF) is outside modular multi-spectral imaging array (MMIA) field of view (FoV), and
there is no optical pulse observed in the photometers. (a) Detected counts in high-energy detector (HED). (b–d) Light curves from the photometers, 337, 777, and
180–230 nm. (e) Map of the area around the International Space Station (ISS) footpoint. The blue square shows the MMIA FoV, the black dots are lightning activity
within ±15 min of the TGF, the magenta stars show lightning activity within a second of the TGF. Figure 5. 3.5. TGF Durations The TGF durations were determined using an algorithm to find a “core duration” defined as the shortest time
interval which includes 90% of the counts (tcore90). The algorithm works by first binning the counts around TGF
time into 40 µs bins and selecting the bin with the most counts as a starting point. Thereafter we go backwards
and include any count that is within 100 µs of the previously included count. The first photon of the TGF is then
found where there is more than 100 µs between the current count and the one before it. This same approach is also
applied from the starting position and forwards in time, to find the end of the TGF. A sliding window is then used
to find the shortest duration that contains 90% of the counts (rounded up) between the start and end counts. As
this method runs the risk of including cosmic showers and solar protons we perform a manual inspection of the SKEIE ET AL. 6 of 14 3.6. Optical Pulse Onsets To determine the onset of the optical pulse we determined several separate
linear fits to different parts of the optical pulse. We used the intersects of
linear fits of the sharpest rise in the optical pulse and the activity before this rise. To make these fits we mainly
used the 777 nm light curves, but for some events a good fit could not be made to the 777 nm light curves, so the
337 nm light curves were used instead. The red light (777 nm) is preferred as it is thought to be mainly associ-
ated to atomic oxygen (OI) which may only exist (after dissociation of O2) in the heated lightning leader channel
(Chanrion et al., 2019), whereas the blue light (337 nm) is associated to the streamer activity, namely N2 second
positive band group radiation caused by excitation by supra-thermal electrons. The 337 band might also detect
some UV emissions, such as from ELVEs. For each event we separate the optical pulse activity into pre-activity and main rise (sharpest increase). One linear
fit was made for the sharpest rise of the optical pulse, while we use three fits for the pre-activity. The pre-activity
is thought to be optical emissions from the propagating lightning leader (Østgaard, Neubert, et al., 2019) and can
be seen in Figure 6 as the relatively slow increase after −1,000 µs and until ∼+100 µs. For some cases we found
it necessary to make two fits to the sharpest increase part, as the pulse was irregular with a separate increase
between the sharpest increase and the pre-activity. The average of the intersects between the main rise fit lines
and the multiple pre-activity fit lines is used as the optical onset time, with the difference between the minimum
and maximum times of these intersects used to define the uncertainties. The uncertainties range from 1 µs to
∼100 µs, with an average of 13 µs. Figure 6 shows an event where we have determined four linear fits. The solid
colored lines show the different linear fits: blue for the sharpest increase in the
optical pulse, magenta, red, and green for different segments of pre-activity. The colored dots show the start and end of the data used in the fit. The dotted
lines show the intersects from the fits of the pre-activity to that of the sharp-
est increase. 3.6. Optical Pulse Onsets As seen in the figure, the pre-activity has a linear trend, but can
be broken into three parts for this particular event. The magenta line shows
the total pre-activity, while the green line uses the data points at what appears
to be a plateau in the pre-activity. Lastly, the red line used the data points
in the last part of the pre-activity where we have a steeper increase than for
the previous parts of the pre-activity. Figure 6. Example of optical pulse onset determination. The solid colored
lines show the different linear fits: green, purple, and red lines show fits for
different parts of the pre-activity data, while the blue line shows a fit to the
sharpest increase of the optical pulse. The dots on the fit lines represent the
start and end of the data used in the fitting. The dotted lines represent the
intersects between the pre-activity fits (green, purple, and red) and the fit for
the sharpest increase. Journal of Geophysical Research: Atmospheres 10.1029/2022JD037128 events for the purpose of removing these counts. To determine the uncertain-
ties in TGF tcore90 durations we chose to add the closest count in time before
and after the determined TGF counts in the method outlined above (these
counts will be more than 100 µs before/after the first/last count). We then
determine the tcore90 duration of this sample and use the difference in duration
compared to the original tcore90 as the uncertainty. Inside FoV (# time
alignments)
Most likely
outside FoV
No optical
pulse
Non-determinable
72 (45)
57
88
4
Note. FoV, field of view. Table 1
Summary of Events Journal of Geophysical Research: Atmospheres Example of an event where the Terrestrial Gamma-ray Flash (TGF) is outside modular multi-spectral imaging array (MMIA) field of view (FoV), and
there is no optical pulse observed in the photometers. (a) Detected counts in high-energy detector (HED). (b–d) Light curves from the photometers, 337, 777, and
180–230 nm. (e) Map of the area around the International Space Station (ISS) footpoint. The blue square shows the MMIA FoV, the black dots are lightning activity
within ±15 min of the TGF, the magenta stars show lightning activity within a second of the TGF. 7 of 14 7 of 14 SKEIE ET AL. SKEIE ET AL. Journal of Geophysical Research: Atmospheres 4. Results and Discussion The median determined delay between onsets is 190 µs,
with only nine events having delays over ∼500 µs. None of the events are observed to have a negative delay, that
is, there are no observed events where the optical pulse onset precedes the onset of the TGF. This has been ambig-
uous in previous work with ASIM data, but has been improved here due to the new and larger sample of events
with better relative timing accuracy and our method of determining that the location of TGFs is inside the MMIA
FoV and association to optical pulses. The sequence of events presented here support the sequence presented in
Østgaard, Neubert, et al. (2019). Our determined delay between onsets is a bit different to what has previously
bins. (c) Histogram of 10 logarithmic bins showing determined delays between TGF and optical signal. show this relationship, where blue dots have the optical onset determined from 777 nm, and the pink dots have
optical onsets determined from 337 nm. The TGFs showing large uncertainties in duration have typically low
number of counts, for which our method of determining the TGF duration is less robust. The expected delay of
the observed optical onsets due to scatting in the cloud was modeled by Luque et al. (2020). Using a uniform
cloud with typical value for particle radius of 10 μm and particle density of 100 cm −3 the delay of the 777 pulse
would be 100 µs for a 6 km thick cloud and 200 µs for 8.5 km thick cloud. Considering a cloud top of 15 km, a
delay of up to 100 µs seems realistic, with expectations that TGFs from these altitudes (above 9 km) are easily
observable. A 200 µs delay due to a 8.5 km thick cloud would place the source very deep in the cloud (∼6.5 km),
making it more difficult to observe all but the most powerful TGFs. 200 µs is therefore used as an upper limit of
delay due to scattering. Figure 7a shows the lines corresponding to these two cloud scattering delays added to the
TGF durations (dotted red and blue lines). The plot shows a tendency for longer-duration TGFs to have longer
optical delays. Panel (c) of the figure shows the distribution of the optical onset delays with respect to the TGF tcore90 start time. 4. Results and Discussion During the period between end of March 2019 and November 2020, 221
events were detected with both MMIA and MXGS data. From these events
88 had no optical pulse within ±5 ms of the TGFs. Using the method outlined
in Section 3 we determined that for 72 of the events the TGFs are inside the
MMIA FoV, and associated to the observed optical signal. Using the light-
ning radio atmospherics from GLD360 we find that the absolute timing can
be improved for 95 of the 221 events, which includes 45 timing corrections
for the events where TGFs are determined to most likely be inside the MMIA
FoV. The mean timing correction was found to be ∼+14 ms, varying from
−11.5 to +40 ms. Going forward, all results refer to the sample of 72 events
determined to most likely be within the MMIA FoV. Figure 6. Example of optical pulse onset determination. The solid colored
lines show the different linear fits: green, purple, and red lines show fits for
different parts of the pre-activity data, while the blue line shows a fit to the
sharpest increase of the optical pulse. The dots on the fit lines represent the
start and end of the data used in the fitting. The dotted lines represent the
intersects between the pre-activity fits (green, purple, and red) and the fit for
the sharpest increase. Using the TGF tcore90 durations and the delay between onsets we plot the
relationship between the two in panel (a) of Figure 7. The blue and pink dots 8 of 14 SKEIE ET AL. 8 of 14 Journal of Geophysical Research: Atmospheres 10.1029/2022JD037128 Figure 7. (a) Plot showing the difference in Terrestrial Gamma-ray Flash (TGF) and optical onset times compared to TGF
durations, with accompanying uncertainties. The blue dots show the events with onsets determined from the 777 nm optical
pulse and the pink dots show the events where onsets were determined from the 337 nm optical pulse. Green arrows refer
to events that are shown in separate Figures. The dotted red line shows the maximum expected optical delay due to cloud
scattering, of 200 µs, and the dotted blue line shows the expected value (∼100 µs). (b) TGF tcore90 durations in 10 logarithmic
bins. (c) Histogram of 10 logarithmic bins showing determined delays between TGF and optical signal. Figure 7. 4. Results and Discussion (a) Plot showing the difference in Terrestrial Gamma-ray Flash (TGF) and optical onset times compared to TGF
durations, with accompanying uncertainties. The blue dots show the events with onsets determined from the 777 nm optical
pulse and the pink dots show the events where onsets were determined from the 337 nm optical pulse. Green arrows refer
to events that are shown in separate Figures. The dotted red line shows the maximum expected optical delay due to cloud
scattering, of 200 µs, and the dotted blue line shows the expected value (∼100 µs). (b) TGF tcore90 durations in 10 logarithmic
bins. (c) Histogram of 10 logarithmic bins showing determined delays between TGF and optical signal. SKEIE ET AL. 9 of 14
show this relationship, where blue dots have the optical onset determined from 777 nm, and the pink dots have
optical onsets determined from 337 nm. The TGFs showing large uncertainties in duration have typically low
number of counts, for which our method of determining the TGF duration is less robust. The expected delay of
the observed optical onsets due to scatting in the cloud was modeled by Luque et al. (2020). Using a uniform
cloud with typical value for particle radius of 10 μm and particle density of 100 cm −3 the delay of the 777 pulse
would be 100 µs for a 6 km thick cloud and 200 µs for 8.5 km thick cloud. Considering a cloud top of 15 km, a
delay of up to 100 µs seems realistic, with expectations that TGFs from these altitudes (above 9 km) are easily
observable. A 200 µs delay due to a 8.5 km thick cloud would place the source very deep in the cloud (∼6.5 km),
making it more difficult to observe all but the most powerful TGFs. 200 µs is therefore used as an upper limit of
delay due to scattering. Figure 7a shows the lines corresponding to these two cloud scattering delays added to the
TGF durations (dotted red and blue lines). The plot shows a tendency for longer-duration TGFs to have longer
optical delays. Panel (c) of the figure shows the distribution of the optical onset delays with respect to the TGF tcore90 start time. The delays are calculated as the duration from the first photon of the TGF tcore90 duration and the onset of the main
optical pulse. The data is binned to 10 logarithmic bins. Journal of Geophysical Research: Atmospheres 10.1029/2022JD037128 been reported using ASIM data. Heumesser et al. (2021) use ASIM data from between June 2018 and October
2019 to determine characteristics of the optical pulses associated to TGFs. Heumesser et al. (2021) shows in
Figure 3, panel (a), events with negative delays, with a mean source time of −10 µs and outliers with more than
4 ms delays. We have shown in this work, using the methods outlined that there are no negative values between
onsets, albeit with a different methodology and somewhat different data set (we only use ASIM data from after
end of March 2019, when the MXGS-MMIA uncertainty was reduced to ±5 µs). However, like Heumesser
et al. (2021) we see the same trend with the majority of events having less than ∼2–300 µs delays between onsets
and a similar group of outliers at around ∼800–1,200 µs. been reported using ASIM data. Heumesser et al. (2021) use ASIM data from between June 2018 and October
2019 to determine characteristics of the optical pulses associated to TGFs. Heumesser et al. (2021) shows in
Figure 3, panel (a), events with negative delays, with a mean source time of −10 µs and outliers with more than
4 ms delays. We have shown in this work, using the methods outlined that there are no negative values between
onsets, albeit with a different methodology and somewhat different data set (we only use ASIM data from after
end of March 2019, when the MXGS-MMIA uncertainty was reduced to ±5 µs). However, like Heumesser
et al. (2021) we see the same trend with the majority of events having less than ∼2–300 µs delays between onsets
and a similar group of outliers at around ∼800–1,200 µs. Panel (b) in Figure 7 shows the TGF tcore90 durations of the 72 events within MMIA FoV, binned to 10 logarithmic
bins. The average tcore90 duration for the 72 TGFs determined to be within MMIA FoV is found to be ∼100 µs,
with a median of 66 µs, which is similar to previously reported ASIM detected TGFs by Østgaard, Neubert,
et al. (2019) and Bjørge-Engeland et al. (2022). All events are found to have the optical signal after the TGF onset, with the events below the dotted blue line
(100 µs scattering delay) being consistent with optical emissions produced simultaneously with the TGF (taking
into account typical delay due to cloud scattering and uncertainties). 4.1. Events Where Optical Onset Delay Is Less Than 100 µs In Figure 7a there are 37 events (∼51%) where the delay is less than 100 µs, meaning that the TGF and the source
of the optical pulse could be simultaneous, when a more realistic delay due to scattering in the cloud is taken
into account. For many short TGFs it is difficult to firmly establish whether the TGFs end before the onset of
the optical pulse or not, due to the uncertainties of the method. For some events with longer duration the TGFs
appear to be continuing after the onset of the optical pulse, suggesting that the conditions for producing runaway
electrons as well as X- and gamma-rays (electric field, voltage drop, and seed particles) could still exist after the
current through the leader channel occurs. However, most of these events have very weak optical signatures, and
we can therefore not make a strong statement based on these outliers. Figure 8 shows one of these events (marked
in Figure 7 with a green arrow). Panel (a) shows the HED and LED detections, with the dotted magenta line show-
ing the first photon of the TGF tcore90 duration. The three lower panels (b–d) show the photometer data from 337,
777, and 180–230 nm. The dotted magenta line in the 777 nm panel shows the average onset determined from the
method described in Section 3.6. As seen in Figure 8 the TGF is not over at the time of the optical pulse onset. The HED counts (black dots) found after the optical pulse onsets are also high energy, so that they cannot be said
to be Compton scattered photons (which can be seen in LED, after the end of the black dots). This means that the
TGF is still ongoing at the onset of the optical pulse, meaning the large electric field needed for TGF production
is still in place when the current surge responsible for the optical pulse starts. Journal of Geophysical Research: Atmospheres There are 37 events (∼51%) which are below
the 100 µs delay line (dotted blue), meaning the TGF and the source of the optical pulse could be simultaneous. Fifteen events (∼21%) are above the 200 µs delay line, where the delay of the observed optical pulses cannot
be explained by scattering from the cloud alone. Twenty events (∼28%) are between the two delay lines, which
means that we cannot rule out that the TGFs and sources of the optical pulses are simultaneous. However, we
think it is unlikely that so many powerful TGFs are detected from so deep in the cloud. The next subsections will
discuss the events that are below the 100 µs delay line (dotted blue) and the events above the 200 µs delay line
(dotted red). 4. Results and Discussion The delays are calculated as the duration from the first photon of the TGF tcore90 duration and the onset of the main
optical pulse. The data is binned to 10 logarithmic bins. The median determined delay between onsets is 190 µs,
with only nine events having delays over ∼500 µs. None of the events are observed to have a negative delay, that
is, there are no observed events where the optical pulse onset precedes the onset of the TGF. This has been ambig-
uous in previous work with ASIM data, but has been improved here due to the new and larger sample of events
with better relative timing accuracy and our method of determining that the location of TGFs is inside the MMIA
FoV and association to optical pulses. The sequence of events presented here support the sequence presented in
Østgaard, Neubert, et al. (2019). Our determined delay between onsets is a bit different to what has previously 9 of 14 SKEIE ET AL. SKEIE ET AL. Journal of Geophysical Research: Atmospheres Journal of Geophysical Research: Atmospheres 10.1029/2022JD037128 Figure 8. Plot showing the detection of a Terrestrial Gamma-ray Flash (TGF) that continues after the onset of the optical
pulse. (a) Shows the high-energy detector (HED) and low energy detector (LED) counts given in energy channels. The dotted
magenta line shows the first photon of the TGF tcore90 duration. Panels (b–d) show the photometer data of 337, 777, and
180–230 nm respectively. The dotted magenta line in the panel (c) shows the determined onset of the optical pulse. Figure 8. Plot showing the detection of a Terrestrial Gamma-ray Flash (TGF) that continues after the onset of the optical
pulse. (a) Shows the high-energy detector (HED) and low energy detector (LED) counts given in energy channels. The dotted
magenta line shows the first photon of the TGF tcore90 duration. Panels (b–d) show the photometer data of 337, 777, and
180–230 nm respectively. The dotted magenta line in the panel (c) shows the determined onset of the optical pulse. 4.2. Events Where Optical Onset Delay Is by More Than 200 µs In Figure 7 there are 15 (∼21%) events that are above the 200 µs delay line (dotted red). This means that the
conditions for TGF production are no longer present and a time interval (few hundreds up to a thousand microsec-
onds) before the leader current occur is observed. Figure 9 shows one of these 15 events, where panel (a) shows
the HED and LED counts of the TGF and the three lower panels (b–d) show the photometer data for 337, 777,
and 180–230 nm. The dotted magenta lines show the onset of the TGF tcore90 (a) and optical pulse (c) and ∼1 ms
delay between the two can be seen. There is ∼600 µs from the end of the TGF to the onset of the optical pulse. The connection between TGF and the leader current is a complex problem. This study shows that TGFs start
before or simultaneous with the optical onset source. The study also shows that there is a group of events where
the TGFs end (∼21%) before the leader current occurs. 10 of 14 SKEIE ET AL. SKEIE ET AL. Acknowledgments
This study was supported by the Research
Council of Norway under contract
223252/F50371 (CoE) and the European
Research Council under the European
Union's Seventh Framework Programme
(FP7/2007–2013)/ERC grant agreement
no. 320839. ASIM is a mission of the
European Space Agency (ESA) and is
funded by ESA and by national grants of
Denmark, Norway, and Spain. Journal of Geophysical Research: Atmospheres Journal of Geophysical Research: Atmospheres 10.1029/2022JD037128 Figure 9. Plot showing the detection of a Terrestrial Gamma-ray Flash (TGF) that has an unusual long delay between onset
of TGF and optical pulse. (a) Shows the high-energy detector (HED) and low energy detector (LED) counts given in energy
channels. The dotted magenta line shows the first photon of the TGF tcore90 duration. Panels (b–d) show the photometer data of
337, 777, and 180–230 nm, respectively. The dotted magenta line in panel (c) shows the determined onset of the optical pulse. Figure 9. Plot showing the detection of a Terrestrial Gamma-ray Flash (TGF) that has an unusual long delay between onset
of TGF and optical pulse. (a) Shows the high-energy detector (HED) and low energy detector (LED) counts given in energy
channels. The dotted magenta line shows the first photon of the TGF tcore90 duration. Panels (b–d) show the photometer data of
337, 777, and 180–230 nm, respectively. The dotted magenta line in panel (c) shows the determined onset of the optical pulse. Data Availability Statement ASIM data used in this study are publicly available from the ASIM Science Data Center (https://asdc.space.dtu. dk). Values determined in this paper and presentations of the 221 events are available at Zenodo: https://doi. org/10.5281/zenodo.6992464 (Skeie, 2022). ASIM data used in this study are publicly available from the ASIM Science Data Center (https://asdc.space.dtu. dk). Values determined in this paper and presentations of the 221 events are available at Zenodo: https://doi. org/10.5281/zenodo.6992464 (Skeie, 2022). Briggs, M. S., Fishman, G. J., Connaughton, V., Bhat, P. N., Paciesas, W. S., Preece, R. D., et al. (2010). First results on terrestrial gamma ray
flashes from the Fermi Gamma-ray burst monitor. Journal of Geophysical Research, 115(A7), A07323. https://doi.org/10.1029/2009JA015242
Briggs, M. S., Xiong, S., Connaughton, V., Tierney, D., Fitzpatrick, G., Foley, S., et al. (2013). Terrestrial gamma-ray flashes in the
Fermi era: Improved observations and analysis methods. Journal of Geophysical Research: Space Physics, 118(6), 3805–3830.
https://doi.org/10.1002/jgra.50205 5. Summary of Results Based on the 72 events where we have optical pulses associated with TGFs, we find the following: 1. All events have TGF onsets before the onset of the optical pulses 1%) of the events are below the 100 µs delay line and therefore have TGF onsets before or simultaneous
h
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ki
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d 2. 37 (∼51%) of the events are below the 100 µs delay line and therefore have TGF onsets before or simultane
with the onset of the optical pulse, taking into account the light scattering in the cloud • Some of these events could have TGFs ending after the onset of the optical pulse 3. 15 (∼21%) of the events (above the 200 µs delay line) have longer delays than can be explained by cloud scat-
tering, which means that the TGFs end before the leader current occurs 4. 20 (∼28%) of the events are between the two delay lines, these events are compatible with TGF onsets before
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flash. Geophysical Research Letters, 37(11), L11806. https://doi.org/10.1029/2010GL043494 Luque, A., Gordillo-Vázquez, F. J., Li, D., Malagón-Romero, A., Pérez-Invernón, F. J., Schmalzried, A., et al. (2020). Modeling lightning
observations from space-based platforms (CloudScat.jl 1.0). Geoscientific Model Development, 13(11), 5549–5566. https://doi.org/10.5194/
gmd-13-5549-2020 Mailyan, B. G., Briggs, M. S., Cramer, E. S., Fitzpatrick, G., Roberts, O. J., Stanbro, M., et al. (2016). The spectroscopy of individual terrestrial
gamma-ray flashes: Constraining the source properties. Journal of Geophysical Research: Space Physics, 121(11), 11346–11363. https://doi. org/10.1002/2016JA022702 Maiorana, C., Marisaldi, M., Füllekrug, M., Soula, S., Lapierre, J., Mezentsev, A., et al. (2021). Observation of terrestrial gamma-ray flash
mid latitude. Journal of Geophysical Research: Atmospheres, 126(18), e2020JD034432. https://doi.org/10.1029/2020JD034432 p
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AGILE MCAL below 30MeV. Journal of Geophysical Research: Space Physics, 119(2), 1337–1355. https://doi.org/10.1002/2013JA019301
Moss, G. D., Pasko, V. P., Liu, N., & Veronis, G. (2006). Monte Carlo model for analysis of thermal runaway electrons in streamer tips in
transient luminous events and streamer zones of lightning leaders. Journal of Geophysical Research, 111(A2), A02307. https://doi. org/10.1029/2005JA011350 f
p y
p
y
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( ),
p
g
Moss, G. D., Pasko, V. P., Liu, N., & Veronis, G. (2006). Monte Carlo model for analysis of thermal runaway electrons in streamer tips in
transient luminous events and streamer zones of lightning leaders. Journal of Geophysical Research, 111(A2), A02307. https://doi. org/10.1029/2005JA011350 ubert, T., Østgaard, N., Reglero, V., Blanc, E., Chanrion, O., Oxborrow, C., et al. (2019). The ASIM mission on the international space station. Space Science Reviews, 215(26). https://doi.org/10.1007/s11214-019-0592-z Neubert, T., Østgaard, N., Reglero, V., Chanrion, O., Heumesser, M., Dimitriadou, K., et al. (2020). A terrestrial gamma-ray flash and ionosph
ultraviolet emissions powered by lightning. Science, 367(6474), 183–186. https://doi.org/10.1126/science.aax3872 Østgaard, N., Balling, J. E., Bjørnsen, T., Brauer, P., Budtz-Jørgensen, C., Bujwan, W., et al. (2019). The modular x- and gamma-ray se
(MXGS) of the ASIM payload on the international space station. Space Science Reviews, 215(23). https://doi.org/10.1007/s11214-018-05 Østgaard, N., Cummer, S. Journal of Geophysical Research: Atmospheres Journal of Geophysical Research: Atmospheres 10.1029/2022JD037128 Carlson, B. E., Lehtinen, N. G., & Inan, U. S. (2007). Constraints on terrestrial gamma ray flash production from satellite observation. Geop
ical Research Letters, 34(8), L08809. https://doi.org/10.1029/2006GL029229 Carlson, B. E., Lehtinen, N. G., & Inan, U. S. (2007). Constraints on terrestrial gamma ray flash production from satellite observation. Geophys-
ical Research Letters, 34(8), L08809. https://doi.org/10.1029/2006GL029229 Celestin, S., & Pasko, V. P. (2011). Energy and fluxes of thermal runaway electrons produced by exponential growth of streamers during
the stepping of lightning leaders and in transient luminous events. Journal of Geophysical Research, 116(A3), A03315. https://doi. org/10.1029/2010JA016260 Chanrion, O., Neubert, T., Lundgaard, R., Stoltze, C., Tcherniak, D., Jessen, N., et al. (2019). The modular multispectral imaging array (MM
of the ASIM payload on the international space station. Space Science Reviews, 215(4), 28. https://doi.org/10.1007/s11214-019-0593-y mmer, S. A., Briggs, M. S., Dwyer, J. R., Xiong, S., Connaughton, V., Fishman, G. J., et al. (2014). The source altitude, electric current, and mmer, S. A., Briggs, M. S., Dwyer, J. R., Xiong, S., Connaughton, V., Fishman, G. J., et al. (2014). The source altitude, electric current, and
intrinsic brightness of terrestrial gamma ray flashes. Geophysical Research Letters, 41(23), 8586–8593. https://doi.org/10.1002/2014GL062196 Demetriades, N., Pohjola, H., Murphy, M., & Cramer, J. (2010). Validation of vaisala's global lightning dataset (GLD360). In Conference paper:
Technical conference on meteorological and environmental instruments and methods of observation. wyer, J. R. (2008). Source mechanisms of terrestrial gamma-ray flashes. Journal of Geophysical Research, 113(D10), D10103. https://doi. org/10.1029/2007JD009248 wyer, J. R. (2012). The relativistic feedback discharge model of terrestrial gamma ray flashes. Journal of Geophysical Research, 117(A2),
A02308. https://doi.org/10.1029/2011JA017160 Dwyer, J. R., Rassoul, H. K., Al-Dayeh, M., Caraway, L., Wright, B., Chrest, A., et al. (2004). A ground level gamma-ray burst observed in
ciation with rocket-triggered lightning. Geophysical Research Letters, 31(5), L05119. https://doi.org/10.1029/2003GL018771 Dwyer, J. R., Schaal, M. M., Cramer, E., Arabshahi, S., Liu, N., Rassoul, H. K., et al. (2012). Observation of a gamma-ray flash at ground
level in association with a cloud-to-ground lightning return stroke. Journal of Geophysical Research, 117(A10), A10303. https://doi. org/10.1029/2012JA017810 wyer, J. R., & Smith, D. M. (2005). A comparison between Monte Carlo simulations of runaway breakdown and terrestrial gamma-ray flash
observations. Geophysical Research Letters, 32(22), L22804. https://doi.org/10.1029/2005GL023848 hman, G. J., Bhat, P. N., Mallozzi, R., Horack, J. M., Koshut, T., Kouveliotou, C., et al. (1994). Discovery of intense gamma-ray flashes of
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level in coincidence with downward lightning leaders. Journal of Geophysical Research: Atmospheres, 123(13), 6864–6879. https://doi. org/10.1029/2017JD027931 Alnussirat, S. T., Christian, H. J., Fishman, G. J., Burchfield, J., & Cherry, M. L. (2019). Simultaneous space-based observations of terrestrial
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nying Elves, detected by ASIM. Journal of Geophysical Research: Atmospheres, 127, e2021JD036368. https://doi.org/10.1029/2021JD036368
Bowers, G. S., Smith, D. M., Kelley, N. A., Martinez-McKinney, G. F., Cummer, S. A., Dwyer, J. R., et al. (2018). A terrestrial gamma-ray
flash inside the eyewall of hurricane Patricia. Journal of Geophysical Research: Atmospheres, 123(10), 4977–4987. https://doi. org/10.1029/2017JD027771 Bjørge-Engeland, I., Østgaard, N., Mezentsev, A., Skeie, C., Sarria, D., Lapierre, J., et al. (2022). Terrestrial Gamma-ray flashes with accompa-
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of Geophysical Research: Atmospheres, 124(24), 14024–14036. https://doi.org/10.1029/2019JD031214 Peterson, M. (2020). Modeling the transmission of optical lightning signals through complex 3-d cloud scenes. Journal of Geophysical Research:
Atmospheres, 125(23), e2020JD033231. https://doi.org/10.1029/2020JD033231 Y., Cummer, S. A., Lyu, F., Briggs, M., Mailyan, B., Stanbro, M., & Roberts, O. (2019). Low frequency radio pulses produced by terrestrial
gamma-ray flashes. Geophysical Research Letters, 46(12), 6990–6997. https://doi.org/10.1029/2019GL082743 Said, R., & Murphy, M. (2016). GLD360 upgrade: Performance analysis and applications. In Conference paper: 24th international lightning
detection conference 6th international lightning meteorology conference. Shao, X.-M., Hamlin, T., & Smith, D. M. (2010). A closer examination of terrestrial gamma-ray flash-related lightning processes. Journal of
Geophysical Research, 115(A6), A00E30. https://doi.org/10.1029/2009JA014835 Skeie, C. A. (2022). Supporting information for the paper: The temporal relationship between terrestrial gamma-ray flashes and associated op
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Het phytopathologisch laboratorium willie commelin scholten van 1895 TOT 1906
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Tijdschrift over planteziekten/Tijdschrift over plantenziekten
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public-domain
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28 -- 28 -- HET PHITOPATHOLOGISCH LABORATORIUIII WILLIE COiilHELIH
SCHOLTEli VAH 1895 TOF 1906, Met ingang van Januari 1906 vrangde ik ontslag als
Directeur van her phytopathologizch laboratorium Willie
Commelin Schelten te Amsterdam, om op to fxeden als
Directeur van een Instituut veer phytopat.hologie, 't wclk
de Reg, eea'J~ng we~rschte to verbinden a.an do Rijks ttoogero
Land-, Tuin- en Boschbouwschool to Wageningen. I/et eexsfgenoemde l~boratorium zal nu eerie geheel
niettwe l~riMe intredcn, -- niet slochts omd,~t her door her
verlies van de f 7000 jaarlijksche tlijk~uhsidie, die her
genoot, op veel meet bescheiden veer zal moeten worden
voortgezet dan in de laatste jtlren her geval was, maar 66k
omdat hot nu zonder twijfel eone gehe~l andere richting zal
uitgaan dan onder mijne dil~ctie, gedurealde ct.e eersto tie0a
jar~n va.n zijn bosf.~an. Hoewel oorspronke]ijk geen ]and;bouwkundige, ben ik
toch dear mijne ongeveer 36-jarige be~.rekking tot her land-
bouwonderwijs met de verschillende takken van bodcmkul-
tuur langen tijd ten naltwste verbondeal gewec~t; trouwe.ns
ik heb van 't begin af aan d.at: ik do nat uurlijke historio
nan eene inrichting veer landbouwqnderwijs te doceeren
kreeg, getracht, naijn yak zooveel, mogelijk dien~stbanr to
maken ann de bGdemkultuur. Her spreekt dus wel van zelf,
dat ik, sexlert ik i~ 1895 aaa~ her hoofd we rd gesteld' van her
phytopa~hologisch ]aboratorium Willie Commelin Scholten,
ook dez~ inriehting in de eersto plaa~s trachtto dlenstbaar te
maken ann de belangen der prakf.ijk. Trouwens ook in de
statuten dezer st;chting was dit op den voorgi~nd gesteld. Nu echfer aan her hoofd van deze stichting eerie dame is --
29
-- komen ~e stalin, 4ie hay studie aun de Universitei.t pa,s
vo.leindigd h,ee~t, dde zieh w61 eea tijd lang onder do ]ei4ing
van mijn' toanmal~ge,n adsistent Dr. van tIall, in zuiver
wete,nsehappelijk mye,ologi.sch werken oefe~de, msar zieh
aan de eiganlijke ziekte~aleer der planten tot du,sver weinig
wijd,de, en die ook stee,d,s buitem a,anraking met de bodem-
kultuur is geblevea~, -- nu zal h.et: phytop.atholog~s;eh
laboratorium Willie Commelin Seholten zieh noodzake-
tijkerwijze op ee~ geheet ande.r gebie,d gaan bew.e,gen. Her
zal meer her karakber van een privaat-labora~orium aanne-
me,n; er zullen do,or de t.aleatvolle Direc.triee o,nc~e,rzoe,-
kingen o.p pl,a.ntkundig, missehien o,ok op i~hygopa.tho~o-
giseh g:ebied wo,rden verrieht; maar de inrieht.ing zal zich
niet, altharLs nie.t 1,e,eht,st.reeks, met do praktijk beano.eien;
al is do mogelijkheid na,tuurlijk niet uitge,sloten d~ som-
mige der al, daar verricht, e onderzoeMl~gen op den duur zullen
blijken, year d,e praktijk niet onn.ut te zijn. HET PHITOPATHOLOGISCH LABORATORIUIII WILLIE COiilHELIH
SCHOLTEli VAH 1895 TOF 1906, Ee~e n~uwe
aansluiting aa.n de pra.ktijk, ean w,erken veer de, praktijk,
mag men van nu ad aan vaa her phytop~th.ologiseh labora-
torium W. C. S. nie4 meet v~rwa:eh~an. En ju~st om4a~ nu m~t mijn a.~treden dit tabera.torium
blijkbaar een gehee] am.de,ran weg ~a.1 uitgaan, zooda$ daar-
mee a. h. w. e ene bepaalde periode vaa zijne gesehiedeais
is afgesleten, is m. i. nu de tijd gekomen, de. gesehiedeais
van deze inriehting van a.f hare stiehting t,ot heden in 't ko.rt
to besehrijven. De droevige aanleiding tot de stiehting van he* phyto-
pathologiseh laboratorium Willie Comme,lin Scholte~ werd
in den eer~tan jaargang van dit Tijdsehrift ui~eengezet. (Zie deel I, bl. 7 tot 12). Als eerste Bestuurders traden
hadst den tIe6r an Mevrouw Scholtea op de Ileere~ Prof. Hugo de gries, F. B. L6hnis 6n Ern.s~ H. Xrelage. 30
I Nada~ onder~eteekende op 4 :Februari 1895 als Diree-
beur was ge,instal]eerd, en du,s a]thans in naam hot ]abor'a-
torium was g eopend -- o,nderget,ee.kende a anvaardde eigen-
tijk zijne betrekking eerst in den zome,r van ,lit jaar en bleef
zoo lung te Wageningen wonen, -- was her ee.rste werk van
he.t Bestuur, n aar den toenmaligen Minister van Binnea~-
la~udsche Zaken, dan Heer M' van Houten, te gaan, om *.e
traehtan eerie sul~sidie voor de stichting te verwerven ; aaa-
gezien de middelen, welke door de stiehters waren besehik-
baar g~steld, onvoldoande waxen om een phytopatkologiseh
laboratorium t~ scheppen, da,t, eenigszins aun ma•
eisehen
kon vol'dee~a. De Minister was niet ganegen, eene zoo hooge
subsidie, te verleenan a]~s word aangevraagd, maar toonde zieh
bereid, in 't vervolg de jaarlijk, sche sore van [ 500, die ik
in den laatsten tijd te Wageningen als pevsoonlijke toe]age
geuoot voor he.t gratis g.even van inliehtingen op phyto-
patho]ogi:sch gebied, van a~ 1896 re, stellan ter be,schikking
van hot p.hys
ltaborato.rium Willie Commelin
Seholte.n. Hij meende evenwet dat dan de Heer LShnis,
die al.s II~speeteur van hot Landbouwonderwijs toe~l ook in
zake subsidies ann partieuliere i.~stellingen ten nutte van
den Landbouw den Minister van advies moest die,nan, niet
langer in her B estuur van de stichting zitting mocht hebben. Doze word door den Heer P..F.L. Waldock te Loesduinen
vervangetu, van wie,ns wer'kzaam leve~ eehter ongelukkige.r-
wijze reM~ na eeal paar jaTeal de draad word afgesaedan. HET PHITOPATHOLOGISCH LABORATORIUIII WILLIE COiilHELIH
SCHOLTEli VAH 1895 TOF 1906, En zo~staken wij in ~en zomer
v,~ 1895 van w al. Her pereeel l~oemer u
n ~
we~d door de familie Schol6en aa~ d~ stichting ve.~'huurd;
her .diende voor mij e~ mijn gezin als woonhus
tevw;.jl
66ne verdiepi~g (be~taaad~ uit twee kamers en suite an ee~
kte;a kamertje) als l aborator~um w~rd ingerieht. Een
p l, an~enkasje van den ruin van de familie Scho]f~n werd bij
den ruin van m~jn hu~s g~trokk.en, en werd be,sternal o,m
di~nen voor h~t he,men van infeetieproe~en. Een aa=tal
objec~n vo,ov onderz.oek en voo~ dem~nst~a.tie bij he,t nni-
versitair .ondevwij,s, d~or mij t~ Wagenimgen ve~zameld,
werden in her laboratorium gedeponeerd ; en ik stelde mijne
vrij uitgebreide phytopathologisehe bibli.otheek ter di, spo-
sitie van de stichting. I)aa~doo.r wet& he~ mogeaijk ge-
maakt, zoowd lint o=derzoek als de college,s ann de. Univ~r-
siteit to bcgi_na~en, zonder da~ eenigszins be~angrijke onkos-
tea veer instal~ati~ noodig waren. l~aboratori.n~ te ~an.vamxle~. En zo~staken wij in ~en zomer
v,~ 1895 van w al. Her pereeel l~oemer u
n ~
we~d door de familie Schol6en aa~ d~ stichting ve.~'huurd;
her .diende voor mij e~ mijn gezin als woonhus
tevw;.jl
66ne verdiepi~g (be~taaad~ uit twee kamers en suite an ee~
kte;a kamertje) als l aborator~um w~rd ingerieht. Een
p l, an~enkasje van den ruin van de familie Scho]f~n werd bij
den ruin van m~jn hu~s g~trokk.en, en werd be,sternal o,m
di~nen voor h~t he,men van infeetieproe~en. Een aa=tal
objec~n vo,ov onderz.oek en voo~ dem~nst~a.tie bij he,t nni-
versitair .ondevwij,s, d~or mij t~ Wagenimgen ve~zameld,
werden in her laboratorium gedeponeerd ; en ik stelde mijne
vrij uitgebreide phytopathologisehe bibli.otheek ter di, spo-
sitie van de stichting. I)aa~doo.r wet& he~ mogeaijk ge-
maakt, zoowd lint o=derzoek als de college,s ann de. Univ~r-
siteit to bcgi_na~en, zonder da~ eenigszins be~angrijke onkos-
tea veer instal~ati~ noodig waren. Intussche~ bleak alra~ dat de ter be.schikking ge,steldo
middel,en ve~l re, geeing w~rem, om bet lab orat~).rium eenigs-
zin~ te doe~ zijn ~ut her incest worden. De ruimte was
ve~l ~e bepe.rkt, voo.ral teen weldva hn de c.olle~tie~ ~n de
bibllotheek (de ~aatste dee~s door a ankoos dee~s door ruiling
van her ~ Tij~chri~t ove~ Plantemziekte,n r te,gen aadere,
recast buite~lan@sche tijd,schriftea e~ ve,rhanddinge,n) zich
sterk uitbmid.dem. Alles ken wddra nie~ m,e~r worden
eeborgen ;de n.o~dige instrumenten konde,n nie,t wovdtm aan-
geschas
~,n omdat de. middel~n ontbraken ~n omdat ex geen
paaa{s veer w.as. HET PHITOPATHOLOGISCH LABORATORIUIII WILLIE COiilHELIH
SCHOLTEli VAH 1895 TOF 1906, Met ingang 1899 erl,angde hog ]abor~to.rium eene zooda-
rtige verhooging van subsidie, da,t hot voor 't vervolg voor de
helft door de Re geering werd beko.sLigd, waarbij eehter de~e
--zooals billijk w~s- zich meet invloed op dan g~ng van
zaken verzekerde en ~evans den phyt,opa.Lhologisehen dienst
in 't loven riep. en ann hot labor~torium verl~and. Pro~. Hugo
de Yri,os, die toeal juist midden in zijne belangrijke onder- --
31
-- zoeking~n omfrealt hot ontstaan der soorien zat, on die
vree.sde, d at ]let Be,sttmrslidmaatschap hem na de uitbrei-
ding, die hot laboratorium zou onde,rgaan, veel meet yam zijn
ko,stbaren tijd zou ontrooven d:an tot du~sve.r he4 geval was,
a,eht|c ]ret raadzaaan toem als Voorzltter van he.t Bes"0uur te
bedanken. Zijne plaats w~rd ing, enomen door Prof. Went
te lN, recht, e,n di~ van den Heer Waldeck door M ~ M. Tyde-
man te Breda. tier past mij hier, e,e~ woo,rd van hulde en
da:nk te bvengen a, an ProL de Yrie,s, zoowel voor hot werk-
zame aande,el, dat hij -- naast de stiehters -- met wijlen den
Heer J. H. Krelag~. heeft gehad in hot tot stared komen va.n
her laboratorium, als voor de aangename wijze, waaxop hij
als Voorzitte,r van hot ]~e,st~ur, st,e,e,cls met mij samenwerktr Toen die Rege,c'ring aan d~e stiehting ee.ne sab,sidie, eerst
van / 500, late,r van / 1000 ver]ee~nde, benoemde zij a]s haren
vertegenwoordi~e.r in he.t B~stuur den beer LShnis; toen
de subsidie tot / 7000 we.rd verh oogd, werde,n twee Re.gee-
ringsgedel(,g, eerd,en in hot Bestuur b,en,oemd, n.1. de tIeeren
Lovink en LShnis, me,t de bevo'egdhe,id de vergade,ringe,n
bij te wmmn e:n (le jaa,rlijksehe rekening van den Pen~ning-
me.ester, den tI.e,er Seholte,n, na te zie,n. ()ok n.a.<l.at eel,st-
g~mo.emde, hot ambl van Direeteur-Gesleraal van den Land-
bouw had aa.nva.ard, b.leef hij, niet~egen,sta:ande, zijne vole
a.ndere v.e,ehmlvatt,ende, werkzaaanheden, als ge.delegeerde der
]{eg'eering zit+ing houden in bet Bestuur. -- Bij (le opricbting w,erd door de s.t.iehters een k.a.pitaal
van / 100,000 g,estomt: een kapitaal,
waarvaxt de re:nte
natuurlijk op venue na nie,t voldoende was om hot, labora-
torture t.ot sta.nd t,e br'e~ngen en te onde.rho.ttden. Da~.r eehter
de. familie Sehol:te:n zich evenwe.1, zij he.t ook niet sehrif-
tdijk, verbonden had om ja~arlijks ongeve.e.r (le, re,nto van
hog eene sore wm/
100,000 bij te passen, had ik geen
1)(,zwaar, de betrekking van Direeteur wm hot to stiehten --32
-- l~aboratori.n~ te ~an.vamxle~. HET PHITOPATHOLOGISCH LABORATORIUIII WILLIE COiilHELIH
SCHOLTEli VAH 1895 TOF 1906, Er m&dd~n zich v~rschille=de pemsone~
san, die, prak~isch in mijn laboratorium wenschten te wee-
ken; nlaar slechts bi] uitzo~deri~g ken aaaa dien we~s.oh
wordea gevolg gegoven, g'aar de ruimto z66 belmrkt wa~ d~t,
op de uren waarop ik zel~s marx 66n enkel porsoon hag om to 33
-- weN;on, mijne eigen werkplaats daarvoor moest win den inge-
mired,
lk herhmcr mij, dat ik in die dagen eens een
l~ez~){'l; kr~,g v;m een vakgonoot, con hekvndc, gels,re'de. Xa.dal wij evn poos haddcn zitten praie~n, g~,f hij mij ziju"
wense}l t.e l.:ennen, mijn lahora.iarium C.pll8 te beziehtigen;
hij keek vle;,md q~, h)en hij hoorde, d,t de suit.e, waarin
ik h',,m had ontvunge,n, zoo o.ng~we<,r mi,j,~ gelw.clo la.!mra-
torium was'.-.- In de evrste j~m,n (retying ik reel bl~z,')eken,
niet deehts van l,'avii~.i en van vakgen(,oien, maar vom'al
ook va.n kijl~grage nieuwsgie.rigen, die gu'~rne ('~'ns de sli,.h-
ting van &, familie Seholteal wensvht~,n h, bezMfligen;
m.e.t, her fond, leiden van (lie pct..,oltell gtng %lke.ns reel van
mi.in ]mslbaren tijd verloren. Ik kan niet aan andv,r(,n
opd,'agen als geleider te funge.eren, omdat ik toen geeu
Ixu'sonee]' had, da{ iels van phyiopaihologisdw zakmt afwist ;
en boven(lien zmt mij dat niet reel hebben gegvven, daar ik
toeh zou hebben moeien werken in helzelfde lokanl, waar
he,t bezoek was. ]-)at de llijkssubsidie van / 500 wel.dra op / 1000 wer,l
gelmwht., lmatt~, nYt vee.1, a;ulgezien de middelen toeh to
beperld l)lcven, .am te krijgen wat ik allereerst noodig had,
n.1. uitln'eiding van lokaliteit en van wetenschaplxqijk ge-
vormd persone~,l. Torn de tleer .~I ~ Sieke.sz, do toenmalig, o I)ireeteur-
Gener;ml van 1,amllmuw, mij d,e opalraeht wihlo verstrekken
mn eene reis naar .Xcm'd-Amerika te oadernemen, hm eindo
na Iv gaan war mlze l~.egeoring ~e doen stond naar aanIeidhlg
w~n hot San aos6 .s(.hihlluisgevaar, wees ik e r gen(u,md(,n
l{e(,r op, dat als ik ,le door (le l~geering to verstrekkea
Ol)dra.(~ht aa.1..vmmhh', he( labm'at.orium tijdelijk moest wnr-
d.(,n gesh)ten, vn dus het ger
van inliehtinge.n aan pra(.Ii~.i,
zoo hmg ik weg bleef, moes~ worden gestaakt. De ]leer
Siekesz, die met mij van meening was, dat eigenlijk reeds 3 m
34 lang de Regeering voor e,en phyto,pa~ho.logiseh Ins6tuut had
mo,eten zorgen, gaf mij de. toezegging, daL hij bet e r zou
tra,ehten hee.n re. HET PHITOPATHOLOGISCH LABORATORIUIII WILLIE COiilHELIH
SCHOLTEli VAH 1895 TOF 1906, leiden, dat aan h.et phytopathotogis, ch labo-
ratorium Willie Commelin ScholLen eeme, .Rijkssubsid{e, zo~
worc~e,n verleend, hoog gealoeg om ee,n adsistent te b,eno,e,men,
die later--wanne.er hij op de hoogte van he* yak zo~ zijn
gekomen -- mij zou kttn~ebn vervangen, als de llege,e'ring
eens later v~eer be,slag op mij wenschLe te leggen, o,f als ik
om ann,dare reden afwe.zig mo,es,~ zijn. Ik wee,s e r den Heer
Sickesz op, dat de be,noeming van ee,n' a:dsistent noodzake-
lijke,rwijze met zieh meebracht uitbre,iding va,n lokaliteit
en van hulpmiddelen van ondc~ozoek, d aar ~n die. e,eale bn de
anderen reeds voor mlj all,ee.n onvoldoende waren. Na mijne
terugkom,st mt Amerika werd afgesproke,n, dat de. tIe.er
Sicke,sz zou voorsielleJa, aan he.t phy~opatholo,gisch labo.ra-
to~rium Willi.e, Commelin Seholte~ eene jaarlijksche sub-
s~die van [ 7000 t e ver scha:ffe.n, wa,arvoor dan de, genoe.mde
stlehting 66k zou moete~n zorgen voor 4e uitoe~enlag van dean
i~tussehen noodig geble.ke,n phytopatho,logischen
dienst. De wetgevende maeht keurde den post van f 7000 voor her
phytopathologisch
laboratorium
goed. De, tteer
Sickesz
wenschte, e ehter niet te tred,en h~ her v erla:nge:n 4er stichters,
o,m a.an de. il~riehting voor eenige ja.ren a ehter66.n e,e~ne, zoo-
danige ,sub,sidle. ~e ve,rlee.nen ; want bij eerie, eventueele orga-
nisati.e van het Laadboawo~derwij.s van den hoogs~en trap
w ensehte hij de handen vrij i~ h ebbea, o m d~i~raaa, als
zulks noo,dig bleek, een phytot~ath.o]ogisch laboratorium t,~
verbinden, als wanneer uit d,r
nard der za~ak de Regeering
~eene ,snbsidie meer zou ver~een,e~ aan e,e~e pa.rticuliero
stiehting. De sub,sidle va,n [ 7000 ging in met 1 :l'a.nna.ri 1899. De. loka.li~eiten van her hboratorium we.rden nn aanzienlijk
uitgebreid, en we]. doordat van nu a~ aan .deze. stichting van 35 den Heer Scholten twee naast dkander g.d,ege,n huizen
h uurde t,ege,n eenen huurprijs van / 3000 pe,r japer,
tte4
e.en~, huis (l~>emer Vissch(~rstra,at n ~ 1) werd g.eheel als
laborat.orium ingerieht, e,n aan di,t huis we,rde'n verb,onden
twev ka.mers van he.t. daa.ra,a.n gre.nze, nd~e pe,ree,e.1 (n ~ 3), het-
we,lk overigens al.s mijn woonhuis funge.erd.e. Her sou.ste,r-
rain va.n h.et labm,a.toriuni
werd als woning van een'
eoneie,rge, ingerieht.. La:aIstg~enoemde trad in 't najaar van 1899 in rune.tie;
de a.dsi.stent (d.e Hem' C. J. J. wu~ Hqll, to,e,nmaals ~,andidaat
in de. plant eu dk!rkunde) aanvaardde zijne betr,ekking op
1 Dcemnb.er 189!1. Met h.et o,og o.p (l.e. HET PHITOPATHOLOGISCH LABORATORIUIII WILLIE COiilHELIH
SCHOLTEli VAH 1895 TOF 1906, be~slomme,ringen, die
ik ia 't v.ervo]g me.t, de.n phytop,a.t,hologi.svh.e.n
dietlst zou
}td)b.e.n, on.~l.e.rging mijn saa'aris e,eno, verho~ging. De,z{,gi~lg
in in d(m zomer 1899 ; tm-aqjl de salari,ss.en wm den adsisl:e.nt
e:n de.n eoneierg.e inginge,n op den ifag, waarop zij hunne
be,trekking aanvaardden. D,c, verho.ogde, t{ijks.subsid, ie koa du,s, voor e,en gedee.lt.e
w,o,rden b.{>steed voor d.e. aansehatfing van de, noodzakelijke
meubelen e,n w.et.en.sehappelijke iitstrume,n•
ee,n. ander
d.e.el ervan werd gebruiM om verande.ringen nan te bre,ng, en
in gas- e.n wate.rle.iding an .a.1 war ve,rder novdig wa,s, om h.et
p.ere,e.e.1 Ro.emer Visseherstra.at n o 1 tot ]abora.torium in te
richten. Van ],u af aan e,ers.t kon her phytopatho.logiseh la.bora-
torium 1,Vii:lie Commelin Seho]te.n, war zijn,e inriehting
b.e.treft, aan billijke, h,,ewcq n, og matige, e,ische.n vohlo,e.n. Door he4. ~,erhoo.o,d.~ budce.t werd ik in sta.a.t ge,steId, i~dor
jaar de. wet.enschagpelijke~ hulpmi.dde,len en d,e b.ibliotheek
tot gro.o,iere uitbreiding te bre,nge,n; er waren a,fzond,e.rlijlm
laborat.oria voor mij en mijnen gd.sist,e,nt, een behomqijk
inge.rieht museum, kame.rtjes voor d.e. biblioth.eek, ee.ne werk-
kame,r voor lie,e,rlingen, e,e,ne donkere, kamer, enz. --
36 Eehter bleef de inriehting c~en greet g(~brek aankleven :
hot ontbreken van bijkans iedere gelegenhcid om besmet-
ting~pro(;ven t~., nemeu. Hot t)lant~c.~nka~je, door den Heer
Seholten aan hot laboratorium afgesta~m, mawr mqnvank(.~]ijk
door hvm met mijne ve, rgunning ia gebruik ~houden w~nr
zijne eigen plan~en, word ook op de.n duur niet o:ni~imd,
]m.ewvl ik op de noodzakelijkheid om een plantenkasjc hu'
b~,sehikldng ~e hebben, herhaaldelijk woes. We] werde~t
l;ogingen aangewend, om met behulp van e~ne a,fzondcrlijke
Rijkssubsidie e,en nieuwe kas te bouwen in den aangr~n-
zenden ruin van den Heer Seholten; maar doze poging+n
mislukten. ],~en reel groo4er bezwaar hog dan hel, gc~mis van een
pla.ni.enka~je, was hot algeheele gebrek "ran fem~in. W:el
l)evond zieh aehter hot labora~oiium e~'n klein ]apje growl,
een Amsterdamseh stad.stuintje; maar besehaduwd als hot
wet;1 d,or tauten en hooge booliwn, was hot veer mijn (l()el
w)lslagen cI~ge.~ehikt. Wcl werdeal door mij in ver,sehil.. lende Mreken des lands proefvclden aangele~d met 't. o~g op
bepaa]de da'u' heerse.hende pluute.nziekten, en ken ik door de
welwi[l.e.ndheid van e(~n paar Wageningsehe lee.raren over
e~nig terrein van de. l~ijks ]toogere I_am(l-, Tuin, en :Bosch-
bouwschool be~(.hil&en, -- maar dringende behoefte bled
beslaan 'ran ('(,n stuk grt)nd in de onmiddellijkc nabijtmid
van hot ]t~boratorium, hetwelk zonder tijdverlies op ieder
uur va.n d~,n dag ken worden bezceht. HET PHITOPATHOLOGISCH LABORATORIUIII WILLIE COiilHELIH
SCHOLTEli VAH 1895 TOF 1906, In dit gemis ken onmogelijl; worden voorzien, zoolang
hot laboratorium in de Roomer u
gevestigd
bleef; want z clfs met opoffering van v~el gehl zou 4aar in
de buurt geen terr~,in te krijgen zijn g~weost. Alleen door
verplaatsing van de inriehting na~r W,~tcrgraa.f.~meer of
naar e.ene der buiienbuurten vaal de gemt~ente Amsterdam
zou men missehien terrein in de onmiddellijko nabijheid 37 vail h,et labo,ratorium hvbbe,n kunn,e,n krijgen; en daar zou-
den e,en laboratorium, evne dlr'ecteurswoning e,n e,e,n/erreln
voor proefneming zeJ~e,r niet mc,er aaln huur ge,koM hebben
dan de twe,e perc,e,Nen z.nd,er terrein in d,e R~<'mer Vissch,er-
stra.at. tHet groot.e ine:onvenient wm h.e,t, gemis wm t,e.rrein en
plan f,enkas da.argdat.en, kon overigeals van af 1899 he~, phy-
topaflMogisch l:abora.torium te Amsterdam, war zij,n,e, inrieh-
tiHg b.etreft, aan billijke hoewcl matige, eisd~en vohlo,en. .~[e~ in:gang van 1 Dec'ember 1S99 was d.e ttc,er van Hail,
candidaat in d.e plant-en dierkunde aan de Universitei~, van
Amsterdanb tot adsi.s~.ent beno,emd,
l)a.ar ,l,e,ze :n e.en.e,
groole sta<l geboren cn gefogen was, wa,s <le ]lmdbouw ~,l
zijne ver'schill.cmd(, v~,rt.akkingen lre,m gehe.e,1 vre,eend geble-
yen ; en he.t sl)r:eek! wel van zelf dat :le. phyt,o.pa,tho]oog met. alleea met 1)oI,a,nis(,he e~t zoi~logisehe ke.nnis kan voi.st.a:an,
ma.ar dat: hi] (ok met de t,>di der vers,qfillende land- en
tuinbouwg.e,wassen ,q~ de hoogte mo.e.t zijn. ],;e:l jong bota. nitieu,s, di(~ gdle.e] w'e.emd .st.a at t.egeaover d.e, pra.ktijk, he.eft
-- al is hij ~,ok uils,l.eken,'l in ziin yak als zoodanig thui,s--
jaren noodig, ,eer hi.i e.en go,e,(] ld~ytopalholoog is gew(,rden. Daa,r(mboven k,on m i.in a,d,sistent zieh n iet :]a(lelijk me{ de
1)orst op h~t,g'o,~,:n hem onlbr'a,k Ioele,ggen, daa,r hij hog m,et
zijn do.ctoraal examen . in de maag" . zal.. ])aa.rna kwaan
h,e,f bewerke,n van zijne dissertat,f,e, waa,rop hij in k[ei 1902
promoveerd,e. In di.en tusschent,ijd had hij e,en t,ijd lang
in her baeleriologis~'h ]~aboratorium van D ~ ~Basenau te
Am,st,erdam gewe:rld, e,n daarmt on,d,er leiding van I?ro~. Beye,rinck t,e, 1),e,lfl. Re,ed,s in 1903 ver'trok D" vaal IIall
naar ~urina.me, waa,r hij op voordra.eh|, van Prof. Went
tot , lnsp.e,eteur van den Imli,lbouw ~J was lwno.emd. Hij
vertr.k dus op c.e." ti,i d, /,.on h;j .juist begon ge:schikt h~,
wor&m, mij van een ge.de,elt,e, mijner ta,ak te, ontlasten. b
38 }Iet volgende jaar werd de tIee r A. W. I)~rost tot Land-
bou~adsisLent a~m ho~ landbouwkundig laboratorimn in
Suriname bencemd ; h ij vertrok daarheen in September 1904. HET PHITOPATHOLOGISCH LABORATORIUIII WILLIE COiilHELIH
SCHOLTEli VAH 1895 TOF 1906, J)e Heer Drost was van de oprieht, ing v:m het phytopatho-
1.cg~sch labora.torlum W. C. S. af nan dez~e inrichting ver-
bo nden geweest. : eerst als amanuensis, ~aler onder den titel
, teehniseh beambte D. Uit de praktijk wm den tuinbouw
voortgesproten, had hij zich ]angzamerhand meer welen-
schappelijke kemfis eigen gemaakt ; en hij was nu veer her
phytopa.thologisch laboratorium een zeer bruikbare, kracht
geworden. Inderfljd werd geschreven dat her veer mij vleieM
was, dat twee ann mijn laboratorium verbonden personen
in beh~ekkingen a.an het nieuw opgerichte landbouwlabo-
ratorium t,e Paramaribo werden benoemd; dit daargelaten,
zal t, oeh moeten worden erkend, d~t hun vertrek, juist teen
zij hunne voorbereidende school hadden doorloopen, oor-
zaak was dat ik veer mijne ]nrichting slecht.s betrekkelijk
weinig val: hunne hulp profiteerde. De I[ee r It. 3i. Qua~ljer, tot opvolger van D ~ van
tfall bermemd, aanvaardde zijne a.dsistenfsbetrekldng in
190~, eel,st nadat hij zijn doet:n'a,al examen in pharmaeie
,'whter den rug had en even w;dr hij zijn praldisch apr~t,he-
k(,rsexamen deed. Deze Heer ken zieh (h's dadelijk gehecl
aan zijne tank wijden, en deed dat voorlaurend met grcoten
ijver. Afkomstig uit eene kleine pleats, had hij reel op
't, platte land verkeerd en belang leeren stellen in al war
het land- en t.uinbouwbedrijf aangaat. De lezers van bet
, Tijdsehrift over Pla.nt,enziekten r hebben reeds met een
paar zijner pmmevruehten kennis gemaakt en zullen spoedig
a.ndere onderzoekingen van hem ]eeren kennen. In plants van den IIeer Drost verleende mij in de laatsto
paar jaren de tIeer C. B. va.n der Zeyde ts Zaa'ada,m hulp
al~ amanuen~sis. m
39
-- Ten slotte, zij her mij vergund met e,en enke,1 woord mijn
jonge,n vriend, de.n Heer W. H. Obreen te gedenke.n, di.e
eerst, e.en tijd ]ang als ijvorig leerling mijne eo,lleg.es volgde
en als labo,ranf in he4 laboratorium werkie, en later-- zonder
ooit ,'flh.ci,ee] a.an d,e inriehting ve.rbond.en fe zijn gewee,st --
zieh voor ha,at zeer verdi.enstelijk maakte, zo.o door he{ doen
van sommigc onderzoekingen, als door hulp bij de bepro.e-
ring van b.e,strijding~smi.dde.len,
do.or hot verva.a,rdigen vaal
photo.'s en door een begin f,e :maken me,t hot eal~a]ogiseerel~,. van d,e. bibliotheel~ der htrielfl,ing e,n her sa.m.~mste,llen van
eene phytopathologis(:he bibliographie. Helaa.s ([,e,ze ijvo-
rige, bereidwillige en bekwame jo:ngding weed t.e mid.den
va,n zij n,e, werkza.aa~he,id door e,e:ne ern.st.ige, ziekte ov.e.rvalle,n,
die hem ten grave sleepte. Zijne nagedaehtenis zal bij mij
in dankbaar aandenken blijven. HET PHITOPATHOLOGISCH LABORATORIUIII WILLIE COiilHELIH
SCHOLTEli VAH 1895 TOF 1906, -- Thans ga ik over tof de b,espreking vaa d,e werkza~am-
he,(]en, ge,durmlde mijn Direetoraat van wege her phy~o-
pa, thologiseh la,b.orah~rium ve.rri~'ht. Yolgens a~:t. 2 van de stieh~ingsacte, wordt dit labora-
{orlum di,e,n,stba,ar gemaakt
: (~ a,an he,t geven van inliehtingen aan d~ beoefenaars
van &m landbouw, den luinbouw e:n d,e, houtte.elt, o,mfrent
de oorzaken van ziekfe.n en besehadigingen vaat voo,r di~
vaklmn behmgrijke g.ewa,ssen, al~mede omtre,n{ de mi(hle.lelt
Oln ,leze, ziekten en be,sehadiginge~l te voorkomen en fe
be~sfrij den ; (c a,an d,e uitbroiding (ler k.e,nnis van plante,nzie]~ten en
sehade,liike die,ren door weien,sehappelijk onde.rzoek; (( a,an de. belaaqgen van h.e,t cnd.erwijs van de. stude,nte,n
in de plant- e,n dierkunde a.a,n :]e, UniversiIeit. van Am,ste*-
dam, en ill 'f a]gemee.n van hot onderwijs in de phylopatho-
logle ~.- --
40
-- War be~reft de aanvrageu om inlichtingen omtreat
pla.nt~enzieI2~en en schaddijkc dim, en e.n ocn.tre.nt &. mid.deleal
let bestrijding, ]~an worden geconstateerd dat deee gedu-
rr
her tienjarig bestaan der inriehting vrij geregeld a.an-
zienlijk zijn toegenomen. Dit kant eenigszias blijken uit her aantal in de versehil-
lendc jaren ingekomen en uitgegane brieven. Dit bedroeg : Ingckomen. Uitgegane bricvcn
in 1895
? 376
1896
553
607
1897
659
756
1898
667
788
1899
753
927
1900
960
1192
1901
975
1156
1902
1169
1355
1903
878
1084
1904
1157
1359
1905
1098
1272 Ik wil daarbij her volgende doen opmcrken : 1 ~ Versehillcnde vragen om inliehtingen werden mij
mondeling ged'aaa ; deze werden bij de uitgegane brieven op-
gel.~.hl, nie.t bij d.e ingekemen brieven. Vo~)r ten deel is he~
d55raa, n toe i.e sehrijve.n, dat-
blijk,ens bove.nsiaande
tabel -- ie.de.r jaar her aaatal uitgegane brieven her aantal
ingekomen brievan hie% onbelangrijk overtrof. 2 ~ In ddn brief we.rden herhaald, elijk ve.rsehillende vr,l-
gen bealttwoord ;ann d.cn anderen kant eehter warener onder
~.le, ingekomen an uitgegane b rieven ook dezulke van admi-
nistratieven aard; zoodat de boven aa.ngegeven ~.ijfers geen
va, wk(',,,'i,/ overzi~'ht ge.ven van d~. uitg~.,br~.idheid van den
inlichling.,:dienst in de ~'erschilJe.n.dc' jaren. Toch geven 2 ~ In ddn brief we.rden herhaald, elijk ve.rsehillende vr,l-
gen bealttwoord ;ann d.cn anderen kant eehter warener onder
~.le, ingekomen an uitgegane b rieven ook dezulke van admi-
nistratieven aard; zoodat de boven aa.ngegeven ~.ijfers geen
va, wk(',,,'i,/ overzi~'ht ge.ven van d~. uitg~.,br~.idheid van den
inlichling.,:dienst in de ~'erschilJe.n.dc' jaren. HET PHITOPATHOLOGISCH LABORATORIUIII WILLIE COiilHELIH
SCHOLTEli VAH 1895 TOF 1906, Toch geven 41 deze ('ijfcrs duide,lijk genoe,g eeno belangrijke toe~eming
a.an van dit ged.e,el~,e, de.r werl~zaamhede.n. 30 Bij he,t. jaar 1898 dient t,e wor&m opgemo~'kt, dat ik
gedurende, dc maa,nde.n April, 5[ei en Juni, -- dus in e.en'
tijd, wann.e,er gewo~nlijk de vragen om inlichtingen her tal-
rijks( inkomen -- op reis naar Amerika was, waaM~or her
laboratorium toen gesloten werd; zoodat her aanta.1 vragen
om inlichtingen
anders zeker aanmerkelijk
grooter wa,s
geweest dan nu her geval was. 40 De aanmerkdijke stijging van her a,an;tal ingekmnen
en uifgega.n.e, brieven van a{ 1900 lig{, fen d,eel.e, in de, invoc-
ring van den phytopathol.ogischen dienst. 5" I)at d.e. inlh~hfings;dienst, in 1903 min,der druk wa.s
dan in he.t voorafga,a.nde, en in h.et volg.ende, jaar, wa.s gelie-
gen ill vers~!hilleltd,e om.staatdighe.:!en. Yoore,erst i.n h.e,t felt,
dat ,le we.e~'si~tvloeden ee,r'st zeer laa.t in 't voorjaar veroor-
loofden, te zaaien; t,e.mvijl de zoo laat g=e,za.ai:le, g.ewas.seab
e,e,mnaa.1 o.pgeko,mcn, zich me,esta! snd o,n~wikkeld,e~n. Te~l
twe.ed.c, was de groot.e vochtigheid van d.en zmner oorzaak,
flat zich b.elrekkei.ik w.einig inseldenpl.agen wm.rd,ede,n; tcr-
wijl h.et weer w.el voehtig, ma.ar Met warm gc:no.eg was, om
v.e.le s~ort,en van pava,sitaire zwammen ~ot st.e.rke, oaflwikke.-
ling en ve.rmcerdering t,e bre,ngcn. Ten flerdc is ook zdler
h.e,t ~'eit. dat D ~" wm I-la.ll w.eldra naar Suriname z, cm ver-
trekken, .e,n dat hij in ve,rba:nd daarmee ottd.e.I~Sdieidt',n r.e,e:is
begonn.e:n corr.espondentie~s .over be.pa.ald,e, p~alltenzidd,en Met
voort.z.r
van invloed op he.t aa.nfal ingeko,me,n en uifg,e-
gano brieven gewee,st. Zo,ov.c.~q l~].ijkt wel, dat gedurend.e.d.e jaren van mijn
Directora.a,l her a a.ntal vragen o,m inlqchtingen vaal den kant
der praetici zo.t!r aanmerke,lijk is toogenomc'n. Vel.e pl'avti~'i
zomh',n g~,w,g:,,hl al
],el n,~q'l,:wa.;mlig~; wal bij l,*'n "!;
't ge,bied van d,c sehad,clijke dieren e:n pla.ntenziekt, en 42
N voorkwam, ~]thans voor zoover het hun onbekend was;
en her aanfal van hen, die vaa i,n]iehtinge,n
van den
kant
van
her
phytopafhologisch
laboratorium
g~bruik
maakten, nam
gele,idelijk
aa.nmerke]ijk
tc,e. ~leer
ell
nmer begon her phyiopathologiseh
laboratorium
W;llie
Commelin Seholten,
althans voor d.e ontwikkelde land-
bouwers en kweekers, en ook voor tie groote grondeige-
n.aren, de geregelde, vraagbaak te word(m op 't ge, bie3
van plantenziekten
en sohade]ijke dieren. Ik ho~)p dat
her Instituut voor phyl.opathologie, dat thans onder mijne
lehling die iaak zal vom~zetten, steed.s mevr in rela.tie me,t
de praetiei moge komen. Va,n her. ideaa.1, dat in smnmige
Staten tier Amerikaansehe I~publiek bereikt is, n.1. HET PHITOPATHOLOGISCH LABORATORIUIII WILLIE COiilHELIH
SCHOLTEli VAH 1895 TOF 1906, t]at de
Staatse.ntomoloog, t}e Staatsphytopatholoog of de deskundige
van bet proefstation (experimeat station) met alle land-
bouwers of ooft.telers in zijn gebied' ge.re~ld in retatie
staat, -- van dat ideaal zullen wij hier te lande hog wel
1,angml tijd ver verwljdertl blijven ; en wel omflat _Xe(lerland,
naast groote landbouwers en groote kweekers, een onnoeme-
lijk aantal kMne boeren en kweekers left, temvijl in de
meeste Staten va.n Amerika het groot grondbezit, ook voor
den ooft.teler, regel is. Maar wanneer wij een.e verge.lijking
in.stelIen tussehen he.t gebruik, (]at in Nederland in de laat-
ste jaren door de praetiei werd gemaakt van her phytopatho-
togiseh labosatorium en dat, hetwelk in andere landen van
Europa van de ~ Auskunfststellen fr
Pflanzensehntz ~ en
dergelijke inriehtingen
gema.~kt wordt, dan staat zekvr
.Nederland in dezen mee bovenaan. En om een ove.rzieht te
hvbben van wat bier te laade tegenwoordig dv phytopatht~-
logie v~r den land- en tuinbouw beteekont, is he.t hie4
vold'tumde, dat men nota neemt va,a bet saiee0s toeinemende
a.anta]: vragen om inliehtingen, d, io va.n de zijde de,. prat.t.M
kwamen bij den Direebeur van lint phytopathologiseh labo- 43 m rato.,'ium te Amsterd,am, -
ook vele ]~ijkshmdlmuwlem'arm~
vn llijkstuinbouwh,(,rar(m
zijn op ,lit gebied met: vru('ht
we,'l~zaam, to,'wiil zij i,, t,,'ijfolavh(igo govallvn mijne hulp
inroepen, en mij verder gvregehl op de h(~gte houden "van
wat ~r in hun ambts~ebied "root merkwmmli~zs op phytoln~-
lhologisch gebied v,mrkomt. Stuns wt'rd dora' welenschal~l~.elijke, buiten de prak-
ti,jk slaan(b, l~]alltkundigen (1(, meening uitgesproken, dat
]wt gewens('hl zmt zijn. &, werk:,;mmheid van den ])irv~'teur
vl.lll het phyl,,palh(~log'isch iaboratorium, war aangaal bet
vers(.hathm van inlichting(,ll aan pra~.tiei, It' b(~l)erlwn,
l'h'n
w(,lmlsdmppelijk g',,vo,'md persoon zml daa.rvom' eigenlijk
(, {t, g,~e.:l ~ zi.in, ziu t iid te I:.ostbaar, daar hi.i diml b(,ier
zou kunn('n lw.st,*d.en. ]I~,rhaahlelijl~ kmm,n tdkc, ns wt,er vra~'(,il onl/l.'t,:~t dt,-
z~,lt',l~, zit~ktt, of h('tzr
s('haddijke dier in; cn hv,t, ;s gt, c'lt
:.lall~(qlalll(, ('l!. OC.ll(, vri.i onvruehtbare taak, allijd weer hei-
zdf& t.e m~'t('n s(,hrijven; p.ers(mml, die wel.enschappelijk
mind('r hong siaan, l<unnen dat werk best doen; de weten-
sdml,p,q:ijk hooge," st~mmle p('rsonml moelen zi(.h met nle.pr
zuiver w(~t.(msdmpl~,'lijl{ werk bezig houden. 11 I, d;!t sian,lpunl .s.taan inde.rdaad x-vrs,.hillen,le l)irve-
leu.ren van lfllyt,qmlo]ogische l:~bm'aiol'ia. Zoo is bet-- of
zoo ,'a.~ hvt althans e(,n l~
.i;~,'en g~,l~,d(m - - geslehl in
h('t . lmb,,ral.oire de Imthologie vdgdt,ah, ~ ond('r d~ d{re~'ti(.,
171111 (l('ll ovm'igens z('t,r v(,rdimlst~,lijken Prof. D" .l';d. Pril-
lieux. HET PHITOPATHOLOGISCH LABORATORIUIII WILLIE COiilHELIH
SCHOLTEli VAH 1895 TOF 1906, Prof..llug'o de Vries g'.:'(fi in ,lt,~,l I van h,,t , L:~nd-
lmuwkundig Tijds(.hrift ~ (18.1.,, hl. 217 (,nz.) vene b.e..~('hri i-
ring van dal lalmralm'ium. Op bl. 220 z('gt hij: , !Iier
(,n ,laar zagr ik kis jes st, aan, waarin monsters van zit'ke
kultuurpIant,
en
ing, ezonden waren. Voor zoover ]t('t ])('-
ken,]~, zi&t(,,~ zi.in, win'den &~ze aan de leecl/,,l~. ~, ,'.'('~
,
t.e.r ,leterminatie en zoo mogelijk ter beantw,;or,ling (1(,1' d,~,u" 44 don inzendcr ge~stelde vragen ~. Men g-ing aan her ]abom-
torium van Pri]lieux blijkbaar vaaa de meening uit dat bij
her geven van inliebtingen aaa praetici, de (lelernfina.lie,
de bepaling van den aard en den naam van den parasiet, clio
de ziekte veroorzaakt, de zaak is, waarop her eigenlijk aan-
komt; en dat wat men dan aan (h,n praticus te schrijvvn
heeft,, in de lrandbo~q~ell kan worden opgezoc.ht. don inzendcr ge~stelde vragen ~. Men g-ing aan her ]abom-
torium van Pri]lieux blijkbaar vaaa de meening uit dat bij
her geven van inliebtingen aaa praetici, de (lelernfina.lie,
de bepaling van den aard en den naam van den parasiet, clio
de ziekte veroorzaakt, de zaak is, waarop her eigenlijk aan-
komt; en dat wat men dan aan (h,n praticus te schrijvvn
heeft,, in de lrandbo~q~ell kan worden opgezoc.ht. Net is waar, dat nu en dan vrage.n omtrent d.ezel'fd,e
ziekte inkomen, die op dezeIfde wijze kunnen wor~len beant-
woord ; en her zou monnike.nwo:rk zijn, wannee,r de ])ireeteur
of een wetensehappelijk gevormde adsisteat pe, rsoonlijk al
de brieven a]s antwoonl daarop ging sehrijven. Een vroeger
aan oen ander adras gesehreven brief kan dan gevoegelijk
d,oor con' amanue.nsis of s~'hrijver wor~len get"Olneer~l, natuur-
lijk evcntu~e] onder aanbrenging van de noodzakdijke ver-
a.nde.ring~n. Maar dat gaat in zeer vele gevallen niet: wan-
neer de bepaliag van den aard der ziekte of be.sehadiging is
afgeloopen, kan op verre na niet altijd al het overigc in de
handboeken wordcn opgezoeht: e.n het laten beantwoorden
wm brievel~ over zi&ten, die vaker voorkwamen, kan dus
lang niet ah.ijd aan leerlingen of ondergesehikte beambten
wordeu overgtflat.en. Imme.rs war men den praetieus ter
bestrijding va,n eene of amlere ziek/e of bc, sehadiging meet
aanraden, hangt op verre na niet uit.sluitend af van den aard
tier ziekte of wm de soort van parasiet, die. haar veroorzaakt. HET PHITOPATHOLOGISCH LABORATORIUIII WILLIE COiilHELIH
SCHOLTEli VAH 1895 TOF 1906, War zou de ]aaldbouwer gezegd hal>
ben, wanneer hem vanwege her phytopat.hologiseh labora-
ratorium de road ware gegeven, 9oft te zaaien[ Ik geloof dat her geheim, waarom in Nederlaaqd meor
saanoa~wea'king tussehen her, phytopathologiseh laboratorium
en <le pm<.tioi }>estaat: d'm in de mc~ste andere ],anden, groo- 46 tendee]s juist d55rin schuilt, dat her geven van inliehtingen
aan de mannen
der p~,a.ktijk niet d,oor mij als een ]ast.lmSt
we.rd aangezien, die. maar tijd cnt~ad~te aaa wete..nsehappe-
lijk werk; maar dat juist (]it gedeelte va.n de werks.aaan-
hede.n steeds al nommer s
vaa her. progra~ma
werd be-
s('houwd (1). Ik weet zeer goed, (lat ik door mij van di(.~
a.dviezen maar walt vlug af te ma.ken of door die grootendee]s (1) D r L. Reh is in een zeer lezenswaardig opstel in . Zeitschrift f~tr
wissensehaftliche Insektenbiologie 9 (1905. IIeft 7) er tegen opgekomen,
dat in Duitschland altijd juist planthundigen aan 't hoofd van phytopa-
thologisehe instituten worden geplaatst ; hij meent dat dierhundigcn
daarop minstens evenveel aanspraak kunnen doen gelden. Zijn ideaal
echter zou d~rin bestaan, dat men phytopathologische instituten
stichtte, ~ an deren Spitze tin Landwirth steht, und an denen wissen-
schaftlich selbstst~indige und einander gleichberechtigte Abteilungen
die botanisehen, zoSlogischen, usw. Aufgaben ilbernehmen, so dass die
ganzen Anstalten doch gemeinsam auf das eine grosse Ziel, die Fiirde-
rung des far unseren Natioualwohlstaad so ntltzlichen Pflanzeaschutzes
hinarbeiteten .. Hij beweert dat echter een dierkundige met evenveel of met meet
reeht aan het hoofd van eea phytopathologisehInstituut kan staan als een
plantkundige, cn gaat aldus voort: 9 Dass die ganze Disciplin dabei nicht
schlechter fahrer wtirde, lehrt uns unter anderen das . Laboratorium
Willie Commelin Scholten . in Amsterdam unter der Leitung des
Zoologen Ritzema Bos, das far den Pflanzenschutz etwa soviel leistet
wie sttmtliche en~sprechende deutsche Anstalten zusammen .. D r Reh kan hier natuurlijk niet her oog hebben op de wetenschappe-
luke onderzoekingen, die aan de Duitsche inrichtingen en die, welke aan
mijn laboratorium hebben plaats gehad ; bet spreekt toch van zelf, dat ~dn
enkel, slechts aan matige eischen voldoend laboratorium zonder kassen
en terreinen, waarin ik gedurende het grootste gedeelte van de tien
jaren van zija bestaan~ 5falleen werkte 5f met geringe hulp, op weten-
schappelijk gebied niet ken praesteeren wat talrijke, veer een gedeelte
zeer goed ingerichte laboratoria met tal van wetenschappelijko perso-
hen er aan verbonden, kunnen doen. HET PHITOPATHOLOGISCH LABORATORIUIII WILLIE COiilHELIH
SCHOLTEli VAH 1895 TOF 1906, De ge~ardheid van den bodem, de ontwikkeling'speriode,
waarin de plant verl~eert, de tijd van 't jaar, de vrueht-
opvolging, de soort of variSteit van 't gewas, d,qt men teelt,
en zoovele andere omsta.ndigheclen zijn van veel bet.eekenis
met bet oog op den raad, dien men moe, t geve,n. Een vom'bevld. I~ermmsl~or{~Sn worden algcm(,~n mt't
sueebs door bespuiting met B~m?~,auxsehe, pap beMl'(~den. Stel nu dat wil&e of Lalhyrus sylvestris Wagneri wordt 45-- ingezonden, aangeta.st, door Pe,.oz~ospora Viciae, en men s{elt
r
iuzending , ter Mdoening ,~ in handen van een lee,fling
~f een minder deskundig beambte, dan zal deze in negen~ig
wm de tmnderd g~vallen adviseerml : be.spuit den akker met
]/ordeauxsehe pap; de landbouwer, die zoo'n advies krijgt,
sehudt her hoofll, en komt misschien later niet mere" tenlg
om advie, s; want de kwaal vetacoont zieh ~rst als 't gewas
re.eds ho(~g is opg~s<'hoten, e.n dan is her onmogelijk z(>o'n
l>eqmiting {e volvoea'm~,- daargela%zt hog dat men liever
de pap niet brengt aver planten, die over niet al te langen
tijd als veevoe.der zullen wordcn gebruikt. Zoo spoedig
mogelijk afmaaien, -- zied.aar her advies, da.t iaa dezen
ge.geven moest zijn; want sleehts bij uitzondering t, ree.dt
&m in de tweed(, sn(~le her kwaad weber op. 't l~mvensta.a.nde is geen gefl'a.geerd geval ; bet heeft zich
werkelijk ii~ mijne, praM, ijk v~orgedaa,n, d.~t een la,boran,t, in
mijn laboralor;um den raad wihle geven, een flink opge-
greet.1 wikkenveld met Bm'deauxsehe pap te be,spuiten. Een ander geval, dat zich eens voordoed, was her vol-
gende. ~I{et, vitriolen ~ van her zaaizaad is bij taru'e een
uit~te.kend midd,el om her opt,rede,n van bra,nd re, voo,rlmmen. Ec, n jongm<msl:h, di~ bij m U we.rkte, wilde ook ~dviseere.n,
d~*, 9c~',*t~ a,htus t,e behamlelen, om her optreden vaaa ger, s,te-
bra,nd tegen te gq,au,
rl'o,en !.k hem att.~nt maa.kte, op her
bozwaar, dot d,e gerstekorre,ls door hare kafjes mnslote,n
blijven, en de bra,ndsporen zieh binnen de kafjes kuo.nen
be.vinden, wilde hij in Mien ernst, d~ gels.t, pellen om ze
daarna te vitrioh'n. (1) D r L. Reh is in een zeer lezenswaardig opstel in . Zeitschrift f~tr
wissensehaftliche Insektenbiologie 9 (1905. IIeft 7) er tegen opgekomen,
dat in Duitschland altijd juist planthundigen aan 't hoofd van phytopa-
thologisehe instituten worden geplaatst ; hij meent dat dierhundigcn
daarop minstens evenveel aanspraak kunnen doen gelden. Zijn ideaal
echter zou d~rin bestaan, dat men phytopathologische instituten
stichtte, ~ an deren Spitze tin Landwirth steht, und an denen wissen-
schaftlich selbstst~indige und einander gleichberechtigte Abteilungen
die botanisehen, zoSlogischen, usw. Aufgaben ilbernehmen, so dass die
ganzen Anstalten doch gemeinsam auf das eine grosse Ziel, die Fiirde-
rung des far unseren Natioualwohlstaad so ntltzlichen Pflanzeaschutzes
hinarbeiteten .. steeds heb ingenomen als Directeur van her phytopathologisch labora-
torium Willie Commelin Scholten, ook bij bevoegde beoordeelaars in
bet buitenland waardeering vindt, niettegenstaande sommige geleer-
den en napraters van dezen er mij wel eens een grief van maakten,
dat ik niet meet . zuiver wetenschappelijk ,, werkte. HET PHITOPATHOLOGISCH LABORATORIUIII WILLIE COiilHELIH
SCHOLTEli VAH 1895 TOF 1906, Waar hij er van spreekt dat mijne
inrichting alleen , [i~r den Pflanzenschutz 9 etwa soviel leistet wie die
samtlichen deutschen Anstalten zusammen, heeft hij zeker her oog ge-
vestigd opwat het phytopathologiseh laboratorium Willie Commelin
Scholten veer de:praktijk is geweest. En moge D r Reh bier ook al over-
drijven~ niet zonder voldoening zie ik dat bet standpunt, 't welk ik - -
,17
- - aan soms n,og onervaran adsi,st,entan of aan leer]ingen oxer ~.~
lagen, meer tijd had overgehouden voor wet~nschappe]djk
onde,rzoek, en a]licht meet naam had! gema,al<t in de, weten-
sehai~pelijk,e, w,eretd ; ma,a,r ik weet o,ok, d'a,t ik o,p die wijse de
phytop,a,t~mlogie niet bij de pract~ci he,t, b~rge,iweeht had
doen verkrij gen, d a,t ik mind~r tot nut vaa de bodemkultuur
i.n Nederla.nd had ge,werkt. En &i,'irom was her mij te doen,
- - niet in de eerste plaats om naam te maken in de weten-
schappelijke werehl. Trouwens her geven van inliehtingen
aan praetici wordt in de st.atuten van bet phytopatTaolo,gisch
laboratorium }V. C. S. her allereerst genoemd onder [le
werkzaamheden van deze inriehting; en stellig ha,d o.ok de
Ilegeering g.een aanleiding gewmden, in de laatste jaren de
helft vau bet jaarlijksche budget voor hare rekening te
nemen, wanneer de stichting niet in de eerste plaats w,or
den Landbouw had gewerkt. -- I~eeds vddr 1895, maar la,t, er i.n s,te,e,ds toe~mraea~de
mate, weed h.erha.ahle.lijk mijn advies do.or de, Re,geering
ge,vraa~gd, ni,et a.tleen in zake phytopaiho~ogisehe, quae.st.ies,
maar in 't algeme.en in allerlei zak.en, de, t.oegepa:s.t.e natuur-
lijke hi,st~orie be.t.reffeade. In 1895 wa,s ik met. dea tte,er
Bult,man afgewmrdigde de,r Nede,rlandsehe t~egeering naar
eene eonfe,rentie t~e. Parijs, waar her. vraa,gst.uk van de, inter-
1rational.e. vo.ge.lbescherming we.rd b.esprokeaL
In 1898 z~.nd
mij d,e Rege,ering naqr d,e Yere,eaigde Staten vaa No~rd-
Amerika, Oill e,oi}_ o,nderzoek in te 8telle,n a,angaande, de
San Jos~ sehildluis en a.angaande, d,e be,t~eke~l~s, die. dit insekt voor oas land e,n in 't algemeen voor Europa. zou
kunnen krijgen,
tier door mij" ultgebraehte rapport w'r-
schecn niet a.]l~n in de Staatscourant, maar werd ook op
last der Reg,,eering in 't Duitseh vertaald. Dat mijn advies ook door her. Bes.tuur van ]let Land-
bouwcomit6, door Besturen van land- en tu~nbouwgenoot-
schappen en hare afdeelingen, d:oor den Directour der Ned. HET PHITOPATHOLOGISCH LABORATORIUIII WILLIE COiilHELIH
SCHOLTEli VAH 1895 TOF 1906, tIeidem.aat,sehappij, door den Inspectenr van her Staatboseh-
beheer, door de Rijkslandbouw- en Ilijkstuinbouwl.ceraren,
door Directeuren van de Rijkslandbouwproefstations en van
onderscheiden inrichtingen voor land- en tuinbouwonder-
wijs herhaa.ldelijk werd gevraagd, spreekt ,,'el van zeLf. 0ok buitenlandsche vakgenoofen wonnen herhaaldelijk
mijn advies in. Een aanbod van een aant.al landbouwers in Suriname,
om ter plaatse een ond.erzoek in to stellen naar den aard der
krullotcn in de cacao en naar de best.rijding der ziektc, werd
door mij na rijp beraad afgewezen, daar ik mij dam'voor
~anger nit Nederland zou moeten verwijdel, c.n, dan met de
belangen mijner bet.rekkingen hier te lande zou zijn in
overeenste.mming to brengen. Herha.ahMijk trad ik bij verschillende tentoonstellin-
gen als jurylid op voor de afdeelingen land- en tuinbouw-
onderwijs (m -onderzoek. Bij gelegenheid vail d~n in 1904
en 1905 te Wageningen gehouden wef]strijd van pu]verisa-
fern's fung~erde ik als voorzltter der jury. Her. nitg(~-
braehf(~ vers]ag over dezen wedstrijd is te vinden in den
XI ~" jaa~'gang van her ~ Tijdse.hrift over Plantenziekten ~,
bl. 81-96. Ilapporteur was de heer S. Lako. In de tweede plants werd, overeenkomstig de staiaien,
her phytopathologiseh labora~orium Willie Commelin Sehol-
ten dienstbaar gemaa.kt m'm de vitbreidin.g der 1,.ennis van --
4 9 ~ pla.tc~ziekte~t cn schadelijke diere~ door wctc~schappel~jk
oml~ r~ocl,!. Op dit gebied heb ik mlnder ge,l,everd &~n ik gaarne
zou hebbea ged,a,a~n. De inliehlingsdienst eischte do org~ans
te re,el ti.jd om ]ang ~ehtere.en aan wetonsehapp,elijke on(ler-
zoekin.o, en te kunne,n werke.n. Toeh zijn ve.rseheiden onderz.o,ekinge.n (~p phytopatho]o-
g~s(.]l g~bied gMaan. Ik voor mij he,b da, a~rbij .c,eMer l~ooit
uit het o.og verloren~ dat ik wefl~z~lam was hi he,t be4ang van
de praktijk. Het kost e, en w et,ellsehappelijk pe,rsoon wel
een~ e,e,nige, zelfopoffe.ring, om de e,e~ne of a,ndere kwe.stie, die
zi, eh g,e.durende e,en o.nc].erzoek w)ordo,et en we,lke uit e,en zui-
vet wc4e.nsehappe,lijk ~Jo.gpunt wel van bel,aag zou zijn verder
te. vervolgo,n, to lat,en rust,o,~l, wanneer blijkt, dat daa.ru[~
voor (~e praktijk 1clefs verder te hqlen zou zijn. Overigens
kunnen weer sores zaken, die uit een zuiver wetensehap-
pelijk oogpunt van weinig belang zijn, voor de praktijk
zeer belangrijk wezen. Of bijv. HET PHITOPATHOLOGISCH LABORATORIUIII WILLIE COiilHELIH
SCHOLTEli VAH 1895 TOF 1906, van eene bepaalde sehihl-
luissoort hog beweeglijke larven op de boomen voorkomen,
wanneer deze in 't najaar de bladeren reeds hebben verloren
of althans w'mneer de bladeren reeds opgehouden hebben te
fungeeren, kan van gr~ot be]ang zijn uit een oeeonomiseh
oogpunt; want de tijd, waarop men de boomen met een
insecticide dient le beslmiten, wordi erdoor bepaald. Do versi,agen, jaar]ijks van v~ege, her phytop~tlmlogiseh
1,aboratorium uitgebraeht, geven een overzieht ()ok van de
wetensehappelijke onderzoekingen, die daar werden verrieht. Daar zij eerst in her (c Landbouwkundig Tijdsehrift ~), later
in bet ~ Tijdsehrift over Plantenziekten ~ werden gepubli-
eeerd, behoeft bier daarop Met uitvo.erig t,e word~n t.erng'-
gekomea. Ik wil tlier sle.ehts de b.e,lsngrijkste, publieaties
even vermehlen. ttet ziju d~ volgenfle : 4 50 Ritzema Bos: over bestrijding van de dennenbastaardrupsen
(Tijdschr. o. rl. z. ~, I, bl. 13-19; ~ Forstlich-natur-
wissensch. Zeitschr. ~, 1895, IIe.ft 4). Ritzema Bos, over ([en, Amerika~mschen kakkerlak ~, scha-
delijk in plantenkassen en de besIrijding wm dit insekt. (,, Tijdsehr. o. Pl.z. ~, II, bl. 22-28). Ritzenm Boa, over inspuiting va,rt bealzine in den bo~lem, als
mi(Mel tege,n scha(lelijke iz~sek~en. (~Tijdschr. o, Pl.z. ~,
II, bl. 28-43;~ 'l, ijdschr, o. Pl.z. D, VII, bl. 37;~ Zeit-
schr. f(ir Pflanzenkrankh. ~, VIII, Ileft 1). 12itzema Bos, ove.r ]lotrytis Douglasii , sehadelijk aa.n kweek-
de~tnen. (~ Tij,lschr. o. Pl.z. ~, IIi, bl. 6; ~( Forstl. nat ul~qssensc.h. Zeitsehr. ~, 1897, Heft 4). H. ,I. Lovink eal Ritze,ma ltos, ond,elwoekingen omtrent her
ge,slach~ Reli,r~ia. ((( Tijclsehr. o. Pl.z. ~, III, bl. 8,3-134 ;
~eve.ns gepublieeerd a]~s uitgave, (le,r Nell. Hei, dema~tseh. en verd.er in cc CenLralb].,a,tt Fir Bacteriologic, u. Para-
sit~nkuaade, ~, II, Abt. 150, 1903, n ~ 8). Ritzenaa B as, ove~" Botrytis Paeo~iae Oud., oorzaak waa ee~m
ziekte bij pmen,en en Canva;]la.ri, a's. (~ Tijdschr. o. Pl.z. ~,lII, bl. 150-157; ~ Zeitsehr. fiir Pflanzen]~rank-
heiten ~, VIII, Heft. 5). Ritzcma Bos, voortz~tting v,'m vroe,gere si.udie.s omtreaat
aaltjeszi~ktc~, o. a. , Tij(.tsc.hr. o. Pl.z. ~, V, bl. 29,
VI, bl. 46; X, bl. 110; XI, bl. 144; ~ Zeitsehr. {(ir
Pfl,~nzenkrankh., XIII Ileft 4 ; XIV lIeft 3. ttandelin-
Hen van h.et achtste Ned. Satuur- en Geneesk. Congres,
gehouden ~ ]h)tterdam; 1902, hi. 180). ]litzema Bos, over eene bacterieziekte de,r syringen ((~ Tijd-
so,hr, o. P].z. ~, V, bl. 177). llitzem,~ Bos, over kmfllo~en e.n heksenbezems in de Cacao-
boomen in Sttriname. (~ Tijdsehr. o. :Pl.z. HET PHITOPATHOLOGISCH LABORATORIUIII WILLIE COiilHELIH
SCHOLTEli VAH 1895 TOF 1906, ~, VI, hi. 65 ;
Zeitsehr. f. Pfla~nzenkr,~'-~kh. ~, XI, Heft 1). --
51
-- Van ][all, over bnclerieziekten in ~!~ ko,ol en l~ij muurbloe-
men. (, Tijd.schr. o. l q.z. ~,, VI, bl. 1(i9). l{iizema Bos, cc Zur I~ebensgeschichte ties Muulwurfs ,,,
Biol. ('.vntralblat.t 9 XVIII, n ~ 2. ltitzema Bos, .vet her vuur in de nareissen en zijn.e In,sir;j-
ding (c, Tijdsdn'. o. lq.z. ,~, YII, bl. 12; ,, Zeilsd, r. fiir
Pilanzo.nkranldleit,,n ,,, lid XIII, 2 lleft). van Hall, over eem, ziekie in de syl.ingon, vel om'zaald do.or
l]olut:i.; c,l:/a,/.*. (,, Tijdschr. ~. lqantenz. ~,, VIII,
hi. 142- l-t-i). Ide <m l{ilzonm Bos, over be.sfrijding van lu, re- en apt)e]-
sdmrit. (,, 'l'ijdsdm o. Plantenz. ,, VIII, bl. 149-177). l{i4,zema. 13os, over het wegblijvo~ en omvallen der tulpen,
vorom'zaakt &~or Botrytis p,rasitiea. (~c Tijdsehr. over
Plantenz. ~, VIII, b]. 177-202; 9 Cenlralblait f/it lla~'-
teriologie u. Parasite.nkunde ,,, II Abt.. Bd X,
190:i,
n ~ 1-8). I{itzema. Bos, over her ontst~qn van giftatoffen in plant en-
deelen, d.ie door parnsitisehe zwammen zijn aangetast,
of door anderc o~rzaken zich nie.f nm'lnaal konden, onf-
wikke]en. (,c .llygienisehe Bluden ~, 1901, n ~ 1, 2 en ;{). vim llal], l)iss0ria.tic, over baefcrie.&~ p]a,nie,nzi.ekie.n, 1902. wul IlalI, c,nqu61e. ,mt rent Ophiobolu.s hm'potrichu.s. (, Tijd-
.~]u'. o. Planfvnz. ,, IX, l)]. 77-97). l{itz~,ma llos, ore.,' Monilia.ziekle.n de.r o.oftboomen. (~ Tijd-
schr. o. Plan~enz. ,), IX, bl. 12.5-1,I7). ]litzema Bos, bijdrage tot de kennis van tie sehurftziekie van
den aardappel. (Zie Itandelinge,n v:m her 9 ~ Xeder]. Natuur- en (~eneosk. Congres, gehouden fe's Graven-
hage, 190:~; bl. 226). vaa~ Ilall, die Sant.t Joharutis krankheit (ler Erb,sen, verur-
saeht yen FusaT'ium t'asi~dectum (, ]~eriehte der deut-
schen botanischen Gesellschaft ~, 1908, Bd. XXI,
Heft 1). --
52
-- l~itzema Bos, ]~ankerstronken en va]lers bij de koo]',, veroor-
za,akt door Phoma olcracea. (, Tijdsehr. o. Plante~z. ,,
X, bl. 53-71, XI, bl. 106-118). Drost, over Pleurococcus vulgaris, a.ls e~dophytiseh ]eve.nde
wier. (, Tijdsehr. o. P]a.ntenz. ~, X, bl. 71-73). Ritzema Bos, Onvruehtbaarheid bij zwarte bessen. (, Tijd-
sehr. o. Pla,ntenz. 7), X, bh 137-145). Ritzema. Bos, over sehade d,oor huisj'esslakken. (, Tijdsehr. o. Plantenz )), X, bl. 145). Ritzema Bos, over de oorzake,n van slechte kiemba.arheid van
in 1903 gew(mnen zaad. (co Tijdsehr. o. Pa.nte~z. ,, X,
5o
bl. L~; XI, bh 124). Quaaje.r an Vfirtheim, onderzoekingea omtre,nt r:ookbeseha-
diging re, Schiodam. (c~ Tijd, schr. HET PHITOPATHOLOGISCH LABORATORIUIII WILLIE COiilHELIH
SCHOLTEli VAH 1895 TOF 1906, o. Plantenz. ~, XI,
bl. 170). H. M. Quanjer, over Plutella cruci/erarum. ((( Tijdschrift
voor enLomologie, deel XLIIX). H. M. Quanjer, over Plutella cruci/erarum. ((( Tijdschrift
voor enLomologie, deel XLIIX). Aan de b.ovensta,ande opsomming van g edane onderzoe-
kinge~ en in '~ lieht gegeven publieaties sluit zich aan eene
opsomming van de volgealdo gedure~de 1895-1906 uitge-
k H. M. Quanjer, over Plutella cruci/erarum. ((( Tijdschrift
voor enLomologie, deel XLIIX). H. M. Quanjer, over Plutella cruci/erarum. ((( Tijdschrift
voor enLomologie, deel XLIIX). Aan de b.ovensta,ande opsomming van g edane onderzoe-
ki
i
' li ht
bli
ti
l it
i h Aan de b.ovensta,ande opsomming van g edane onderzoe-
kinge~ en in '~ lieht gegeven publieaties sluit zich aan eene
opsomming van de volgealdo gedure~de 1895-1906 uitge-
geve,n werken : Ritzema Bos, q Ziekten en Be,sehadigingen der Kultuur-
g~wass.en )), 2 deepen. (Groninge,n bij J. B. Wolfe,s;
1897 e n 1898); 2 d~ d~*uk o~der den titel 9 Ziekten en
Besehadigingen der Landbouwgewa.s~sen ~. 2 deelen. (Groningen, J. B. Wolters; 1902). Ritzema Bos, 9 Zi.ekten en Be schadJigingen dev oo~boo-
men ,, 4 d,ee.]en. (Groningen, bij 5. t3. Wo]ters; 1905). Ritzema Bos, 9 Zi.ekten en Be schadJigingen dev oo~boo-
men ,, 4 d,ee.]en. (Groningen, bij 5. t3. Wo]ters; 1905). Rite,zem,~ Bos, a Sehoolplate~l van voor den akke,rbo,uw seha-
delijke dieren ~, met Handleiding. (Groningen, bij
J. B. Wolter.s; 1905). Rite,zem,~ Bos, a Sehoolplate~l van voor den akke,rbo,uw seha-
delijke dieren ~, met Handleiding. (Groningen, bij
J. B. Wolter.s; 1905). Ritzema Bos, 9 Zoologie ~iir I,a~dwirte ,. (Berlin; Paul
Parey), 2 ~ druk 1896, 30 druk 1900, 40 druk 1905. --
53
-- Van de Engelsche uitgave van (lit werk,vertauld door Prof. Aiu~worth Davi~, vc.rscheen de 1" (lruk ill 1894, de 2 ~ in 1900. l~itz.ema Bt~s, ~ Schet.am uit het ;..even de.r Vogels ~, (Am-
sler(lam, Va.n den ]h'oeeke, 1897.) Van de Engelsche uitgave van (lit werk,vertauld door Prof. Aiu~worth Davi~, vc.rscheen de 1" (lruk ill 1894, de 2 ~ in 1900. l~itz.ema Bt~s, ~ Schet.am uit het ;..even de.r Vogels ~, (Am-
sler(lam, Va.n den ]h'oeeke, 1897.) In de derde l)laal,s moes|, volge.ns de statuten, het l)hy -
topafllologis(.h labm'atoriunl Willie Commelin Scholteu
wor(len (liensibaar genmakt , a.an (lc belal)gen van her,
(mdcrwijs van tle stu(lenten in de I)lant.- eli dierkunde aan
de l'niversiteii van 3.m,ier(]mn, en in "t algeme.en van het
onderwijs in de phylol)athologie ~. Met. HET PHITOPATHOLOGISCH LABORATORIUIII WILLIE COiilHELIH
SCHOLTEli VAH 1895 TOF 1906, uitzondering van twe~- jaren, waarin doer mij ge(m
college werd gegeven, werden door mij in iede.ren em.'~us in
t wee uren l>er week onderseheiden hoofdstukken uit de zie.k-
tenleer (ler t)hmt.en belmndeld. Aanvankelijk wa.s (te phyit;-
patho]ogie veto' nicmand een examenvak ; later werd zij dat
veer hen, die het do(.toraal ex'unen in de 1)|ant- en (lierl~un(le
wihIen afleggen. Ten ein.te (1(, examens niet te reel t(,
verzwaroll, W('l'd c.ahter ge.woonlijk sleehts over een vrij
1x,p<.rli~ g'o,h'elte va,n de t)hyi.opathologie ge~xaminecr(l. Zoo k~os (I~ een h(~t m~(le, rwerp : galvormi.ng, - - eeu amler
3e zield.e.n, d.,,.or Ne.mai.oderL en Phyr
t.e.~ree.g'ge.bn~.eht:
--- ~,e.u d(.,.rde, de sehad.e.lijke zoogdiel'en en voge]s, --- e(,n
vierde de [Tredineiql en Uslil'agine~n (roest- en bra.ndzwarn-
men), -- weer andel,en k(.zen de I)|ani(~nziek~n ver<)orza-
kende. Pyrenomyceten en ])iscomyeete.n; een koos de door
zwmnmen ver(,orzaakh, ziek%n van (~onife.re.n on V;lll graan-
gewassen : enz. Mijn<~ <'oll(~ge,~ werden in de verschillmlde, jaren door
(x~n z~er ver.s<'hil]end o'etal studenten bezoeht; terwijl (Vt
ge.tal (,en pa.ar jarpn ('('it 25-;I0 tal lx, droeg, waren er ook
jaren van sleehts 5 hfn)rders; de mee.ste jaren bedrocg hun
aantal (mgevoer (,en 12 tal. --
54 De meeste hoorders waren studenten en candidaten ;n
de plant- en die::kunde.; verder werden de colleges gevolgd
door cenige pharmaceutcn, door en]:ele medic:i, alsmede door
een p,'uar or~derwijzers en een' tuinbouwkundi~, die nict a.]s
studenten waren ingesehrc:e.n. In aalrsluiting aan de colleges we,rden eenige malen in
verschille,nde declen des laad~ i)hytopathologische excursion
gemaakt. -- ttoewd over be]angs~elling van de zijde dcr stu.dcnten
in 't geheel niet te k~agen viel, moet toch worden erkend,
dat de colleges niet in alle, deelen aan hun doe1 l:on.den
bca_ntwoorden. En gcc.n wonder l De studenten in de plant-
en dierkun(le volgden de colleges om hunne na.tuurhisto-
rische kenni~ t>e vermeer(tcren ; maa.r daar zij in 't algemeeu
niet in de landbouwwetcnsehap thuis waren, kon de toepaz-
sing van de natuurhis.torisehe studie op de bodemk:tltuur
niet voldoende tot haar recht komen. De phytopathdogie
is nu cenmaa] eenc toe.~Dqste wetenschap, wetker doel is,
de t)roductiviteit vaa: den bodcm is vermeerderen door (le
kwalen der kultuurplanten te bestrijden; zij baseert zich
66k op de plantkundc cn de dierkunde, ma.ar 56k op natuur-
en seheikund.e, op de leer van de plantenteelt, en op de erva-
ring der plantentclers. HET PHITOPATHOLOGISCH LABORATORIUIII WILLIE COiilHELIH
SCHOLTEli VAH 1895 TOF 1906, De phytopathologie is gecn tak van
de botanic, zooals vele plantkundigen schijnen tc mccncn,
die zich voorstclI,en dat een bonanicus, wanneer hij e(m-
voudig war extra, werk maakt van zwammen, zoo dadelijk
phytopatholoog is geworden. Zij is evenmin een tak van
de botanic, als zij een tak is van de zoologic, do physiea
of de ch.emie. Die vakken zijn al te gadc.r grondleggea:d,>
weterrschappen voor do phytopathologie, wdke zich zoowel
op deze vakken baseert als op de praktisehe ervaring dcr
kult, nurmannen. ~N'u zijn studcnten in de plant- en die.rkunde in de mecstc 55
-- gevallen te weinig met de praktijk van de verschillende
takken van plantenteelt o p de hoogte, om de phytopatho-
logie met vrucht te bestudeeren ; en voor de praktische resul-
tat,en dezer to.egepa,ste wetenschap voelen zij dikwijls ook
Met de wa.re be]angstelling. De studie de~' phytopatho,logie, knn a lleen tot ha ar 1~eeht
komen aml ee,ne inrM~ting van Itooge,r Landbouw~o,nderwijs
(Hoehsehule fiir Bodenkul~ur). Dit toegepaste yak te d,oce,e-
ren aan eerie Unive~siteit,waaraan geon ]!and b,ouwin,s~dtuut is
verbonden, komt ong.eveer op 't zelfde neer als : een college
in ophthahnologi.e, te g.e.ven aan e,ea.e. IYniver'sit~eit zonder
medisehe faeulteit. De Pro~essor voor dat v, ak zou voor lie
stud,ent.e,n, die hoo.fdzakelijk de zoiilo.gie en de verg,elijkende
physiologic, tot stndievak hebhea geko.ze.n, eea a.antal col-
l.eges kun.n.en geven over de a.na,tomie en physio.l.ogie van her
oog, waarva.n zij heel war nut zouden kunnen hebben;
e, ll ook de studenten in de. physiea z,ouden met nut ao,odanig
college van den ophthalmoloog kunnen volgen ;--maar noeh
de. zoSlogea~ no.eh de physiei zouden door 't vo.lge.n va.n zo,o-
da.nig college ophtha,lmologen wordea. Om o,phthMlnoloog
te word.en, dient men eers,t mede,eljne;n te studee,ren ; daarna
ka.n me.n zivh in e,ene b,epaaht,e riehting speeialiseeren. Om
phyt,opatho.loog te wor3en, dient men vooraf pl:antenteelt te
hebben bestude,erd, of althans op de ee.ne o.f a.ndere ma,nie.r
zieh de kennis van de teelt wm de 'kultuurgewassen te hebben
eige,n gemaaJd. De,n b.ota.nieu,s, die phytopathologie wil
studeeren, ontbreeld ook meestal te veel de belangsIelling in
de voordeelen, welke dit. yak den praeiieus trae'ht te ver-
sehaffen; bij hem is de wetensehappelijke sfudie nommer
66n, en belangstelling wmr de praktijk heeft hij maar' al te
vaak alleen zoo lang hij denkt, uit de ervaringen der prak-
tijk aanknoopingspunten te vinden, die vtmr zijne studie
van belang zijn. HET PHITOPATHOLOGISCH LABORATORIUIII WILLIE COiilHELIH
SCHOLTEli VAH 1895 TOF 1906, --
56
-- Ik heb dan ook meer ea meet mijne, colleges aan do
Amsterdamsehe U.niversiteit, bij allo betlaagst~lling, die
door HI{. ~.~oucle~nfen wer.d betoond, leeren besch, ouweal a~s
eerie aanvulling bij hunne aa.tuurhisl,ori~che studia, -- ~liet
al.s e~.ne opleiding l,ot phytopathologea; da.arvoor moe.~ ik
de U~iversiteiten, zooals zij bij (ms zijn in~,,'ieht, ong'c-
sehikl, a,ehten. Dat neemt natuurlijk niet weg, da.t zieh
tamer de student, en aan eene Universiteitt personem ktmne~l
bevinden, die door hunnen natuurlijken a.aaHeg, door
hmmo belangst.elling in, en door de door h~n opgedano
kelmis v~m (le versehilleade takken der t)laatenteell, wel
dege.lijk geschiktt zijn, om goede phytopathologen te wof
den. -- ~%devl, mijn veri.rek n.aar Amsterdam had ik aan do
toenma]igo Hoog~,re Lind- en ]3oschbouwschoot t(~ Wagc-
ningen her onderwijs in de ken_his der schudelijko dieren
behouden, en ]afer, toen de wetenschappelijke cursus aan de
Rijksfuinbouwsehool werd ingerieht, ham ik aaa die afdoc-
ling her onderwijs in de gehede phytol~thologie op m;j. Dit, onderwijs bleef ik g,e.ven gedurende mijn tie.njarig ver-
blijf to Amsterdam. -- Ten slotfo meende ik, dat her ook de taak van he~
phyl,opathologiseh ]a.bo~,n.torium behoorde, de kennis der
plantenziekfen en schadelijkc dieren o.nder ]andbouwers,
kweekers, ooft- en groeafe.telers en boschbouwcrs Iv verbre;-
den, en de be]aagste]ling in de phytopathologie bij de prac-
'ciei t,e wekke,n. En ik lmb de e~'varing r
dnt her
le.vende woord in deaen reel vermag. Da.t de phytx~patho-
logie m.eer e.n m e.er bij de praet~ei hier {e. laa,le bet, burger-
,~.5t hee.ft verk~gen, moet .,eer zeker 66k voor eea decl
worden toegesehreven aan de voordrachten, die deor her
personeel der stichting horhaa.lde]ijk in onderseheiden dee-
len dcs lands werden gehou&m. u
ble:'k het. heel nuttig - -
5 7
n te zljn, wann eer na afloop der ]ezing de gelegenhe~d werd
opengesteld,
d.en spreker
om inlichtingen
te
vrageu
omtrent phytopathologische kwesties. In stre ken, vaa waar
vroeger nooit inze,ndingen kwamen, werden dikwijls na
eene gehoud.en l ezing ve,rscheide,n trouwe corre,spoaden/en
van bet phytopathologisch laboratorium aangeworven. Gedurende bet tijdperk van Sept. HET PHITOPATHOLOGISCH LABORATORIUIII WILLIE COiilHELIH
SCHOLTEli VAH 1895 TOF 1906, 1895 t.ot Januari 1906
werden door mij voordra.chten gehoud,en te Am~sterdam
(3 maal), tIaarl,em, Zaandam (2 maal), in den Bangerd, te
Levuw0,rden, Groningen (2 ma,al), Oldehove, Warffum,
Loppersum, Appingedam, Beerta, Winschoten, Seheemda,
Zuidbro,eL Zuidlare.n, Nieuw Buinen, Gm~sse], (Iro.en]o,
Wint~erswijk, u
Ze]hem, Brummen,
Zutphen,
Yo,rst, Twello, Apehloorn, Nijkerk, Wageningen, Arnhe,m,
Nijmege:n, Tiel, Buren, Cul,~.mborg, ]lijsenburg, Z,eist,
Utre.eht, Ba,arn, Hilversum, Bussum, AaJ~sme.e,r (2 maul),
Bre.uke.l.e.u, Leid.en, Bo,skoop, 's-Oravenhage, Rotterdam,
Axel en Tilburg. -- Do,or D" vaa Ha].l werde~n voordrachten
gehoude,n te Am.sterd'lm, Baarn, tIeinkenszand en Kruinin-
gen ; door A. W. Drost te Ba.arn en te Bus.sum. D.e vo.ordra.chten werden d,oo.rgaans gehond~e.n bf met
st,eun van de Nedea']and, sche phytopat.hologische Ve,reeni-
gMg, voor we]ke ook D r He.[nsius e.enige ]ezingen hie.ld, bf
met st~un van de e.ene of aadere land- of tuinbouwmaat-
schappij. -- Ten .sloit.e moet.en al~ een,e, hoo,gst, be~angrijke werk-. zaamheid va,n her phytopathologisch labora,torium worden
be.schouwd de be,zoeken, do.or he.t t)ers, on.eel afgelegd bij d,e
hutdbouwers, kwe,ekers, ooftt.e,lers, enz. ]),aar, o,p bet ar-
beid,ste,'rein der onderseh.ei&n praetiei, is ge]egenhehl te
(~ver niet, alle.ea om inlichtinge~ {,e v.e,rstre,kken omtrmd de
daar voorkomemle, ziekt.en en beschadigingen en mnirent
hare be,strijding, maar .er is ook ge]egenheid, om kc~nni:, to 58
-- maken met. ziekten en bcsehMiging~.n, (lie den bezoeker tot
dttsver onbekend waren. Ook doer men daar reel speeiale
]/ennis op omtrent de ku]tuur der gewasscn onder verschil-
lende omstand.,igheden : een eersie vereischte voor hem, die
met vrueht als phyt()patholoog wil werkzaam zijn. Yan d(.'
gelegenheid om praetiei te beT, oeken en de voorl~omende
zk'ktcn en besehadigingen in l.oeo waar te nemen, werd dan
ook veelvuhlig gebruik gemaakt. Wanneer ik aan he~ einde va.n mijne werkzaamheid a]s
Direeteltr van her phyiopa.thologisch la.boratorium Willie
Commelin Scholten hog eens e,en blik sla. op (le tien jaren,
die achier mij lighten, dan kan ik met eenig~ vohl~ening
getuigen dat her uitgezaai(le za.ad wortel hecft geseholen. ])e phytopathologie heeft zieh eene plaais wet~n te veroveren
bij hen, die zi(.'h in h'ede.rland met (te bo(lemkul~uur bezig
hou(len. De opriehting van bet: Rijks Insiituuf voor phyfo-
pt~t.hologie, verbonden aan (le Rijks Hoogere Land-, Tuin-
en Bcsehbouwsehool te Wageningen, zal mij in staat stellen,
omler gunstiger omsfau.dighe,.lea te werken dan tot (lusver. HET PHITOPATHOLOGISCH LABORATORIUIII WILLIE COiilHELIH
SCHOLTEli VAH 1895 TOF 1906, Ik hoop dat her mij gege.ven moge zijn, aan deze nieuwe
inriehting nog eenige jaren in her belang wm de v~3erhl.nd-
sehe bo(lemku}{uur we.rkzaam te zijn, zoowel (loor her geven
va.n adviezen en d~)or her geven van omlel~vijs in engeren en
ruimeren kring, alsook door wetensehappelijk onderzoek. pp
j
J. Rn'z~.:~a., Bos. Wageningen, Maart. 1906. Wageningen, Maart. 1906.
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Bioremediation efficacy—comparison of nutrient removal from an anaerobic digest waste-based medium by an algal consortium before and after cryopreservation
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Cronfa - Swansea University Open Access Repository This is an author produced version of a paper published in:
Journal of Applied Phycology Cronfa URL for this paper:
http://cronfa.swan.ac.uk/Record/cronfa36103 p
Silkina, A., Nelson, G., Bayliss, C., Pooley, C. & Day, J. (2017). Bioremediation efficacy—comparison of nutrient
removal from an anaerobic digest waste-based medium by an algal consortium before and after cryopreservation.
Journal of Applied Phycology, 29(3), 1331-1341.
http://dx.doi.org/10.1007/s10811-017-1066-x * Alla Silkina
a.silkina@swansea.ac.uk; alla.silkina@gmail.com Paper: p
Silkina, A., Nelson, G., Bayliss, C., Pooley, C. & Day, J. (2017). Bioremediation efficacy—comparison of nutrient
removal from an anaerobic digest waste-based medium by an algal consortium before and after cryopreservation. Journal of Applied Phycology, 29(3), 1331-1341. http://dx.doi.org/10.1007/s10811-017-1066-x Silkina, A., Nelson, G., Bayliss, C., Pooley, C. & Day, J. (2017). Bioremediation efficacy—comparison of nutrient
removal from an anaerobic digest waste-based medium by an algal consortium before and after cryopreservation. Journal of Applied Phycology, 29(3), 1331-1341. http://dx.doi.org/10.1007/s10811-017-1066-x This item is brought to you by Swansea University. Any person downloading material is agreeing to abide by the terms
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repository. http://www.swansea.ac.uk/library/researchsupport/ris-support/ J Appl Phycol (2017) 29:1331–1341
DOI 10.1007/s10811-017-1066-x Bioremediation efficacy—comparison of nutrient removal
from an anaerobic digest waste-based medium by an algal
consortium before and after cryopreservation Alla Silkina1 & Graham D. Nelson1 & Catherine E. Bayliss1 & Craig L. Pooley1 &
John G. Day2 Received: 28 September 2016 /Revised and accepted: 17 January 2017 /Published online: 5 February 2017
# The Author(s) 2017. This article is published with open access at Springerlink.com Abstract An algal consortium was isolated from an integrat-
ed steelmaking site at TATA Steel Strip Products Ltd. in Port
Talbot, UK, and its bioremediation capacity tested. Excellent
Bbioremediation^ was observed when the mixed culture was
Bapplied^ to diluted effluent from an enhanced anaerobic di-
gestion plant at Dŵr Cymru Welsh Water at Port Talbot, UK. After 5 days of cultivation in a 600-L photobioreactor, 99% of
the total nitrogen (initial level, 4500 μmol L−1) and total phos-
phorus (initial level, 690.4 μmol L−1) were removed from the
waste stream. The consortium was deposited in the Culture
Collection of Algae and Protozoa (CCAP), an international
depository authority for microalgal patents, as CCAP 293/1. This material has been successfully cryopreserved using a
two-step cryopreservation protocol with dimethyl sulphoxide
(5% v/v) used as a cryoprotectant. On recovery of samples
after 3 months storage at −196 °C, the specific bioremediation
activity of the revived consortium was tested. The capacity of
the revived culture to bioremediate effluent was not signifi-
cantly different (p < 0.05) from a non-cryopreserved control,
with 99% of total nitrogen and phosphorus remediated by day
4. Although non-axenic algal cultures have previously been
cryopreserved, this is the first report of the successful cryo-
preservation of mixed algal consortium, with validation of its
ability to bioremediate after thawing comparing non-
cryopreserved cultures with a revived post-thaw algal consortium. The study also highlights the need to ensure the
long-term security and the requirement to validate the func-
tionality of conserved inocula with biotechnological/
bioremediation potential. Keywords Alga . Algal consortium . Bioremediation . Cryopreservation Keywords Alga . Algal consortium . Bioremediation . Cryopreservation 2
The Culture Collection for Algae and Protozoa, Scottish Association
for Marine Science, Scottish Marine Institute, Oban PA37 1QA, UK 1
Centre for Sustainable Aquatic Research (CSAR), Swansea
University, Swansea SA2 8PP, UK Introduction There is a long tradition of bioremediation of waste streams by
microalgae with early work undertaken by Oswald and col-
leagues in the 1950s (Oswald et al. 1953a, b, 1957b) leading
to the subsequent widespread use of high rate algal ponds
(HRAPs) for effluent treatment in many countries. In addition
to the capacity of algae in HRAPs to utilise nutrients, includ-
ing dissolved nitrates and phosphates, some taxa can degrade
toxicants such as high molecular weight polycyclic aromatic
hydrocarbons (Juhasz and Naidu 2000). Furthermore, algae
can adsorb heavy metals and other toxicants in aquatic envi-
ronments (Gadd 2009). Current technology for algal wastewa-
ter treatment uses HRAPs; however, low algal productivity
(generally ∼10 t ha−1 year−1) and requirement for expensive
processes for algal harvest limit the commercial exploitation
of algal biomass (Craggs et al. 2011). With the development of
novel technologies, this process has become more efficient
and cost effective, for example the use of shallow,
paddlewheel-mixed, HRAPs can result in productivities
>30 t ha−1year−1 and better consistency of nutrient removal
(Craggs et al. 2011). Furthermore, considerable amounts of
research have been focused on the development of
photobioreactors (PBRs) over the past two decades and a wide
range of different designs and configurations have been built
and tested around the world (Pulz 2001; Singh and Sharma 1332 J Appl Phycol (2017) 29:1331–1341 2012). Successful PBR cultivation systems, with high bio-
mass productivity, have been demonstrated in high latitudes
(Fuentes-Gruenewald et al. 2015). These have used semi-
continuous cultivation approaches with resultant high nutrient
removal rates employing Porphyridium purpureum and
Scenedsmus sp. (Ruiz-Marin et al. 2010). to the algal uptake, phosphorus precipitation enhances the
remediation process (Laliberte et al. 1997). A further advan-
tage is that a mixed consortium has better ability to uptake
carbon, as oxygen evolution by the algae facilitates aerobic
bacterial growth and in addition many of the algae involved
can directly sequester carbon via mixotrophic, or heterotro-
phic, growth (Oswald et al. 1957a; Day and Turner 1992). The waste material from an anaerobic digestion (AD) plant
was used in this study. This effluent contains high concentra-
tions of nitrogen (in the form of ammonia) and phosphorus
and may cause environmental problems if discharged without
treatment (Li et al. 2011). Introduction The reduction of nutrient levels in
effluent is required both to avoid environmental damage on
discharge and to conform to the local and domestic legal re-
quirements (Welsh Government DfEFaRA 2014). These re-
quirements can be performed by microalgae because of their
capacity to use nitrogen and phosphorus in their metabolism
(Pittman et al. 2011). However, the efficiency of the algal
bioremediation process primarily depends on the algal strain
selected or the constituents of a microbial consortium in-
volved. To date, the majority of studies that have focused on
remediation have utilised uni-algal cultures (Wang et al. 2010;
Lizzul et al. 2014; Praveenkumar et al. 2014; Whitton et al. 2016; Schulze et al. 2017). The use of algal consortia has been
less extensively studied, although a number of recent reports
have indicated that this strategy can outperform pure culture
applications (Dalrymple et al. 2013; Samorì et al. 2013). The maintenance of a functionally stable, reproducible mas-
ter stock culture, in this case, an algal consortium, is a pre-
requisite for sustainable remediation, as it is in any other bio-
technological process employing microorganisms. Furthermore,
the ex situ maintenance and deposition of the microorganism(s)
in an international depository authority (IDA) under the terms of
the Budapest Treaty (Anonymous 1977) are absolute require-
ments on submission of a patent. This raises specific challenges
as IDAs are contracted to maintain the organisms, without loss
of their desired patent-related characteristics for in excess of
15 years. Whilst for some algae, serial transfer may suffice and
stability of metabolite production may be maintained, as in the
case of pigment mutants of Parachlorella kesslerii (Müller et al. 2007), for other algal taxa, it may result in loss of key characters/
capabilities (Day and Fleck 2015). Serial transfer cannot abso-
lutely guarantee retention of key characteristics, and although
there are no published data on the retention or loss of bioreme-
diation capability of algal consortia that have been held under
laboratory conditions for extensive periods, alternative conser-
vation strategies that do not require maintaining growing cul-
tures may be optimal. In addition to the possibility of loss or
reduction in efficacy of the characteristics relevant to the patent
application, serial transfer by its nature has the potential to result
in human-error-induced mistakes such as mislabelling or con-
tamination on transfer. Introduction Therefore, in addition to issues associated
with functional stability, alternative long-term preservation
methods that minimise handling/manipulation are needed to
guarantee that materials remain Bfit for purpose^. Cryopreservation, storage at ultra-low temperatures (normally
−80 °C for prokaryotes and <−120 °C for eukaryotes), is widely
accepted as the optimal method for the conservation of patented
strains, although for many bacterial taxa, freeze-drying
(lyophilisation) may also be applicable (Day and Stacey 2008). G
ll
l
l
lt
h ld i
h l b
t
i
l pp
(
y p
)
A key aspect of this study has been the use of a mixed algal
bacterial consortium from a polluted ecosystem, which was
the basis of a patent submission (Silkina and Nelson 2014). The stability and sustainability of mixed cultures has previ-
ously been reported (Kumar and Goyal 2009; Van Den Hende
2014; Van Den Hende et al. 2014a, 2014b, 2015, 2016). Furthermore, isolated mixed consortia from polluted ecosys-
tems have already adapted to survive on the waste stream as
the natural selection process has already been undertaken. Thus, there is a high probability that the consortium will be
more stable than an artificially formulated algal/bacterial mix-
ture. Additionally, due to the high level of tolerance of pollut-
ants, it is probable that mixed consortia should acclimate more
quickly than a monoculture culture. Studied mixed consortia
composed of tolerant species, most commonly chlorophytes,
have previously been reported for their capacity to remediate
waste water (Khan et al. 2008; Ruiz-Marin et al. 2010; Wang
et al.2010). In these studies, the mixed consortia had higher
rates of uptake of nitrogen and phosphorous, as the different
species could utilise different uptake mechanisms. Algae are
known to have mechanisms for the uptake of various forms of
nitrogen, namely, ammonia, nitrate and amino acids; these are
highly variable within the algal community and individual
algal strains may have differing nutrient uptake capacities
(Olguın 2003; Barsanti and Gualtieri 2006; Cai et al. 2013). The growth of the mixed consortia results in a rapid increase
in pH of the growing culture. Under this condition, in addition Generally, algal cultures held in research laboratories, cul-
ture collections or by commercial organisations for biotech-
nological applications are maintained as uni-algal
(monocultures) but not necessarily axenic cultures (Lorenz
et al. 2005). Furthermore, the commensal bacteria may be vital
to maintaining healthy functional cultures (Prakash et al. 2011; Amaral et al. 2013). Algal consortium and cultivation The ACCOMPLISH algal consortium was isolated by taking
water samples from an integrated steelmaking site in spring
2012 (Port Talbot, UK) and cultivating these on a defined
freshwater algal medium based on the nutrient levels
employed in f/2 medium (Guillard 1975). The most robust
consortium, i.e. where no obvious change in the algal mix
was observed on successive transfers, has been used in a bio-
remediation study using nutrient media based on the waste
stream from an enhanced anaerobic digestion plant (Port
Talbot, UK) (Silkina et al. 2015). Since February 2012 this
consortium has been maintained in the Bioscience
Department microalgal culture collection (Swansea) in a
freshwater algal medium based on the f/2 nutrient profile
(Guillard 1975) under 18 °C, 100 μmol photons m−2 s−1 with
16:8 light/dark cycle. The material was deposited in the
Culture Collection of Algae and Protozoa as a patent deposit
and allocated the unique identifier CCAP 293/1 (note: patent
strains are not in the public domain and cannot be directly
obtained from the CCAP). A bench scale trial was conducted using 2-L flasks exter-
nally illuminated on one side by a twin florescent tube (Natural
daylight Osram tube). Each flask was sealed using a nitrile
rubber bung with two separate holes drilled for glass tubes to
be inserted, one for aeration and sampling and the other for
venting the exhaust gas. The room temperature was maintained
at 18.0 ± 3 °C. Lighting from the florescent tubes was provided
by an 18:6 light cycle at 200 μmol photons m−2 s−1 with sam-
ples taken at the end of the light cycle. The flask was contin-
uously sparged with ambient air at 0.1 vvm, with the addition
of 0.03% (v/v) CO2 during the light cycle. The pH was main-
tained by the addition of 10 mg L−1 sodium bicarbonate. Daily samples (15 mL) were aseptically taken from the
cultures, their pH immediately measured, then cell concentra-
tion and biovolume were assessed by Coulter Counter
(Multisizer 4). Samples for water chemistry analysis were tak-
en every 24 h and centrifuged for 15 min at 3000×g. Supernatant samples were then passed through GF/F
Whatman filters and stored frozen at −20 °C for 1 week prior
to analysis. The predominant algal species in the consortium was iden-
tified by Banco Espanol de Algaes (BEA), Grand Canarias,
Spain, as Franceia amphitricha. Introduction Cryopreservation has been widely
employed to maintain algal cultures; however, for the majority
of the protocols employed, axenicity has been the key to suc-
cessful recovery of a healthy algal culture (Taylor and Fletcher
1998; Day and Fleck 2015). Where cryopreservation has been
applied to non-axenic algal cultures, in some cases, additional
procedural steps may be required to reduce available carbon J Appl Phycol (2017) 29:1331–1341 1333 Table 1
Taxonomic designation of algal taxa in CCAP 293/1
consortium
Species
Family
Class
Franceia amphitricha
Oocystaceae
Trebouxiophyceae
Scenedesmus sp. Scenedesmaceae
Chlorophyceae
Chlorella sp. Chlorellaceae
Trebouxiophyceae
Chlamydomonas sp. Chlamydomonadaceae
Chlorophyceae
Desmodesmus sp. Scenedesmaceae
Chlorophyceae Table 1
Taxonomic designation of algal taxa in CCAP 293/1
consortium released as a result of cell lysis that may result in a bacterial
Bbloom^ and subsequent algal death (Heesch et al. 2012;
Amaral et al. 2013). This study explored a scenario where
there was a functionally stable, mixed, algal-bacterial flora. The objective was to demonstrate that cryopreservation could
be used to conserve this undefined algal consortium and to
validate that the application of a standard cryopreservation
approach could be employed to ensure functional stability of
the consortium. released as a result of cell lysis that may result in a bacterial
Bbloom^ and subsequent algal death (Heesch et al. 2012;
Amaral et al. 2013). This study explored a scenario where
there was a functionally stable, mixed, algal-bacterial flora. The objective was to demonstrate that cryopreservation could
be used to conserve this undefined algal consortium and to
validate that the application of a standard cryopreservation
approach could be employed to ensure functional stability of
the consortium. Material and methods assess optical density, a direct light measurement was selected
in the NIR (near-infrared) spectrum, at a wavelength of
750 nm, so that colour influences would not affect the optical
density values. The supernatant was then autoclaved at 121 °C
for 20 min and, after cooling, stored in hermetically sealed
containers at 4 °C for no longer than 1 month. Dilutions of
this material to final concentrations of 1, 5 and 10% (v/v) were
made to test their suitability in media formulations. Algal consortium and cultivation Other algal taxa present in
the consortium included Scenedesmus sp., Chlorella sp.,
Chlamydomonas sp. and Desmodesmus sp. (Table 1). Bioremediation assessment Scale-up cultivation experiments were performed using two
600-L capacity horizontal tubular photobioreactors (BioFence,
from Varicon Aqua Solution manufacturing) located in a heat-
ed greenhouse, Swansea, UK. The duration of experiment was
a 12-day consecutive period. The abiotic conditions were tem-
perature 18–25 °C and 2% CO2 injection, which was regulated
by pH measurements (i.e. CO2 addition when the medium was
above pH 7.5). The natural (greenhouse) light conditions were
variable over the experimental period ranging from 300 to
1000 μmol photons m−2 s−1, with a light/dark cycle of
16:8 h. During the cultivation period, waste-based medium
[10% (v/v), 100 mL of prepared waste solution for 1 L of
culture] was tested against a control f/2 medium. The control medium employed was a freshwater algal medium
based on the nutrient levels employed in f/2 medium (Guillard
1975). The waste-base nutrient medium was prepared using
Dwr Cymru Welsh Water’s (DCWW) anaerobic digested
(AD) effluent sampled directly after the digester gravity belt
filters. Fresh samples of DCWW’s AD waste effluent were stored
in a fridge at 4 °C before being treated. The samples had high
levels of turbidity and were passed through a 100-μm bag
filter and then filtered through a 0.2-μm hollow fibre cartridge
(GE Healthcare, USA) to improve the effluent’s clarity. To 1334 J Appl Phycol (2017) 29:1331–1341 Cryopreservation of the algal consortium Cryopreservation was performed according to Day and
DeVille (1995). Dimethyl sulphoxide (DMSO) (Sigma-
Aldrich Ltd., UK) was filter sterilised in a sterile f/2 freshwater
algal medium to a final concentration of 10% (v/v) using a
0.20-μm sterile syringe filter (Iwaki, Japan). An aliquot
(10 mL) of the 10% (v/v) DMSO solution was aseptically
added to 10 mL of an early stationary phase culture of the
algal consortium CCAP 293/1 in a sterilised Universal bottle
(25 mL). The Universal was inverted several times to ensure
complete mixing and the algal culture in 5% (v/v). DMSO was
dispensed in 1.0 mL aliquots into cryovials (Greiner Bio-One
GmbH, Germany). These were then incubated at room tem-
perature (∼20 °C) for 20 min prior to cryopreservation to Cell growth Every 24 h, cell concentration, cell size and biovolume mea-
surements were performed by Coulter Counter Multisizer 4,
Beckman, USA, to quantify culture growth as described by
(Mayers et al. 2013). After 24 h, and again after 3 months, storage triplicate
samples were transferred in LN2 from the cryostorage facility
to the lab. They were then thawed by direct immersion in a
preheated water bath at 40 °C and were removed as soon as all
visible ice had melted. Immediately after thawing, the samples
were aseptically inoculated into tissue culture flasks contain-
ing 20 mL of the sterile f/2 freshwater medium. The flasks
were wrapped in aluminium foil to prevent the possibility of
light-induced stresses/metabolic uncoupling and incubated at
20 °C in the dark for 36 h before removal of the aluminium. For culture regrowth, samples were incubated under a 12:12 h
light/dark regime, irradiance ∼30 μmol photons m−2 s−1 PAR
for 3 weeks and periodically visually assessed and examined
by phase contrast microscopy to confirm regrowth. The cul-
tures (after cryopreservation) derived from samples which had
been stored for 3 months were then dispatched by post to
Centre for Sustainable Aquatic Research (CSAR), Swansea,
UK, to undertake post-preservation functional/bioremediation
stability assessment. The growth rate was calculated based on biovolume using
the formula provided by Levasseur et al. (1993): Growth rate : K0 ¼ Ln N 2=N 1
ð
Þ= t2−t1
ð
Þ where N1 and N2 = biovolume (measured by Coulter counter)
at time 1 (t1) and time 2 (t2), respectively. Sample analyses enable the cryoprotectant to enter the cells. The cryovials were
then transferred to a controlled rate cooler (Kryo 360 3.3,
Planer plc, UK). The samples were cooled at −1 °C min−1
between 20 and −40 °C, with auto-ice nucleation at −5 °C,
and then held for a further 15 min at −40 °C. The cryovials
were then rapidly removed from the cooler unit, plunged into
liquid nitrogen (LN2) and transferred to the CCAP cryobank
for storage in liquid phase liquid nitrogen (−196 °C). Statistical analysis Statistical analysis of data was conducted using R software,
using two- or three-factor analysis of variance allowing us to
assess the differences between each treatment (i.e. medium;
strain) and the data evolution in time. The analysis was con-
ducted on the following variables: cell density, cell size and
biovolume. When a significant difference was found, a post
hoc Tukey test was used at a confidence level of 95%. The
statistical interaction between factors was also assessed by the
Spearman correlation test. Remediation N and P uptake The nutrient levels in the algal media were analysed every
other day using an automated segmented flow analyser
(AA3, Bran Luebbe, Germany). The automated procedure
for the determination of nitrate (NO3) and nitrite (NO2) uses
the process whereby nitrate is reduced to nitrite by a copper-
cadmium reductor column (Armstrong et al. 1967). The nitrite
then reacts with sulfanilamide under acidic conditions to form
a diazo compound. This compound then couples with N-1-
naphthylethylene diamine dihydrochloride to form a purple
azo dye. Nitrate/nitrite analysis was performed, which along
with ammonia, measured by the Berthelot reaction, in which a
blue-green coloured complex is formed that is measured at
660 nm, gave total nitrogen (TN). The automated procedure
for the determination of ortho-phosphate is based on the col-
orimetric method in which a blue colour is formed by the
reaction of ortho-phosphate, molybdate ion and antimony
ion followed by reduction with ascorbic acid at a pH < 1. The reduced blue phospho-molybdenum complex is read at
880 nm. Bioremediation using the algal consortium in a 2-
and 600-L photobioreactor c Bioremediation—re-
moval of phosphorous (error bars
indicate the standard deviation,
the number of independent
replicates = 3)
J Appl Phycol (2017) 29:1331–1341
1335 1
10
1
2
3
4
5
6
7
8
Cell concentration (x106 cell mL-1)
Time (day)
0
1000
2000
3000
4000
5000
6000
7000
0
1
2
3
4
5
6
7
8
Concentration (Total N µmol L-1)
Time (day)
0
5
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15
20
25
30
35
40
0
1
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Concentration (Total P µmol L-1)
Time (day)
(c)
(b)
(a)
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8
Cell concentration (x106 cell mL-1)
Time (day)
0
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Concentration (Total N µmol L-1)
Time (day)
0
5
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15
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25
30
35
40
0
1
2
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4
5
6
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Concentration (Total P µmol L-1)
Time (day)
(c)
(b)
(a)
1335 1
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1
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3
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7
8
Cell concentration (x106 cell mL-1)
Time (day)
0
1000
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3000
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7000
0
1
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Concentration (Total N µmol L-1)
Ti
(d
)
(b)
(a) 1
10
1
2
3
4
5
6
7
8
Cell concentration (x106 cell mL-1)
(a) (a) 1
2
3
4
5
6
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8
Time (day)
0
1000
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3000
4000
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6000
7000
0
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Concentration (Total N µmol L-1)
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0
5
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40
0
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Concentration (Total P µmol L-1)
Time (day)
(c)
(b) (b) 0
1000
0
1
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8
Conce
Time (day)
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8
Concentration (Total P µmol L-1)
Time (day)
(c) 0.35 day−1 in freshwater f/2; however, the duration of the
exponential phase was 3 days in the cultures grown on waste
nutrients and 5 days in f/2. The growth observed in media
supplemented with 1 and 5% of waste effluent was lower,
and statistical analysis indicated that the medium formulation
used had an impact of the growth estimated by cell density
(p < 0.05) (Fig. 1a) (Table 2). Fig. 1 Growth and nutrient
uptake of the ACCOMPLISH
algal consortium in 250-mL flask
under f/2 (black circle), 1% (white
circle), 5% (black square) and
10% (white square) of effluent
nutrients. a Cell density. b
Bioremediation—removal of ni-
trogen. c Bioremediation—re-
moval of phosphorous (error bars
indicate the standard deviation,
the number of independent
replicates = 3) Bioremediation using the algal consortium in a 2-
and 600-L photobioreactor The algal consortium was successfully grown on media sup-
plemented with Bwaste nutrients^ at 1, 5 and 10% (v/v) and
control f/2 (Fig. 1a). Adaptation to the new nutrient regime
was observed over the first 2 days under all waste dilutions
tested and the best growth observed was in a medium supple-
mented with 10% waste effluent. The exponential growth rate
in the 10% growth medium was 0.4 day−1, in comparison to J Appl Phycol (2017) 29:1331–1341 1335 1
10
1
2
3
4
5
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7
8
Cell concentration (x106 cell mL-1)
Time (day)
0
1000
2000
3000
4000
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6000
7000
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Concentration (Total N µmol L-1)
Time (day)
0
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40
0
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3
4
5
6
7
8
Concentration (Total P µmol L-1)
Time (day)
(c)
(b)
(a)
Fig. 1 Growth and nutrient
uptake of the ACCOMPLISH
algal consortium in 250-mL flask
under f/2 (black circle), 1% (white
circle), 5% (black square) and
10% (white square) of effluent
nutrients. a Cell density. b
Bioremediation—removal of ni-
trogen. Bioremediation using the algal consortium in a 2-
and 600-L photobioreactor The interaction between both
factors was significant for both the cell density and the
biovolume (p < 0.05), demonstrating that there was a signifi-
cant difference in growth between both media; however, that
difference was not the same over the time course of the exper-
iment. Indeed, the cell density results indicated that there were
no apparent differences between the two media at the begin-
ning of the experiment, but there were differences after a few
days of incubation. Furthermore, the post hoc Tukey test in-
dicated that employing the control medium resulted in higher cell density and biovolume. Finally, the correlation test
showed that the three variables (cell density, cell size and
biovolume) were correlated, demonstrating that under all con-
ditions tested that when a high cell density was observed, cells
were larger and total biovolume higher (Table 2). The nutrient uptake by the algal consortium is shown in
Fig. 1b, c. Nitrogen was gradually removed from the media by
the algal cultures under all the experimental conditions tested. Phosphate uptake was more rapid and by day 4, all the phos-
phorus in the media had been assimilated by the algal cultures
under all nutrient conditions tested. The waste medium used
had an impact on the nitrogen uptake rate (p < 0.05) and the
results of post hoc tests (Fig. 1). The interaction between the
concentration of both factors was significant for the cell den-
sity and the biovolume (p < 0.05) indicating that there was a
significant difference in performance between media; J Appl Phycol (2017) 29:1331–1341 1336 Table 2 Biovolume, cell
concentration and cell size of
algal consortium growing on
different nutrient conditions and
scale (± indicates the standard
deviation, the number of
independent replicates = 3)
Treatment
Biovolume
(× 108 μm3 mL−1)
Cell concentration
(× 106 cell mL−1)
Cell size (μm)
1% effluent (250 mL)
2.3 ± 0.3
2.7 ± 0.3
5.2
5% effluent (250 mL)
5.2 ± 0.5
3.6 ± 0.3
5.5
10% effluent (250 mL)
2.8 ± 0.1
7.7 ± 0.1
5.5
Control (f/2) (250 mL)
1.8 ± 0.2
5.5 ± 0.4
5.4
10% effluent (600 L)
4.8 ± 0.5
3.7 ± 0.1
7.7
Control (f/2) (600 L)
5.8 ± 0.5
6.5 ± 0.3
7.1 The algal consortium grown on waste medium had slow
phosphorus uptake during the first 3 days; after that, the
phosphorus uptake accelerated. Bioremediation using the algal consortium in a 2-
and 600-L photobioreactor By day 6, all the phosphorus
had been removed by the algal consortium showing rapid
waste remediation, comparable to the control (freshwater
f/2) treatment. however, that difference was not related to the time of sam-
pling. The cell density results indicated that there were no
apparent differences between culture nutrient uptake in the
control and experimental culture media at the beginning of
the experiment, but differences were observed after a few days
of incubation, due to the adaptation to the media. The algal consortium was successfully grown in PBR
remediating a waste-based medium (Fig. 2a), (Table 2). The
lag phase, adaptation to the reactor and nutrient conditions,
was observed for the first 2 days for both control and waste
nutrient conditions; the growth rate in this period was
0.1 day−1. The subsequent exponential phase lasted 4 days
with a growth rate of 0.35 day−1 for the culture grown on
AD waste nutrients. This was significantly different
(p > 0.005) from the control (f/2) medium where the culture
grew at a slower rate and entered stationary phase after 5–
6 days of cultivation. The highest final cell density (3 × 107
cells mL−1) was observed in the waste water-grown culture
(Fig. 2a) (Table 2). Statistical analysis demonstrated that both
the time of experiment and the medium used influence the
three studied variables: cell density, cell size and biovolume
(p < 0.05). Furthermore, the interaction between both factors
was significant for the cell density and the biovolume
(p < 0.05) indicating that there was a significant difference in
culture growth between both media (Table 2); however, that
difference was not dependent on the time of sampling. Indeed,
the cell density data indicated that there were no apparent dif-
ferences between adaptations in the two different media for-
mulations at the beginning of the experiment, but there were
differences after a few days of incubation. Furthermore, the
post hoc Tukey test demonstrated that using the waste medium
resulted in higher cell density and biovolume. Finally, the cor-
relation test indicated that the three variables were correlated,
demonstrating that at higher cell density, there were also bigger
cells and thus greater total biovolume (Table 2). Validation of bioremediation capacity
of the cryopreserved algal consortium On thawing of samples after 24 h and 3 weeks storage under
liquid nitrogen, relatively rapid recovery and regrowth of the
consortium were observed in all replicate cultures. Within 2–
3 weeks, the cultures were comparable in density and appear-
ance to the culture prior to cryopreservation. No overgrowth
of the algal culture by the commensal bacteria was observed,
and on microscopy, all replicates appeared to be effectively
identical with respect to algal morphotypes present and were
indistinguishable by light microscopy from the non-
cryopreserved control samples that had been maintained in
parallel under the standard cultivation regime. On testing the growth of the two cultures, the non-
cryopreserved algal consortium and the algal consortium after
cryopreservation, over 13 days, good growth levels were ob-
served (Fig. 3). No significant difference (p < 0.05) was ob-
served between growth rates of the algal consortium before
and after cryopreservation, which were 0.6 and 0.7 day−1,
respectively (Fig. 3a). The nutrient remediation functionality
(N and P uptake rate) was very similar in comparison with the
previous experiment, and by day 4, 99% of the total nitrogen
and total phosphorus were remediated by both of the cultures
studied (Fig. 3b, c). Table 2 Biovolume, cell
concentration and cell size of
algal consortium growing on
different nutrient conditions and
scale (± indicates the standard
deviation, the number of
independent replicates = 3) Discussion c Bioremediation—re-
moval of phosphorous (error bars
indicate the standard deviation,
the number of independent
replicates = 3)
J Appl Phycol (2017) 29:1331 1341
1337 1
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Time (day)
(a)
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Concentration (Total N µmol L-1)
Time (day)
(a)
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Concentration (Total P µmol L-1)
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(c) 1
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(a) 1
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Cell concentration (106 cell mL-1)
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9
10
11
Concentration (Total N µmol L-1)
Time (day)
(a)
(b) Fig. 2 Growth and nutrient
uptake of the ACCOMPLISH
algal consortium in a 600-L PBR
under (black diamond) f/2 and
(white diamond) 10% of effluent
nutrients. a Cell density. b
Bioremediation—removal of ni-
trogen. c Bioremediation—re-
moval of phosphorous (error bars
indicate the standard deviation,
the number of independent
replicates = 3) (a) 0
1
2
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0
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Concentration (Total N µmol L-1)
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(b) Time (day) Concentration (Total N µmol L-1) (b) (
y)
0
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Concentration (Total P µmol L-1)
Time (day)
(c) consortium, where the dominant groups of algal species belong
to either the Chlorophyceae or Trebouxiophyceae. Discussion The nitrogen uptake observed was not significantly dif-
ferent (p < 0.05) between waste and control (f/2) conditions
(Fig. 2). By day 6, total nitrogen was remediated, i.e. re-
moved from media by the algal consortium (significantly
different (p < 0.05) than f/2 medium grown consortium). The phosphorus uptake for control and waste-based media
growth conditions were significantly different (p < 0.05). Conventional wastewater treatment technologies (e.g. anaero-
bic digestion) still have technical-economic limitations, mainly
caused by their high energy requirement and relatively poor
nutrient removal (de Godos et al. 2010). The development of
algal technology for bioremediation applications has the poten-
tial to overcome these constraints as they can provide a system 1337 J Appl Phycol (2017) 29:1331–1341 1
10
100
0
1
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3
4
5
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9
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11
Cell concentration (106 cell mL-1)
Time (day)
0
1500
3000
4500
6000
0
1
2
3
4
5
6
7
8
9
10
11
Concentration (Total N µmol L-1)
Time (day)
(a)
(b)
0
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80
100
120
140
160
180
0
1
2
3
4
5
6
7
8
9
10
11
Concentration (Total P µmol L-1)
Time (day)
(c)
Fig. 2 Growth and nutrient
uptake of the ACCOMPLISH
algal consortium in a 600-L PBR
under (black diamond) f/2 and
(white diamond) 10% of effluent
nutrients. a Cell density. b
Bioremediation—removal of ni-
trogen. Discussion Taxa belong-
ing to these classes, particularly Chlorella and Scenedesmus,
have been demonstrated to have high bioremediation potential
(Chinnasamy et al. 2010a; 2012 Su et al.; Shene et al. 2016). However, F. amphitricha has not previously been reported in
bioremediation studies (Tsarenko and John 2011). capable of high levels of nutrient removal, with the capacity to
remediate a wide spectrum of waste nutrients by an environ-
mentally friendly alternative to conventional methods (Boelee
et al. 2011; Posadas et al. 2013; Polishchuk et al. 2015). The
advantages of the use of mixed microalgal consortium have
been the focus of recent research (Kerckhof et al. 2014;
Mahapatra et al. 2014; Lahel et al. 2016). Furthermore, similar
to the results of this study, high levels (above 90%) of remedi-
ation of inorganic nutrients have been reported in studies using
both mixed natural or artificially formulated algal consortia
(Fergola et al. 2007; Chinnasamy et al. 2010b; Renuka et al. 2013) . In this study, we have used a natural algal mixed The use of municipal waste nutrient source was one of
the objectives of this study and the remediation of
4500 μmol L−1 (64 mg L−1) of N and 690 μmol L−1
(21 mg L−1) of P by a reduction of 99% was achieved using
the algal consortium. Other research studies (Woertz et al. 1338 J Appl Phycol (2017) 29:1331–1341 1
10
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1000
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Cell concentration(106 cell mL-1)
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Concentration (Total N µmol L-1)
Time (day)
(b)
(a)
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80
ation (Total P µmol L-1)
(c) 1
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Cell concentration(106 cell mL-1)
Time (day)
(a) (a) (a) Cell concentration(106 cell mL-1) asamy et al. 2010b; Silva-Benavides and
2; Su et al. 2012) have reported comparable
remediation capacities of 41–100% f
for phosphorus. Discussion However, there
are few reports on the retention of functionality of cryopre-
served microbial consortia for environmental remediation in-
cluding Augustynowicz et al. (2008) who demonstrated the
capacity of a revived cryopreserved mixed bacterial commu-
nity to degrade petroleum-derived environmental
contaminants and Kerckhof et al. (2014) who successfully
optimised the conservation of a methanotrophic co-culture
(MOB), with potential for mitigation of greenhouse gas emis-
sions, environmental pollutant removal and bioplastics pro-
duction, as well as an oxygen-limited autotrophic
nitrification/denitrification (OLAND) biofilm, with enhanced
economic and ecological benefits for wastewater treatment. These consortia both retained good levels of functionality,
although the preservation of the community structure (as de-
termined by 16S rRNA gene sequencing) was incomplete. In
our study community structure, beyond the presence of the
key algal taxa, this was not determined, as functionality was
the primary requirement. this is achievable at pilot-scale, rather than lab-scale (0.25–
5 L) volume. The cultivation of the algal consortium with
waste nutrient removal in a 600-L PBR in greenhouse
reflected real environmental conditions (variable light,
pH, nutrients availability, temperature) similar in any po-
tential industrial site, rather than in laboratory. All these
parameters influence the consortium behaviour and biore-
mediation capacity; however, although the remediation
process in PBR was slightly longer than in the lab-scale
experiment, as has previously been reported, the culture
was more stable (Bordel et al. 2009). Overall, the scalabil-
ity, nutrient removal capability and cell density of the algae
produced demonstrated the potential of an integrated sys-
tem capable of both bioremediation and the production of
algal biomass. In the biotechnological application of algae in phyco-reme-
diation, as in any other industrial application, stability of the
alga(e) and/or the mixed consortium is a pre-requisite for en-
suring sustainable results/production. In case of successful
waste remediation by algae, the main challenges are to main-
tain the long-term effectiveness and long-term homeostasis of
the mixed consortium. (Gonçalves et al. 2016). The use of
cryopreserved master stock cultures provides insurance for
the sustainability of production. Furthermore, it provides prac-
titioners with an option to ensure consistency in batch-based
processes, where inoculum build-up may be initiated for each
batch from the master stock culture. Discussion However, this study h
1
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Cell concentration(106 cell mL-1)
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500
600
700
0
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8
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Concentration (Total N µmol L-1)
Time (day)
(b)
(a)
0
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80
0
1
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Time (day)
(c) Time (day) 0
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0
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Concentration (Total N µmol L-1)
Time (day)
(b) Concentration (Total N µmol L-1) (b) (
y)
0
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0
1
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Concentration (Total P µmol L-1)
Time (day)
(c) (c) 2009; Chinnasamy et al. 2010b; Silva-Benavides and
Torzillo 2012; Su et al. 2012) have reported comparable 2009; Chinnasamy et al. 2010b; Silva-Benavides and
Torzillo 2012; Su et al. 2012) have reported comparable remediation capacities of 41–100% for N and 12–100%
for phosphorus. However, this study has demonstrated that 1339 J Appl Phycol (2017) 29:1331–1341 Fig. 3 Growth and nutrient uptake for the ACCOMPLISH algal
consortium before and after cryopreservation in flask (250 mL) system;
black diamond—ACCOMPLISH mixed consortium before
cryopreservation, white diamond—ACCOMPLISH mixed consortium
after cryopreservation. a Cell density. b Bioremediation—removal of
nitrogen (flask system). c Bioremediation—removal of phosphorous
(error bars indicate the standard deviation, the number of independent
replicates = 3)
R capacity post-thaw of Porphyridium cruentum to produce ze-
axanthin and beta-carotene and for the cyanobacterium
Planktothrix to produce a cytotoxin. In addition, Nakanishi
et al. (2012) have demonstrated that the chlorophyll content
of Nannochloropsis oculata ST-4 and Tetraselmis tetrathele
T-501 was not significantly changed after 15 years of
cryostorage. More recently, Hipkin et al. (2014) reported the
successful cryopreservation of the transgenic diatom
Thalassiosira pseudonana CCAP 1085/23, which
overexpressed a GFP-tagged nuclear localised protein, pre-
and post-cryopreservation as a proxy for a biotechnological
product. To date, there have been no previous reports on the
cryopreservation of a mixed algal consortium. Discussion Cryopreservation is also
an important tool for the Biological Resource Centre (BRC)
community to hold patented strains, minimising risks associ-
ated with alternative procedures such as serial transfer and
facilitating the exploitation of non-traditional biological re-
sources (Stacey and Day 2014). Under the patent procedure,
the strain deposited should retain the trait/capacity of commer-
cial relevance. As part of the procedure, on the request of the
depositor/owner of the strain, the cryopreserved samples may
be revived, dispatched and re-tested for their biotechnological
capabilities. There are numerous reports on the conservation
of algae by cryopreservation for biotechnological or aquacul-
ture use, highlighting the importance of minimising risks as-
sociated with losing, contaminating or the potential loss of
traits by genetic drift (Cañavate and Lubińn 1995; Day et al. 2005; Rhodes et al. 2006). However, despite the importance of
having evidence based on the retention of biotechnological
potential, there are few commercially relevant reports in the
literature. Hédoin et al. (2006) demonstrated the retention in In this study, we have confirmed the integrity of mixed
consortium after the cryopreservation and demonstrated its
capacity to bioremediate effluent effectively. This has signifi-
cant implications to the algal biotechnology sector as a whole,
where in reality, mixed/non-axenic cultivation will be a pre-
requisite for future economic success. In addition, this paper
provides a successful model of a procedure that has allowed
the protection of intellectual property (IP) associated with a
mixed consortium. This is in the vanguard of the BRC com-
munities’ attempts to service the challenges associated with
rapid developments in algal/protistan biotechnology. In conclusion, microalgae, with their photoautotrophic ca-
pabilities, are able to uptake the waste nutrients using solar
energy and carbon dioxide and can thus convert these nutri-
ents to valuable biomass. This capability will, in the opinion
of the authors, be a major component in a more integrated
bioeconomy that will help to manage pollution worldwide,
resulting from expansion of the global population and indus-
trial activities. Underpinning these capabilities by guarantee-
ing the functional stability of conserved consortia is a key
component in ensuring sustainability and long-term biotech-
nological exploitability. 1340 J Appl Phycol (2017) 29:1331–1341 Acknowledgements
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use, distribution, and reproduction in any medium, provided you give
appropriate credit to the original author(s) and the source, provide a link
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A
p = 9.6e-1
0.00
0.25
0.50
0.75
1.00
0
10
20
30
40
50
Time
Overall Survival
CBTN (H3K27-altered DMG)
PNOC003 (H3K27-altered DIPG)
22
11
4
1
1
1
28
19
4
0
0
0
PNOC003
CBTN
0
10
20
30
40
50
Time
Number at risk
PNOC003+CBTN A
p = 9.6e-1
0.00
0.25
0.50
0.75
1.00
0
10
20
30
40
50
Time
Overall Survival
CBTN (H3K27-altered DMG)
PNOC003 (H3K27-altered DIPG)
22
11
4
1
1
1
28
19
4
0
0
0
PNOC003
CBTN
0
10
20
30
40
50
Time
Number at risk
PNOC003+CBTN TP53
mut
wt
(N=35)
(N=15)
2.3
reference
(1 08 - 5 1)
0.032 *
E
p = 5.2e-4
0.00
0.25
0.50
0.75
1.00
0
10
20
30
40
50
Time
Overall Survival
TP53mut;10del
TP53mut;10wt
TP53wt;10wt
14
4
1
0
0
0
20
14
1
0
0
0
14
11
6
1
1
1
TP53mut;10wt
TP53mut;10wt
TP53mut;10del
0
10
20
30
40
50
Time
Number at risk
PNOC003+CBTN
p = 2.4e-2
0.00
0.25
0.50
0.75
1.00
0
10
20
30
40
50
Time
Overall Survival
15
6
1
0
0
0
7
5
3
1
1
1
TP53wt
TP53mut
0
10
20
30
40
50
Time
Number at risk
TP53mut
TP53wt
CBTN
p = 9.6e-1
0.00
0.25
0.50
0.75
1.00
0
10
20
30
40
50
Time
Overall Survival
CBTN (H3K27-altered DMG)
PNOC003 (H3K27-altered DIPG)
22
11
4
1
1
1
28
19
4
0
0
0
NOC003
CBTN
0
10
20
30
40
50
Time
Number at risk
PNOC003+CBTN
4
5
D
p = 1.3e-1
0.00
0.25
0.50
0.75
1.00
0
10
20
30
40
50
Time
Overall Survival
10del
10wt
8
3
1
0
0
0
13
8
3
1
1
1
10wt
10del
0
10
20
30
40
50
Time
Number at risk
CBTN
F
Supplemental fgure 1. 10del
Supplemental fgure 1. D
p = 1.3e-1
0.00
0.25
0.50
0.75
1.00
0
10
20
30
40
50
Time
Overall Survival
10del
10wt
8
3
1
0
0
0
13
8
3
1
1
1
10wt
10del
0
10
20
30
40
50
Time
Number at risk
CBTN
Supplemental fgure 1. A
p = 9.6e-1
0.00
0.25
0.50
0.75
1.00
0
10
20
30
40
50
Time
Overall Survival
CBTN (H3K27-altered DMG)
PNOC003 (H3K27-altered DIPG)
22
11
4
1
1
1
28
19
4
0
0
0
PNOC003
CBTN
0
10
20
30
40
50
Time
Number at risk
PNOC003+CBTN D A COHORT
CHR10
TP53
PNOC003
CBTN
del
wt
mut
wt
(N=28)
(N=22)
(N=15)
(N=34)
(N=35)
(N=15)
1.2
reference
2.3
reference
2.3
reference
(0.64 - 2.2)
(1.13 - 4.8)
(1.08 - 5.1)
0.57
0.022 *
0.032 *
# Events: 49; Global p-value (Log-Rank): 0.0022
AIC: 280.5; Concordance Index: 0.65
Hazards ratio
E
p = 5.2e-4
0.00
0.25
0.50
0.75
1.00
0
10
20
30
40
50
Time
Overall Survival
TP53mut;10del
TP53mut;10wt
TP53wt;10wt
14
4
1
0
0
0
20
14
1
0
0
0
14
11
6
1
1
1
TP53mut;10wt
TP53mut;10wt
TP53mut;10del
0
10
20
30
40
50
Time
Number at risk
PNOC003+CBTN
-5
p = 1.3e-1
0.00
0.25
0.50
0
10
20
30
40
50
Time
Overall Survival
8
3
1
0
0
0
13
8
3
1
1
1
10wt
10del
0
10
20
30
40
50
Time
Number at risk
F COHORT
CHR10
TP53
PNOC003
CBTN
del
wt
mut
wt
(N=28)
(N=22)
(N=15)
(N=34)
(N=35)
(N=15)
1.2
reference
2.3
reference
2.3
reference
(0.64 - 2.2)
(1.13 - 4.8)
(1.08 - 5.1)
0.57
0.022 *
0.032 *
# Events: 49; Global p-value (Log-Rank): 0.0022
AIC: 280.5; Concordance Index: 0.65
Hazards ratio
B
C
E
p = 5.2e-4
0.00
0.25
0.50
0.75
1.00
0
10
20
30
40
50
Time
Overall Survival
TP53mut;10del
TP53mut;10wt
TP53wt;10wt
14
4
1
0
0
0
20
14
1
0
0
0
14
11
6
1
1
1
TP53mut;10wt
TP53mut;10wt
TP53mut;10del
0
10
20
30
40
50
Time
Number at risk
PNOC003+CBTN
p = 2.4e-2
0.00
0.25
0.50
0.75
1.00
0
10
20
30
40
50
Time
Overall Survival
15
6
1
0
0
0
7
5
3
1
1
1
TP53wt
TP53mut
0
10
20
30
40
50
Time
Number at risk
TP53mut
TP53wt
CBTN
p = 9.6e-1
0.00
0.25
0.50
0
10
20
30
40
50
Time
Overall Surv
22
11
4
1
1
1
28
19
4
0
0
0
PNOC003
CBTN
0
10
20
30
40
50
Time
Number at risk
-log10 P
Overall Survival
0
1
2
3
4
5
-10
-16
-12
-17
-13
-11
+1
-18
-14
-5
-19
p = 1.3e-1
0.00
0.25
0.50
0
10
20
30
40
50
Time
Overall Survi
8
3
1
0
0
0
13
8
3
1
1
1
10wt
10del
0
10
20
30
40
50
Time
Number at risk
F B
p = 2.4e-2
0.00
0.25
0.50
0.75
1.00
0
10
20
30
40
50
Time
Overall Survival
15
6
1
0
0
0
7
5
3
1
1
1
TP53wt
TP53mut
0
10
20
30
40
50
Time
Number at risk
TP53mut
TP53wt
CBTN E
p = 5.2e-4
0.00
0.25
0.50
0.75
1.00
0
10
20
30
40
50
Time
Overall Survival
TP53mut;10del
TP53mut;10wt
TP53wt;10wt
14
4
1
0
0
0
20
14
1
0
0
0
14
11
6
1
1
1
TP53mut;10wt
TP53mut;10wt
TP53mut;10del
0
10
20
30
40
50
Number at risk
PNOC003+CBTN B E C
-log10 P
Overall Survival
0
1
2
3
4
5
-10
-16
-12
-17
-13
-11
+1
-18
-14
-5
-19 C COHORT
CHR10
TP53
PNOC003
CBTN
del
wt
mut
wt
(N=28)
(N=22)
(N=15)
(N=34)
(N=35)
(N=15)
1.2
reference
2.3
reference
2.3
reference
(0.64 - 2.2)
(1.13 - 4.8)
(1.08 - 5.1)
0.57
0.022 *
0.032 *
# Events: 49; Global p-value (Log-Rank): 0.0022
AIC: 280.5; Concordance Index: 0.65
Time
F F # Events: 49; Global p-value (Log-Rank): 0.0022
AIC: 280.5; Concordance Index: 0.65 Hazards ratio
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Inactivated ostreid herpesvirus-1 induces an innate immune response in the Pacific oyster, Crassostrea gigas, hemocytes
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Brief Research Report
28 April 2023
DOI 10.3389/fimmu.2023.1161145
TYPE
PUBLISHED
OPEN ACCESS
EDITED BY
Daniela Melillo,
National Research Council (CNR), Italy
REVIEWED BY
Alejandro Romero,
Spanish National Research Council (CSIC),
Spain
Junfa Yuan,
Huazhong Agricultural University, China
*CORRESPONDENCE
Lizenn Delisle
lizenn.delisle@Cawthron.org.nz
Anne Rolton
anne.vignier@cawthron.org.nz
07 February 2023
ACCEPTED 18 April 2023
PUBLISHED 28 April 2023
RECEIVED
CITATION
Delisle L, Rolton A and Vignier J (2023)
Inactivated ostreid herpesvirus-1 induces
an innate immune response in the Pacific
oyster, Crassostrea gigas, hemocytes.
Front. Immunol. 14:1161145.
doi: 10.3389/fimmu.2023.1161145
COPYRIGHT
© 2023 Delisle, Rolton and Vignier. 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.
Inactivated ostreid herpesvirus-1
induces an innate immune
response in the Pacific oyster,
Crassostrea gigas, hemocytes
Lizenn Delisle 1*, Anne Rolton 1* and Julien Vignier 2
1
Biosecurity Group, Cawthron Institute, Nelson, New Zealand, 2 Aquaculture Group, Cawthron
Institute, Nelson, New Zealand
Infectious diseases are a major constraint to the expansion of shellfish
production worldwide. Pacific oyster mortality syndrome (POMS), a
polymicrobial disease triggered by the Ostreid herpesvirus-1 (OsHV-1), has
devastated the global Pacific oyster (Crassostrea gigas) aquaculture industry.
Recent ground-breaking research revealed that C. gigas possess an immune
memory, capable of adaption, which improves the immune response upon a
second exposure to a pathogen. This paradigm shift opens the door for
developing ‘vaccines’ to improve shellfish survival during disease outbreaks. In
the present study, we developed an in-vitro assay using hemocytes – the main
effectors of the C. gigas immune system – collected from juvenile oysters
susceptible to OsHV-1. The potency of multiple antigen preparations (e.g.,
chemically and physically inactivated OsHV-1, viral DNA, and protein extracts)
to stimulate an immune response in hemocytes was evaluated using flow
cytometry and droplet digital PCR to measure immune-related subcellular
functions and gene expression, respectively. The immune response to the
different antigens was benchmarked against that of hemocytes treated with
Poly (I:C). We identified 10 antigen preparations capable of inducing immune
stimulation in hemocytes (ROS production and positively expressed immunerelated genes) after 1 h of exposure, without causing cytotoxicity. These findings
are significant, as they evidence the potential for priming the innate immunity of
oysters using viral antigens, which may enable cost-effective therapeutic
treatment to mitigate OsHV-1/POMS. Further testing of these antigen
preparations using an in-vivo infection model is essential to validate promising
candidate pseudo-vaccines.
KEYWORDS
immune priming, Ostreid herpesvirus-1, pseudo-vaccination, innate immune memory,
flow cytometry, reactive oxygen species, droplet digital PCR
Frontiers in Immunology
01
frontiersin.org
Delisle et al.
10.3389/fimmu.2023.1161145
OsHV-1 infection in natura. More recently, Fallet et al. (36) showed
that early life exposure of C. gigas to ‘microorganisms’ provided
inter-generational protection against recurring OsHV-1 infections,
indicating a potential trained immunity via epigenetic
modifications. In bivalves, hemocytes play a central role in
immunity, identifying and destroying pathogens through
phagocytosis, oxidative stress, apoptosis, and autophagy –
functions that can be characterized using flow cytometry and
molecular analyses (37–43). Transcriptome analyses in oysters
primed with Poly (I:C) have identified several pattern recognition
receptors (PRRs) involved in antiviral signaling. These include, for
example, retinoic acid-inducible-gene-I and Toll-like-receptors
homologs of the Jak-Stat pathway, stimulator of interferon genes,
interferon regulatory factors, and many IFN-stimulated genes (i.e.,
Viperin or ADAR), which are all implicated in the detection of virus
and antiviral functions (44–48).
We aim to evaluate the potency of inactivated OsHV-1
preparations (antigens) to elicit an antiviral response in C. gigas
hemocytes. This research is undertaken in the context of developing
new strategies of immune priming to improve oyster resilience
to POMS.
1 Introduction
The occurrence of mass mortality events and the emergence of
infectious diseases affecting marine organisms have increased
dramatically in recent years, exacerbated by a changing
environment (1–3). These outbreaks can have disastrous
consequences on biodiversity and cause rapid population declines,
particularly in cultured livestock (4, 5). Diseases are the major
limiting factor for the expansion of the aquaculture industry, with
losses attributed to infectious microbial diseases alone exceeding US
$ 6 billion per annum (6). One striking example is Pacific oyster
mortality syndrome (POMS), which is associated with the detection
of the Ostreid Herpes virus-1 (OsHV-1) and its variants. Over the
last 15 years, OsHV-1 has decimated Pacific oysters, Crassostrea
gigas, worldwide (7; see for review 8–10). The virus induces an
immune-compromised state in infected oysters, which evolves
toward subsequent bacteremia by opportunistic bacterial
pathogens, leading to mortality rates of up to 100% in juveniles
(11). Recorded for the first time in France in 2008, OsHV-1 µvar
rapidly spread along the European coastline (9, 10, 12), and
closely related variants of the virus were further detected during
mortality events in Australia (13), New Zealand (14), Korea (15),
and more recently in California (16). The inability to contain the
rapid spread of the virus combined with an absence of therapeutic
treatments resulted in huge losses of aquaculture stocks. Selective
breeding to improve resistance to POMS (or OsHV-1) has shown
potential as a prevention strategy, with moderate to high heritability
for survival achieved during OsHV-1 infection (17–20).
Implementation of a breeding program and access to selectively
bred stocks can, however, be economically challenging for many
end-users, prompting the need for new, accessible, and
complementary mitigation strategies to reduce the impact
of diseases.
For example, vaccination and immune priming have proven to
be an effective preventative measure for many major diseases
affecting livestock, including fish, and more recently invertebrates,
such as crustaceans (21–25).
Invertebrates lack a conventional adaptive immune system (i.e.,
lymphocytes or antibodies) and instead rely on innate immunity to
prevent the infection of invading pathogens (26, 27). Numerous
studies have reported that invertebrates also possess diverse forms
of immune ‘memory’ in which a potentiated immune response
(resulting in a reduction of host susceptibility to the infection) has
been recorded following a secondary exposure to a pathogen (28–
31). For instance, in the scallop Chlamys farreri, a first short
exposure to the pathogen Vibrio anguillarum increased
phagocytosis, acid phosphatase activity, and survival following a
second encounter to the pathogen (32). Pacific oysters stimulated
primarily by heat-killed Vibrio splendidus also displayed stronger
immune responses at cellular and molecular levels when they were
subjected to a secondary challenge with the live bacteria (33). (34,
35) showed that injection of Polyinosinic: polycytidylic acid or Poly
(I:C), a synthetic analog of double stranded RNA with
immunostimulant properties, induced a long-lasting antiviral
response in Pacific oysters, protecting them against subsequent
Frontiers in Immunology
2 Material and methods
2.1 Preparation of OsHV-1 antigens
In June 2021, the experiment was conducted to (1) screen
multiple preparations of inactivated OsHV-1 (hereafter referred
to as antigens) by measuring immune-related functions of
hemocytes using flow cytometry (FCM, repeated three times), and
(2) confirm the potency of a selected subset of antigen preparations
to stimulate immunity using FCM (cytotoxicity and ROS) and
molecular analyses (immune- related gene expression, repeated
three times, Figure 1).
2.1.1 OsHV-1 stock
The OsHV-1 suspension stock was produced in October 2019,
as described in Camara et al. (18), from diseased oysters infected
with OsHV-1 during a lab challenge (49). Briefly, tissue from high
virus load oysters was homogenized, purified by serial filtrations
down to 0.22 mm and cryopreserved according to Kirkland et al.
(50). On the 3 June 2021, cryopreserved OsHV-1 stock was
defrosted by dipping in a 22°C water bath for 10 min. Viral
suspension was titrated using qPCR (51) and diluted in 0.22 µmfiltered sterile seawater (SSW) to reach a final concentration of
9.0.105 copies. µl-1. Prepared virus was then inactivated using the
methods described below.
2.1.2 BEI inactivation
A 0.2M Binary ethylenimine (BEI) solution was prepared by
cyclization of 0.2M 2-bromoethylamin-HBr in 0.2M NaOH at 37°C
for 1 h (52). The BEI solution was added to OsHV-1 suspension to
either a concentration of 0.1% (v/v) and incubated at 22°C for 1 h,
4 h, or 6 h, or to a concentration of 0.04% (v/v), and incubated at
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FIGURE 1
Schematic overview of the in-vitro experimental design, (A) refers to the live infectious OsHV-1, (B) whole virus killed by chemical (BEI,
formaldehyde), heat inactivated, or exposed to freeze-thaw cycles, (C) OsHV-1 DNA, (D) OsHV-1 whole protein extract, (E) Poly (I:C, positive
control), (F) sterile filtered seawater (negative control). ROS stands for ‘reactive oxygen species’, ORF to ‘open reading frame’. Figure was created
using BioRender.com.
22°C for 4 h, 6 h, 9 h, 18 h, or 22 h. Inactivation reactions were
stopped by addition of sodium thiosulphate 1M (neutralizing agent)
to reach a final concentration of 10% (v/v). Obtained inactivated
viral suspensions were stored at 4°C until use. Suspension of
neutralized 0.1% (v/v) BEI diluted in filtered (0.22 µm) SSW was
used as the vehicle control (VC), (Supplementary Table 1).
2.1.4 Heat inactivation
For preparation of heat-inactivated OsHV-1, viral suspensions
were incubated for 1 h at 45°C, 50°C, or 52°C, or for 30 min at 54°C,
56°C, and 60°C using a dry bath, and then stored at 4°C until use
(Supplementary Table 1).
2.1.5 Freeze-thaw cycles
Viral suspensions were placed at -80°C for 12 h, transferred to
-20°C for 12 h, and then maintained at 4°C. After complete thawing,
viral suspensions were immediately re-frozen at -80°C for 12 h. The
freeze-thaw cycles were repeated twice (for Thawing 1) or three
times (for Thawing 2), and the antigen suspensions were stored at 4°
C until use (Supplementary Table 1).
2.1.3 Formaldehyde inactivation
Formaldehyde solution 37% (w/v) was added to the OsHV-1
suspension to a final concentration of 5%, 0.3%, or 0.01%, and
incubated at 22°C for 2 h, 4 h, or12 h (5%); for 4 h, 8 h, 12 h, 24 h,
48 h, or 60 h (0.3%); or for 12 h, 24 h, or 60 h (0.01%), respectively.
Virus inactivation was stopped by addition of 35% sodium
bisulphite to reach a final concentration of 0.035%, and
suspensions were then stored at 4°C. Suspension of 5% (v/v)
neutralized formalin diluted in filtered (0.22 µm) SSW was used
as the VC (Supplementary Table 1).
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2.1.6 OsHV-1 DNA
Total viral DNA was extracted from 1 ml of the OsHV-1
stock suspension using blood and tissues kit (QIAGEN)
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immediately added to a 1.5 mL Eppendorf, previously kept on ice.
Individual samples were checked under a light microscope (40X
magnification) to confirm purity. When ≈16 mL of pure, clean
hemolymph had been collected from multiple individuals,
hemolymph samples were pooled, diluted (1:4 v/v) with
autoclaved 0.2 µm-filtered sterile seawater (FSSW) for flow
cytometry or kept undiluted for molecular analyses, and stored
on ice until exposure.
according to the manufacturer’s protocol. Extracted DNA was
resuspended in 10 mM Tris-HCl buffer (pH 7.4) to reach a final
concentration of 10 ng DNA µl-1 and stored at -20°C until use. The
solution 10 mM Tris-HCl buffer (pH 7.4) was used as a VC
(Supplementary Table 1).
2.1.7 Viral proteins
Total viral proteins were extracted from 5 ml of the OsHV-1
stock suspension via bead beating for 10 min at 1500 rpm and 4°C
using a 1600 MiniG automated tissue homogenizer (SPEX Sample
Prep, Metuchen, NJ). Proteins were then solubilized for 45 min by
adding 2 ml of extraction buffer (100 mM potassium phosphate, 50
mM NaCl, 0.1 mM EDTA-Na2, 1% polyvinyl pyrrolidone, 2 mM
phenylmethylsulfonyl fluoride and 0.1% TritonX-100; pH 7.5 at 4°
C). Solubilized proteins were extracted by centrifugation at 13,500
rpm for 15 min at 4°C and ultra-filtered (10 kDa molecular weight
cut-off, Amicon Ultra-0.5 10K, Merk-Millipore, Burlington, USA)
following manufacturer’s specifications. The semi-purified proteins
were reconstituted in 200 µl of sterile Phosphate Buffer Saline (PBS).
Total protein content of the lysate was quantified by the Lowry
protein assay (53), diluted in sterile PBS to reach a final
concentration of 0.2 mg.ml-1, and stored at -80°C until use.
Protein extracts were either pure (Protein 1) or diluted at 1:10 (v/
v) in PBS (Protein 2). Sterile PBS was used as a vehicle control
(Supplementary Table 1).
2.4 In-vitro exposure
Before experimental exposure, each antigen preparation and
vehicle control were diluted (1:100 v/v) with FSSW. Polyinosinicpolycytidylic acid [Poly (I:C)], a synthetic analogue of doublestranded RNA (dsRNA), was used as positive control (0.05 mg.
ml-1 in SSW; 54) and FSSW was used as a negative control. For flow
cytometry, antigens preparations or controls were added (1:80 v/v)
to (1:4 v/v) diluted hemolymph (detailed above) and incubated at
room temperature (22°C) for 1 h. The expression of five immunerelated genes and the viral gene ORF 87 was evaluated by adding 20
µl of the diluted antigen preparations (1:100 (v/v) or controls to 400
µl of pure hemolymph in 1.5 ml Eppendorf tubes and incubated at
22°C for 1 h.
Following experimental exposure, hemocyte reactive oxygen
species (ROS) production and viability were determined (detailed
below). This process was repeated daily for 3 consecutive days to
obtain n = 3 independent replicates.
Based on ROS production in hemocytes and an absence of
cytotoxicity, a subset of promising antigens was selected for further
FCM validation and molecular analyses in a second experiment.
Specific antigen preparations which induced significantly less ROS
production were also included in the second experiment to
maintain a range of contrasted immune responses and improve
validation. This second in vitro challenge was repeated daily for 3
consecutive days to obtain n = 3 independent replicates.
2.2 Oysters
The experiment was performed using hemolymph of hatcherybred juvenile C. gigas [8 months old, mean live weight 6.7 ± 3.1 g].
These oysters were the offspring of naïve wild stocks and therefore
were expected to be highly susceptible to POMS. Prior to sampling,
oysters were maintained in flow-through seawater (10 µm filtered)
at ambient conditions (10 – 22°C and a salinity of 35 ± 1) and fed ad
libitum with hatchery-grown algal food. Experimental oysters were
considered naïve to POMS/OsHV-1 due to their rearing with
continuous supply of UV-sterilized seawater (80 mJ cm-2) and
maintenance under strict biosecurity management to ensure they
remained OsHV-1-free. This status was confirmed prior to the
experiment by the absence of significant mortality and OsHV-1
DNA detection in tissue (n = 10) using qPCR (51). Oysters were
starved for 24 h prior to hemolymph collection to minimize algal
contamination of the hemolymph.
2.5 FCM assessments
Hemolymph samples that had been exposed to different antigen
preparations for 1 h were analyzed using a Guava® EasyCyte™
5HT flow cytometer equipped with a blue laser (488 nm) and green
(525/30 nm), yellow (583/26 nm), and red (695/50) detectors (EMD
Millipore, USA). Samples were mixed at medium speed and
acquired at a flow rate of 0.24 µL s-1 for 30s.
The production of intracellular ROS was measured using 2’,7’Dichlorodihydro-fluorescein diacetate (DCFH-DA, Sigma Aldrich,
D6883) according to Donaghy et al. (55). Following 30 min of
experimental treatments incubation, DCFH-DA was added at a
final concentration of 10 µMand incubated in the dark at room
temperature (22°C) for another 30 min until analysis (= total 1 h
exposure to treatments). Relative ROS production was expressed as
the level of green (FL1) fluorescence.
2.3 Hemolymph collection
Hemolymph was sampled from between 22 and 35 oysters
collected and pooled daily for experimental exposure. A small notch
was made in the shell using wire cutters, and oysters were bled from
the adductor muscle sinus using a 25G 1.5-inch needle with 1 mL
syringe, previously kept on ice. Between 150 and 1500 µl of
hemolymph was withdrawn from each individual and
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The viability of hemocytes was measured using Fluorescein
diacetate (FDA, Invitrogen, F1303) according to Rolton et al. (56).
Following 50 min of experimental treatments incubation, FDA was
added at a final concentration of 1.25 mg L-1 and incubated in the
dark at room temperature for 10 min until analysis (= total 1 h
exposure to treatments). Hemocytes were divided into those with
high FL1 (corresponding to metabolically active/viable cells) and
those with low green fluorescence (non-viable).
3 Results
3.1 79% of the antigenic preparations did
not affect the viability of oyster hemocytes
Of the 33 antigenic preparations tested, 26 had no cytotoxic
effect on oyster hemocytes (as measured by viability). However,
hemocytes exposed for 1 h to the following antigen preparations –
OsHV-1 previously inactivated using BEI 0.04% for 4 h and 6 h
(Figure 2A), formaldehyde 5% for 12 h, formaldehyde 0.3% for 8 h
or 24 h, or formaldehyde 0.01% for 12 h (Figure 2C), and heated at
52°C for 1 h (Figure 2D) – showed a significant reduction in
viability compared to hemocytes exposed to SSW. The viability of
hemocytes that had been heat killed (negative control) was very low
(10.2 ± 5.2% mean ± SD, n = 3) compared to hemocytes exposed to
SSW (91.3 ± 8.0%, p = 1.7e-13) (Figure 2).
2.6 Molecular analyses
RNA was extracted from 400 µl of hemolymph previously exposed
to 20 µl of antigen preparation, SSW (negative control), Poly I:C
(positive control), or live infectious OsHV-1 using the Quick RNA/
DNA Miniprep plus kit (Zymo Research) according to the
manufacturer’s protocol. RNA was eluted in 50 µl DNAse/RNasefree water. As described in Delisle et al. (49), samples were treated with
DNAse I (TURBOTM DNase, Invitrogen), the absence of DNA in the
samples was confirmed by a 16S PCR assay, purity of the isolated RNA
was assessed, and DNAse-treated RNA was transcribed into cDNA.
Finally, droplet digital PCR (ddPCR) was conducted in an automated
droplet generator (QX200 Droplet Digital PCR SystemTM, Bio-Rad)
to determine the expression of five genes (Jak, Stat6, Viperin, IRF2,
Myd88) related to oyster innate immunity (44, 57, 58), as well as the
ORF 87, an OsHV-1 gene selected from the 39 ORFs described by
Segarra et al. (59). Each ddPCR reaction included 1 µl of 3 µM of the
primers (Jak, Stat, Viperin, Myd88) or 10 µM (IRF2, ORF87), 10 µl
ddPCR Supermix for Evagreen (Bio-Rad), 2 µl cDNA, and 8 µl sterile
water for a total reaction volume of 21 µl. As described in Delisle et al.
(49), ddPCR was performed using the following cycling protocol: hold
at 95°C for 5 s, 45 cycles of 95°C for 30 s, 60°C for 1 min, 4°C for
5 min, and a final enzyme deactivation step at 90°C for 5 min. The
plate was then analyzed on the QX200 instrument (Bio-Rad). For each
ddPCR plate run, at least one negative control (RNA/DNA-free water;
Life Technologies), and one positive control (C. gigas DNA or Gblock
for ORF87 diluted 1/10,000) were included.
3.2 Antigenic preparations induced ROS
production in hemocytes
Hemocytes of C. gigas that had been exposed to 10 antigenic
preparations of inactivated OsHV-1 and to the positive control
(Poly I:C) showed increased ROS production (Figure 3).
Specifically, preparations of virus inactivated using BEI 0.04% at
9, 18, and 22 h (Figure 3A), formaldehyde 5% for 4 h, formaldehyde
0.01% for 24 h (Figure 3B), heat shock at 50°C for 1 h, at 60°C for
0.5 h, or three freeze-thaw cycles (Figure 3C), as well as viral protein
extracts pure or diluted 1/10 (v/v, Figure 3D), all significantly
increased hemocyte ROS production compared to those exposed
to SSW (p < 0.001).
Based on an absence of cytotoxicity and high levels of ROS
production (using FCM), 10 antigen preparations, as well as eight
additional antigen preparations that induced a limited subcellular
immune response (Figures 2, 3), were selected for validation (using
FCM and molecular analysis).
2.7 Statistical analyses
3.3 All the selected antigen
preparations induced upregulation of
immune-related genes
Statistical analyses were computed using R 4.2.1 (https://www.rproject.org/) and the packages ‘ggpubr’ (60) and ‘rstatix’ (61). One way
ANOVA and t tests were performed to evaluate the effects of each
antigen preparation on ROS production, hemocytes viability, and gene
expression, in comparison to the effects of SSW exposure (negative
control); p-values were adjusted with Holm correction. For gene
expression, a heatmap was constructed using Multiple Experiment
Viewer software (62; http://mev.tm4.org/#/datasets/upload). For all
analyses, the threshold significance level was set at 0.05.
All the selected antigen preparations induced the upregulation
of at least one of the immune- related genes of C. gigas. Exposure of
hemocytes to the Poly (I:C) at 0.05 mg. ml-1 for 1 h induced a
significant upregulation of MyD88, Viperin, and Stat6 (Figure 4).
Exposure of hemocytes to OsHV-1, which had been inactivated
using BEI 0.04% for 22 h, resulted in a significant upregulation of
all five of the immune- related genes analyzed. Vehicle controls did
not induce the expression of the immune- related genes in
hemocytes, except the phosphate buffered saline (VC protein,
Supplementary Table 1), which induced a significant upregulation
of Stat6, Viperin, IRF2, and MyD88. As expected, the expression of
ORF87 was only detected in hemocytes exposed to live infectious
OsHV-1 (Figure 4).
2.8 Ethics approval
The study was conducted according to the guidelines of the
Declaration of Helsinki and approval of the Animal Ethics
Committee was not applicable for the use of oysters.
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A
B
C
D
FIGURE 2
Viability (in percent) of Crassostrea gigas hemocytes exposed for 1 h to OsHV-1, which had been inactivated with different preparations of (A) Binary
ethylenimine BEI, (B) formaldehyde, and (C) temperature treatments. (D) shows viability of hemocytes exposed to two viral protein concentrations
and purified viral DNA (see Supplementary Table 1 for details of the different treatments). Box plots indicate the median, upper, and lower quartiles.
Whiskers indicate the highest and the lowest percent viable hemocytes and dots indicate outliers. Significance levels are expressed by asterisks: * (p
< 0.05), ** (p < 0.01), ***(p < 0.001), **** (p < 0.0001). N = 3/6 replicates. ‘SW’ stands for seawater, ‘VC’ for vehicle control.
33 antigen preparations tested in our study, only seven induced a
significant decline in hemocyte viability. Incomplete neutralization
of the denaturing agent could explain this observed cytotoxicity.
The residual infectivity post-inactivation of the preparations tested
was verified by the absence of the open reading frame 87 (ORF87)
expression in the hemocytes following 1h of antigen exposure. This
ORF codes for an apoptosis inhibitor protein and is expressed
during the first few hours post OsHV-1 infection in oysters (42, 59).
A 1-hour exposure of hemocytes to 0.5 mg.ml-1 of Poly (I:C)
was sufficient to induce cellular ROS production – a proxy of
immune response in bivalves (64) – and an upregulation of the
genes coding key antiviral effectors: MyD88, Stat 6, and Viperin,
validating our screening approach. Increased ROS indicates a
stimulation of an immune response, as ROS production is
associated with internal chemical destruction of engulfed
pathogens or foreign particles within hemocytes (65, 66). The
mechanisms of hemocyte activation in oyster immune defense,
however, are still largely unknown. In response to foreign
particles, oyster hemocytes can: secrete effectors extracellularly;
phagocytose particles, where they are destroyed by ROS and
4 Discussion
In the present study, we were able to stimulate an antiviral
response in juvenile Crassostrea gigas hemocytes using inactivated
OsHV-1 and viral extracts, as shown by ROS induction and
upregulation of antiviral response-related genes. A previous study,
in which C. gigas hemocytes were exposed to live OsHV-1, also
resulted in the expression of genes involved in immune-related
functions (42); however, this study is the first time inactivated
OsHV-1 preparations have been shown to elicit an immune
response in-vitro.
Virus inactivation transforms antigens from being infectious to
non-infectious, and it is important to determine any cytotoxicity of
the antigen preparations prior to determining their potency (52). In
the absence of bivalve cell line cultures in which to determine
cytotoxicity, here, we used a FCM-based assessment of hemocyte
viability for rapid in-vitro screening of the OsHV-1 inactivated
preparations. Morga et al. (63), similarly used FCM to determine
hemocyte viability following an in-vitro exposure of C. gigas
hemocytes to the protozoan parasite Bonamia ostreae. Among the
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A
B
C
D
FIGURE 3
Reactive oxygen species (ROS) production (expressed in arbitrary unit, a.u) in Crassostrea gigas hemocytes exposed for 1 h to OsHV-1, which had
been inactivated with different preparations of (A) Binary ethylenimine BEI, (B) formaldehyde, and (C) temperature treatments. (D) shows ROS
production in hemocytes exposed to two viral protein concentrations and purified viral DNA (see Supplementary Table 1 for details of the different
treatments). N = 3/6 replicates, box plots indicate the median, upper and lower quartiles; whiskers indicate the highest and the lowest values of the
dataset and dots indicates outliers. Significance levels are expressed by asterisks: * (p < 0.05), ** (p<0.01), ***(p<0.001), **** (p<0.0001). ‘SW’ stands
for seawater, ‘VC’ for vehicle control.
had been stimulated for 8 h and 3 h, respectively, with 50 µg.ml-1
of Poly (I:C), a much higher concentration than was used in the
present study (68, 69). Upregulation of Viperin – an interferoninducible antiviral protein – and Stat 6 – a signal transducer and
activator of transcription – have also been observed in oyster
hemocytes primed with Poly (I:C), in comparison to those
exposed to seawater (46, 70).
defense molecules stored in granules; and following stress or
recognition of foreign particles by soluble and cellular pattern
recognition receptors, induce the expression of specific immune
genes (67). An induction of MyD88 gene, an essential
signal transducer in the interleukin-1 and Toll-like receptor
signaling pathways, was also observed in mussel (Mytilus
galloprovincialis) and scallop (Pecten maximus) hemocytes that
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activity as human Viperin against Dengue virus in-vitro (46, 80).
Consequently, its expression in hemocytes could constitute a good
indicator of the potency of the antigen preparations.
Besides chemical agents, physical methods were used to
inactivate OsHV-1 in this study. Thermal inactivation of OsHV-1
at 50°C and 60°C and long thawing cycles caused an increase in
ROS production, while the associated molecular responses appeared
moderate. These variations in the results could probably be
explained by the broad range of stressors able to induce ROS
production in bivalves (81), in comparison to the specificity of
the antiviral response. Nonetheless, we obtained a good correlation
overall between immune-related markers measured via flow
cytometry (ROS production) and qPCR analyses (immune-gene
expression), with the ranking of the 10 best antigen preparations
maintained when upregulation of immunity genes was considered.
Of note, protein extracts obtained from OsHV-1 and
resuspended in PBS also induced a marked positive expression of
MyD88, IRF2, and Viperin; however, the confounding effect of the
vehicle control PBS on the hemocytes cannot be excluded. Similar
induction of immune response following PBS exposure has been
reported (82, 83).
To conclude, these findings are significant as they evidence for
the first time the potential for stimulating oyster’s innate immunity
using OsHV-1 antigens, which may enable cost-effective
therapeutic treatment to mitigate the economic impacts of OsHV1/POMS. For instance, chemical inactivation using the Binary
ethylenimine at 0.04% for 22 h was identified as the best
candidate preparation requiring additional research. However, it
is essential to test these antigen preparations further using in-vivo
infection models to validate promising candidate pseudo-vaccines.
Nonetheless, we demonstrated that the use of flow cytometry-based
cellular assays was an effective and rapid screening tool to select
treatments for ‘pseudo-vaccine’ development.
FIGURE 4
Heatmap focusing on the expression of 5 genes related to the
innate immune response in Crassostrea gigas and ORF87, an OsHV1 ORF expressed early after infection. The intensities of the colors
indicate the magnitude of the differential expression (log2 foldchange), N = 3 replicates. The fold-changes were calculated by
comparing the expression of each gene in hemocytes exposed to
Poly (I:C), live infectious OsHV-1, or OsHV-1 inactivated, with its
expression in negative control (hemocytes exposed to SSW).
Significance levels are expressed by asterisks: * (p < 0.05), ** (p <
0.01), ***(p < 0.001), **** (p < 0.0001).
The interaction between OsHV-1 and the oyster host cells has
not been fully elucidated, and the functions of proteins coded by
OsHV-1 genome are largely unknown. However, antibody blocking
and pull-down assays suggest the potential implication of three
putative OsHV-1 membrane proteins (ORF 25, ORF 41, and ORF
72) in the virus/host interaction by binding of host cytoskeleton (71,
72). Interestingly, antigen preparations resulting from BEI
inactivation induced the strongest immune and antiviral response.
Binary ethylenimine is an aziridine preparation commonly used in
veterinary vaccine production as an inactivating agent (73). At 1
mM, BEI induces an alkylation of the nucleic acids without
damaging proteins (74). In the present work, high ROS
production coupled with upregulation of the five tested genes
were recorded when using OsHV-1 inactivated with BEI at
[0.04%] for 22h, supporting the role of viral proteins in the
stimulation of antiviral response in oysters. Conversely, we
observed a reduced potency of antigen preparations that were
inactivated by formaldehyde, one of the most widely used
inactivating agents, for an extended exposure time (12 and 48h),
suggesting an irreversible denaturation of proteins induced by
formaldehyde (75).
Three antigen preparations inactivated with BEI induced a
strong upregulation of the transcript coding for Viperin. Viperin
is a highly conserved evolutionary host protein (76), which restricts
the replication of a range of RNA and DNA viruses [e.g., human
cytomegalovirus (77), immunodeficiency virus (78), and Hepatitis
C virus (79)], by interacting with viral protein and altering the site
of virus budding. In C. gigas, Viperin has been reported to be one of
the earliest and most regulated genes in response to OsHV-1
exposure (54, 80), and it also exhibits the same level of antiviral
Frontiers in Immunology
Data availability statement
The original contributions presented in the study are included
in the article/Supplementary Materials. Further inquiries can be
directed to the corresponding authors.
Author contributions
LD, AR, JV, conception and design. LD and AR lab trial and
data analyses. All authors, writing, revision/editing. LD, JV funding.
All authors contributed to the article and approved the
submitted version.
Funding
This work was supported by the Cawthron Shellfish
Aquaculture Research Platform (contract no. CAWX1801) funded
by the New Zealand Ministry of Business, Innovation
and Employment.
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Acknowledgments
Publisher’s note
We would like to thank Ruth Garcia Gomez, Hayley Chapman,
Carol Peychers, and Karthiga Kumanan for their technical assistance
and Kirsty Smith for her advice. Thank you also to Nick King and
Seumas Walker for reviewing this manuscript. We are also grateful to
William McKay and Jim Dollimore at Biomarine Ltd for the provision
of wild oysters, and the hatchery team at the Cawthron Aquaculture
Park for maintaining and growing the experimental animals.
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
Supplementary material
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.
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/
fimmu.2023.1161145/full#supplementary-material
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Assessment of the association of the rs12329760 polymorphism of the <i>TMPRSS2</i> gene with acute coronary syndrome in patients with new coronavirus infection
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Оценка ассоциации полиморфизма rs12329760 гена TMPRSS2 с острым
коронарным синдромом у пациентов, перенесших новую коронавирусную
инфекцию В.А. Козик1, Л.А. Шпагина1, И.С. Шпагин1, С.В. Максимова1, 2, Н.Г. Ложкина3,
В.Н. Максимов1, 2 1 Новосибирский государственный медицинский университет
Министерства здравоохранения Российской Федерации
Россия, 630091, г. Новосибирск, Красный пр., 52 Козик В.А., Шпагина Л.А., Шпагин И.С., Максимова С.В., Ложкина Н.Г., Максимов В.Н., 2024 А Т Е Р О С К Л Е Р О З / A T E R O S C L E R O Z А Т Е Р О С К Л Е Р О З / A T E R O S C L E R O Z 2024 Т. 20, № 1 Научно-практический журнал DOI 10. 52727/2078-256Х-2024-20-1-35-41 Аннотация Обоснование. Сердечно-сосудистые заболевания (ССЗ) остаются на первом месте по
уровню заболеваемости и смертности как в Российской Федерации, так и во всeм мире. С
конца 2019 – начала 2020 г. планету охватила пандемия новой коронавирусной инфекции
(НКИ), которая внесла свой вклад в развитие и течение ССЗ. Поэтому стали рассматривать
молекулярно-генетические маркеры как факторы, потенциально влияющие на ССЗ, их развитие
и тяжесть течения. Цель исследования – оценить ассоциацию варианта нуклеотидной последо-
вательности rs12329760 гена ТMPRSS2 с острым коронарным синдромом (ОКС) у пациентов,
перенесших НКИ. Материал и методы. В исследование включены 100 пациентов (женщин –
50, мужчин – 50) с ОКС и перенесенной НКИ, которые отбирались на основе положитель-
ного ПЦР-теста на наличие SARS-CoV-2 в анамнезе, госпитализированных в региональный
сосудистый центр № 7 Городской клинической больницы № 2 г. Новосибирска. Возраст
женщин составил 59,5 ± 7,2 года, мужчин — 53,5 ± 9,3 года. Диагноз ОКС устанавливался
по совокупности критериев, предложенных и установленных Российским кардиологическим
обществом и в соответствии с обновленными клиническими рекомендациями Министерства
здравоохранения Российской Федерации от 2020 г. Пациентам проводились клинико-
инструментальное обследование, коронароангиография со стентированием, предусмотрен-
ные стандартами оказания медицинской помощи и клиническими рекомендациями. У па-
циентов определяли вариант нуклеотидной последовательности rs12329760 гена TMPRSS2 с
помощью ПЦР с последующим анализом полиморфизма длин рестрикционных фрагментов. Группу сравнения составили 200 пациентов с ОКС без перенесенной НКИ (нет положи-
тельного ПЦР-теста, нет антител к SARS-CoV-2). Результаты. Носительство генотипов СС,
CT, TT rs12329760 гена TMPSS2 не ассоциировано с повышенным риском развития ОКС у
пациентов, перенесших НКИ. При сравнении частот генотипов rs12329760 гена TMPSS2 в
группах с ОКС и НКИ и ОКС без НКИ не получено статистически значимых различий. В
группе с ОКС и НКИ чаще встречается носительство гомозиготного генотипа СС. Заключение. Вариант нуклеотидной последовательности rs12329760 гена TMPRSS2 не ассоциирован с ОКС
у пациентов, перенесших НКИ. Ключевые слова: острый коронарный синдром, новая коронавирусная инфекция, вариант
нуклеотидной последовательности, ген TMPRSS2, генетические маркеры, rs12329760. Ключевые слова: острый коронарный синдром, новая коронавирусная инфекция, вариант
нуклеотидной последовательности, ген TMPRSS2, генетические маркеры, rs12329760. Конфликт интересов. Авторы заявляют об отсутствии конфликта интересов. Финансирование. Молекулярно-генетический фрагмент исследования выполнен в рамках
бюджетной темы, рег. № 122031700094-5. Автор для переписки: Козик В.А., e-mail valiyta90@mail.ru
Для цитирования: Козик В.А., Шпагина Л.А., Шпагин И.С., Максимова С.В., Ложки-
на Н.Г., Максимов В.Н. Оценка ассоциации полиморфизма rs12329760 гена TMPRSS2 с
острым коронарным синдромом у пациентов, перенесших новую коронавирусную инфекцию. Атеросклероз, 2024; 20 (1): 35–41. doi: 10. Assessment of the association of the rs12329760 polymorphism of the TMPRSS2
gene with acute coronary syndrome in patients with new coronavirus infection
V.A. Kozik1, L.A. Shpagina1, I.S. Shpagin1, S.V. Maksimova1, 2, N.G Lozhkina3, V.N. Maksimov1, 2 1 Novosibirsk State Medical University of Mivzdrav of Russia
52, Krasny av., Novosibirsk, 630091, Russia
2 Research Institute of Internal and Preventive Medicine –
Branch of the Institute of Cytology and Genetics, Siberian Branch of Russian Academy of Sciences
175/1, Boris Bogatkov str., Novosibirsk, 630089, Russia
3 Federal Research Center for Fundamental and Translational Medicine
2, Timakov str., Novosibirsk, 630117, Russia Abstract The authors declare no conflict of interest. Funding. The molecular genetic fragment of the study was carried out within the framework of
the budget topic reg. No. 122031700094-5. Conflict of interest. The authors declare no conflict of interest. Funding. The molecular genetic fragment of the study was carried out within the framework of
the budget topic reg. No. 122031700094-5. Conflict of interest. The authors declare no conflict of interest. Funding. The molecular genetic fragment of the study was carried out within the framework of
the budget topic reg. No. 122031700094-5. Funding. The molecular genetic fragment of the study was carried out within the framework of
the budget topic reg. No. 122031700094-5. g
p
g
Correspondence: Kozik V.A., e-mail valiyta90@mail.ru Correspondence: Kozik V.A., e-mail valiyta90@mail.ru Citation: Kozik V.A., Shpagina L.A., Shpagin I.S., Maksimova S.V., Lozhkina N.G, Maksimov V.N. Assessment of the association of the rs12329760 polymorphism of the TMPRSS2 gene with acute cor-
onary syndrome in patients with new coronavirus infection. Ateroscleroz, 2024; 20 (1): 35–41. doi:
10.52727/2078-256X-2024-20-1-35-41 Abstract Introduction. Cardiovascular diseases (СVD) rank first in terms of morbidity and mortality not
only in the Russian Federation but throughout the world. Since the end of 2019 a pandemic of Se-
vere Acute Respiratory Syndrome (SARS) or new coronavirus infection (NCI) has spread throughout
the world, which contributed to the development and course of CVD. Therefore, genetic markers
began to be considered as factors potentially influencing CVD, its development and severity. Objec-
tive: To evaluate the association of the nucleotide sequence variant rs12329760 of the TMPRSS2
gene with acute coronary syndrome in patients who have had a new coronavirus infection. Material
and methods. The study included 100 patients (women – 50, men – 50) with ACS and previous
NCI, who were selected on the basis of a positive PCR test for the presence of SARS-CoV-2 in
the anamnesis, hospitalized at the regional vascular center No. 7 of the City Clinical Hospital No. 2 of the city of Novosibirsk. Women age was 59.5 ± 7.2 years, men age was 53.5 ± 9.3 years. The
diagnosis of ACS was established according to a set of criteria proposed and established by the Rus-
sian Society of Cardiology and in accordance with the updated clinical recommendations of the
Ministry of Health of the Russian Federation of 2020. Patients underwent clinical and instrumental
examination, coronary angiography with possible stenting, as provided for in the standards of medical
care and clinical guidelines. The nucleotide sequence variant rs12329760 of the TMPSS2 gene was
determined in patients using PCR with further analysis of restriction fragment length polymorphism. The comparison group consisted of 200 patients with ACS without previous NCI (no positive PCR
test, no positive antibodies). Results. Carriage of CC, СТ, TT, genotype variants of the nucleotide
sequence rs12329760 of the TMPSS2 gene are not associated with an increased risk of developing
ACS in patients with previous NCI. When comparing the frequencies of the rs12329760 genotypes of
the TMPSS2 gene in groups with ACS with NCI and ACS without NCI, no statistically significant
differences were obtained. In the group with ACS with NCI, carriage of the homozygous genotype
was more common (p = 0.011). Conclusions. Variants of the nucleotide sequence rs12329760 of the
TMPSS2 gene are not associated with ACS with previous NCI. Keywords: acute coronary syndrome, new coronavirus infection (NCI), nucleotide sequence vari-
ant (NSV), TMPRSS2, genetic markers, rs12329760. Conflict of interest. Аннотация 52727/2078-256Х-2024-20-1-35-41 © Козик В.А., Шпагина Л.А., Шпагин И.С., Максимова С.В., Ложкина Н.Г., Максимов В.Н., 2024 35 Атеросклероз. Т. 20. № 1. 2024 / Ateroscleroz. Vol. 20. N 1. 2024 Введение но, несмотря на это, смертность от ССЗ оста-
ется крайне высокой [2]. Свой вклад в развитие
данной патологии внесла новая коронавирусная
инфекция (НКИ), что обусловливает актуаль-
ность данной проблемы [3]. Уже известно, что
НКИ поражает сердечную мышцу, активирует
гиперкоагуляцию и способствует микротромбо-
зам, вызывая тем самым как в госпитальном,
так и в постгоспитальном периоде ОКС, мио- Как в Российской Федерации, так и во всем
мире сердечно-сосудистые заболевания (ССЗ)
остаются на первом месте по уровню заболева-
емости и смертности [1]. Новые лекарственные
препараты, новые технологии лечения внесли
значительный вклад в течение острого коронар-
ного синдрома (ОКС) и прогноз для пациентов, 36 В.А. Козик, Л.А. Шпагина, И.С. Шпагин и др. Из исследования исключались пациенты
с любой онкологической патологией, тяжелой
печеночной
и
почечной
недостаточностью,
инсулинзависимым сахарным диабетом 1 и 2
типа, воспалительными заболеваниями печени,
почек, эндокринных желез, половых органов,
психическими расстройствами, алкоголизмом,
токсикоманией, ВИЧ. От каждого пациента
получено информированное согласие на участие
в исследовании в соответствии с этическими
требованиями
ВОЗ. Исследование
одобрено
локальным этическим комитетом ФГБОУ ВО
«Новосибирский
государственный
медицин-
ский университет» Минздрава России (протокол
№ 152 от 25.05.2023). кардит, инфаркт миокарда (ИМ) с подъемом и
без подъема сегмента ST [4]. Особое внимание
ученые всего мира уделяют молекулярно-гене-
тическим маркерам как факторам, потенциаль-
но влияющим на ССЗ, их развитие, тяжесть те-
чения. Так, например, определенные пациенты
лучше реагируют на лечение и кардиореабили-
тацию, а у кого-то чаще случаются осложнения. тацию, а у кого-то чаще случаются осложнения. Ген трансмембранной сериновой протеа-
зы-2 (transmembrane serine protease 2, TMPRSS2)
кодирует одноименный белок из семейства се-
риновых протеаз [5], широко распространен-
ных в природе и составляющих около трети
всех известных протеолитических ферментов,
которые участвуют в различных процессах, та-
ких как пищеварение, оплодотворение, акти-
вация комплемента, апоптоз, иммунный ответ
[6]. Ген TMPRSS2 ассоциирован с раком про-
статы и повышенным риском инфицирования
коронавирусами SARS-CoV и SARS-CoV-2. В
результатах большого количества исследований
определяются ассоциации варианта нуклеотид-
ной последовательности (ВНП) rs12329760 гена
TMPRSS2 с различными ССЗ [7]. Целью насто-
ящего исследования стала оценка ассоциации
ВНП rs12329760 гена TMPSS2 с ОКС у пациен-
тов, перенесших НКИ. Первую запись ЭКГ пациенту проводила
бригада скорой медицинской помощи, а за-
тем – дежурная служба регионального сосудис-
того
центра. При
выполнении
настоящего
исследования по ЭКГ, которую регистриро-
вали в 12 стандартных отведениях с помощью
шестиканального аппарата Megacart (Simens-
Elema АВ, Германия), определяли динамику,
локализацию и стадию ОКС. Введение Селективную ко-
ронарографию выполняли на 1–3-и сутки от
развития симптомов острого ИМ на ангиогра-
фической установке Optima IGS 330 (General
Electric, США) с фиксацией изображения на
компьютере. Всем пациентам ставились коро-
нарные стенты с лекарственным покрытием си-
ролимус BioMime (Meril Life Sciences, Индия),
система доставки быстрой смены rapid exchange
(RX) с раздуваемым полуподдатливым балло-
ном, минимально выходящим за пределы стен-
та. Всем пациентам с ОКС и НКИ в анамнезе
при поступлении были проведены холтеровское
мониторирование ЭКГ и все остальные лабора-
торные исследования согласно стандарту обсле-
дования ОКС. Материал и методы Дизайн исследования: сравнительное опи-
сательное
исследование
двух
параллельных
групп с ретроспективным компонентом. Ос-
новную группу составили 100 больных с ОКС
и перенесенной НКИ (наличие положительного
ПЦР-теста на наличие SARS-CoV-2 в анамне-
зе, повышенное содержание IgG в крови), 50
женщин (возраст 59,5 ± 7,2 года) и 50 мужчин
(возраст 53,5 ± 9,3 года). В группу сравнения
вошли 200 пациентов с ОКС без перенесенной
НКИ в анамнезе, сопоставимых по полу и воз-
расту, она была набрана на базе ГБУЗ НСО
«Городская клиническая больница № 1» г. Но-
восибирска и включала больных с острым ИМ с
подъемом сегмента ST, последовательно посту-
павших в отделение реанимации и интенсивной
терапии регионального сосудистого центра № 1. Больные основной группы (с НКИ в анамне-
зе)
поступали
в
региональный
сосудистый
центр № 7 ГБУЗ НСО «Городская клиниче-
ская больница № 2» г. Новосибирска. Диагноз
ОКС устанавливался по совокупности критери-
ев, предложенных и установленных Российским
кардиологическим обществом и в соответствии
с обновленными клиническими рекомендация-
ми Министерства здравоохранения Российской
Федерации от 2020 г. ДНК из образцов венозной крови выделялась
с
помощью
фенол-хлороформного
метода. Всем
исследуемым
пациентам
определяли
ВНП rs12329760 гена TMPRSS2 с помощью
полимеразной цепной реакции с последующим
анализом полиморфизма длин рестрикционных
фрагментов. Для амплификации использовали
праймеры:
5'-ctacggaccaaacttcatccttccg-3'(F)
и
5'-gtctgttactgtcactcggcgggt-3'(R). Смесь для ПЦР
объемом 25 мкл включала: Трис-HCI (pH 9,0)
75 мM, (NH4)2SO4 20 мM, Тween-20 0,01 %,
3,0 мМ MgCl2, по 2,0 мкл каждого праймера,
0,5 мМ смеси dNTP, 2 мкг ДНК, 1 единицу
Тaq-полимеразы, вода до объема 25 мкл. Ам-
плификацию проводили в следующем тем-
пературном режиме: 33 цикла, включающих
денатурацию 95 °С 30 с, отжиг праймеров
60 °С 30 с и элонгацию 72 °С 30 с. Рестрикцию 37 Атеросклероз. Т. 20. № 1. 2024 / Ateroscleroz. Vol. 20. N 1. 2024 циентов), генотипа CC – 60 % (30 пациентов). В группе сравнения у мужчин частота генотипа
ТТ составила 5,2 % (6 пациентов), частота ге-
нотипа СТ – 26,1 % (30 пациентов), частота ге-
нотипа СС – 68,7 % (79 пациентов). У женщин
в группе с ОКС и НКИ частота генотипа ТТ
составила 14 % (7 пациентов), частота геноти-
па СТ – 40 % (20 пациентов), частота генотипа
СС – 46 % (23 пациента). В группе сравнения
у женщин частота генотипа ТТ составила 8,2 %
(7 пациентов), частота генотипа СТ – 35,3 %
(30 пациентов), частота генотипа СС – 56,5 %
(48 пациентов). Материал и методы Таким образом, носительство
гомозигот как в исследуемой группе, так и в
группе сравнения было существенно выше
гетерозигот
в
процентном
соотношении. Статистически значимых различий выявлено не
было. проводили с 10 ед. активности рестриктазы
Msp I («СибЭнзим», Новосибирск) при 37 °С
в течение 16 ч. Размер продукта амплификации
160 п.н. Детекцию продуктов амплификации
и рестрикции осуществляли методом электро-
фореза в 4 % полиакриламидном геле с после-
дующим окрашиванием бромистым этидием. После проведения рестрикции при генотипе ТТ
детектировался продукт 160 п.н., при генотипе
СС – продукты 137 п.н. и 23 п.н., при гетеро-
зиготном генотипе все перечисленные продук-
ты: 160 п.н., 137 п.н., 23 п.н. проводили с 10 ед. активности рестриктазы
Msp I («СибЭнзим», Новосибирск) при 37 °С
в течение 16 ч. Размер продукта амплификации
160 п.н. Детекцию продуктов амплификации
и рестрикции осуществляли методом электро-
фореза в 4 % полиакриламидном геле с после-
дующим окрашиванием бромистым этидием. После проведения рестрикции при генотипе ТТ
детектировался продукт 160 п.н., при генотипе
СС – продукты 137 п.н. и 23 п.н., при гетеро-
зиготном генотипе все перечисленные продук-
ты: 160 п.н., 137 п.н., 23 п.н. При выполнении статистического анализа
рассчитывали частоту качественных признаков,
средние значения количественных показателей
(М) и их стандартное отклонение (SD). Срав-
нение
групп
по
качественным
признакам
проводили с использованием критерия χ2, ко-
личественных – с помощью критерия Стью-
дента. Оценивали отношение шансов (ОШ) с
95%-м доверительным интервалом (95 % ДИ)
для каждой переменной. Клинико-анамнестическая
характеристика
пациентов основной группы и группы сравнения
представлена в табл. 2, из которой видно, что у
больных ОКС с НКИ чаще, чем в отсутствие
последней, наблюдались однососудистое пора-
жение коронарных артерий, артериальная гипер-
тензия, чрескожные коронарные вмешательства
со стентированием, смерть родственников от
ССЗ и дислипидемия в анамнезе (табл. 2). Результаты Всем пациентам определяли ВНП rs12329760
гена трансмембранной сериновой протеазы-2 с
помощью полимеразной цепной реакции, по
результатам которого в группе c ОКС и НКИ
генотип TT выявлен у 9 (9 %) пациентов,
генотип СT – у 38 (38 %), генотип СС – у 53
(53 %). В группе сравнения: генотип TT – у 13
(6,5 %) пациентов, генотип CT – у 60 (30 %),
генотип СС – у 127 (63,5 %) пациентов соот-
ветственно. Частоты генотипов представлены в
табл. 1. Обсуждение В настоящем исследовании чаще всего
наблюдалось носительство генотипа CC гена
TMPRSS2 rs12329760. Не выявлено связи ВНП
с развитием ОКС у пациентов, перенесших
НКИ, в отличие от данных зарубежных работ, в
которых ВНП изучались на больших выборках
пациентов. Так, например, ученые Бразилии,
включившие
в
исследование
402
пожилых
пациента с подтвержденным COVID-19 [6], по- С разделением по полу в исследуемой
группе у мужчин частота генотипа TT составила
4 % (2 пациента), генотипа СT – 36 % (18 па- Т аблица 1
Частоты генотипов rs12329760 гена TMPRSS2 с острым коронарным синдромом у пациентов, перенесших новую
коронавирусную инфекцию и группы сравнения
T able 1
Frequencies of rs12329760 genotypes of the TMPRSS2 gene with acute coronary syndrome in patients who had a new
coronavirus infection and comparison groups
TMPRSS2, rs12329760
ОКС и НКИ /
ACS with NCI
Контрольная группа с ОКС /
Control group with ACS
p
n
%
n
%
Генотип TT / Genotype TT
9
9
13
6,5
р = 0,432
Генотип СТ / Genotype CT
38
38
60
30
р = 0,207
Генотип СС / Genotype CC
53
53
127
63,5
р = 0,104
Всего / Total
100
100,0
200
100,0 ц
Частоты генотипов rs12329760 гена TMPRSS2 с острым коронарным синдромом у пациентов, перенесших новую
коронавирусную инфекцию и группы сравнения
T able 1 Частоты генотипов rs12329760 гена TMPRSS2 с острым коронарным синдромом у пациентов, пере
коронавирусную инфекцию и группы сравнения s12329760 гена TMPRSS2 с острым коронарным синдромом у пациентов, перенесших новую
коронавирусную инфекцию и группы сравнения 38 В.А. Козик, Л.А. Шпагина, И.С. Шпагин и др. Обсуждение Т аблица 2
Клинико-анамнестическая характеристика ОКС у пациентов, перенесших НКИ, и группы сравнения
T able 2
Clinical and anamnestic characteristics of acute coronary syndrome in patients who suffered a new coronavirus infection
and comparison group
Показатель /
Characteristic
ОКС с
НКИ /
ACS with
NCI
(n = 100)
ОКС без
НКИ /
ACS with-
out NCI
(n = 200)
p
ОШ [95% ДИ] /
OR [95% CI]
Возраст, лет/ Age, years
55,5 ± 9,5
56,5 ± 7,2
0,933
–
Сроки поступления: до 2 ч /1 сут, n (%) /
Timing of patient admission: up to 2 hours/1 day, n (%)
79/21
(79/21)
118/82
(59/41)
<0,001
2,614
[1,497; 4,566]
Тромболизис, n (%) /
Thrombolysis, n (%)
8 (8)
14 (7)
0,755
1,155
[0,468; 2,853]
Предшествующая стенокардия напряжения,
функциональный класс 2–3, n (%) /
Previous angina pectoris, functional class 2–3, n (%)
23 (23)
56 (28)
0,4354
0,768
[0,439; 1,343]
Острый ИМ в анамнезе, n (%) /
History of acute MI, n (%)
7 (7)
12 (6)
0,738
1,179
[0,449; 3,094]
Чрескожные коронарные вмешательства в анамнезе,
n (%) /
History of percutaneous coronary interventions, n (%)
9 (9)
6 (3)
0,025
3,198
[1,105; 9,254]
Аортокоронарное шунтирование в анамнезе, n (%) /
History of coronary artery bypass grafting, n (%)
1 (1)
4 (2)
0,524
0,495
[0,055; 4,488]
Однососудистое поражение по
коронароангиографии, n (%) / Single-vessel lesion ac-
cording to coronary angiography, n (%)
72 (72)
68 (34)
<0,001
4,992
[2,951; 8,442]
Многососудистое поражение по
коронароангиографии, n (%) /
Multivessel lesion according to coronary angiography, n (%)
28 (28)
51 (25,5)
0,644
1,136
[0,662; 1,950]
Артериальная гипертензия, n (%) /
Arterial hypertension, n (%)
72 (72)
104 (52)
0,001
2,374
[1,415; 3,981]
Подтвержденный сахарный диабет 2 типа, n (%) /
Confirmed type 2 diabetes mellitus, n (%)
21 (21)
44 (22)
0,843
0,942
[0,524; 1,694]
Курение, n (%) /
Smoking, n (%)
34 (34)
77 (38,5)
0,447
0,823
[0,498; 1,360]
Смерть родственников от ССЗ, n (%) /
Relatives death’s from CVD, n (%)
20 (20)
72 (36)
0,005
0,444
[0,252; 0,785]
Дислипидемия в анамнезе, n (%) /
History of dyslipidemia, n (%)
17 (17)
57 (28,5)
0,030
0,514
[0,280; 0,941]
Жизнеугрожающие нарушения ритма, n (%) /
Life-threatening rhythm disturbances, n (%)
11 (11)
32 (16)
0,244
0,649
[0,312; 1,349]
Кillip > II, n (%)
12 (12)
38 (19)
0,126
0,581
[0,289; 1,170] Т аблица 2
я характеристика ОКС у пациентов, перенесших НКИ, и группы сравнения Т аблица 2
Клинико-анамнестическая характеристика ОКС у пациентов, перенесших НКИ, и группы сравнения
T able 2 Т
Клинико-анамнестическая характеристика ОКС у пациентов, перенесших НКИ, и группы сравн предыдущих исследований этого варианта в
популяциях европейского происхождения, ко-
торые предполагали, что носительство данного
аллеля, наоборот, является защитным факто-
ром от тяжелого течения НКИ. Список литературы/ References 1. Barbarash
O.L.,
Duplyakov
D.V.,
Zateish-
chikov D.A., Shakhnovich R.M., Yavelov I.S., Ya-
kovlev A.N., Abugov S.A., Alekyan B.G., Arkhi-
pov
M.V.,
Vasilieva
E.Yu.,
Galyavich
A.S.,
Ganyukov V.I., Gilyarevskiy S.R., Golubev E.P.,
Golukhova E.Z., Gratsianskiy N.A., Karpov Yu.A.,
Kosmacheva E.D., Lopatin Yu.M., Markov V.A.,
Nikulina N.N., Pevzner D.V., Pogosova N.V., Pro-
topopov A.V., Skrypnik D.V., Tereshchenko S.N.,
Ustyugov S.A., Khripun A.V., Shalaev S.V., Shpe-
ktor A.V., Yakushin S.S. Acute coronary syndrome
without ST segment elevation electrocardiogram. Clinical guidelines 2020. Russ. J. Cardiol., 2021; 26
(4): 149–202. (In Russ). doi: 10.15829/1560-4071-
2021-4449 [Барбараш О.Л., Дупляков Д.В., Затей-
щиков Д.А., Шахнович Р.М., Явелов И.С., Яков-
лев А.Н., Абугов С.А., Алекян Б.Г., Архипов М.В.,
Васильева Е.Ю., Галявич А.С., Ганюков В.И.,
Гиляревский С.Р., Голубев Е.П., Голухова Е.З.,
Грацианский Н.А., Карпов Ю.А., Космачева Е.Д,,
Лопатин Ю.М., Марков В.А., Никулина Н.Н.,
Певзнер Д.В., Погосова Н.В., Протопопов А.В,,
Скрыпник Д.В., Терещенко С.Н., Устюгов С.А.,
Хрипун А.В., Шалаев С.В., Шпектор А.В., Яку- 1. Barbarash
O.L.,
Duplyakov
D.V.,
Zateish-
chikov D.A., Shakhnovich R.M., Yavelov I.S., Ya-
kovlev A.N., Abugov S.A., Alekyan B.G., Arkhi-
pov
M.V.,
Vasilieva
E.Yu.,
Galyavich
A.S.,
Ganyukov V.I., Gilyarevskiy S.R., Golubev E.P.,
Golukhova E.Z., Gratsianskiy N.A., Karpov Yu.A.,
Kosmacheva E.D., Lopatin Yu.M., Markov V.A.,
Nikulina N.N., Pevzner D.V., Pogosova N.V., Pro-
topopov A.V., Skrypnik D.V., Tereshchenko S.N.,
Ustyugov S.A., Khripun A.V., Shalaev S.V., Shpe-
ktor A.V., Yakushin S.S. Acute coronary syndrome
without ST segment elevation electrocardiogram. Clinical guidelines 2020. Russ. J. Cardiol., 2021; 26
(4): 149–202. (In Russ). doi: 10.15829/1560-4071-
2021-4449 [Барбараш О.Л., Дупляков Д.В., Затей-
щиков Д.А., Шахнович Р.М., Явелов И.С., Яков-
лев А.Н., Абугов С.А., Алекян Б.Г., Архипов М.В.,
Васильева Е.Ю., Галявич А.С., Ганюков В.И.,
Гиляревский С.Р., Голубев Е.П., Голухова Е.З.,
Грацианский Н.А., Карпов Ю.А., Космачева Е.Д,,
Лопатин Ю.М., Марков В.А., Никулина Н.Н.,
Певзнер Д.В., Погосова Н.В., Протопопов А.В,,
Скрыпник Д.В., Терещенко С.Н., Устюгов С.А.,
Хрипун А.В., Шалаев С.В., Шпектор А.В., Яку- /j
p
5. Kozik
V.A.,
Shpagin
I.S.,
Zolotukhina
E.V.,
Panacheva L.A., Shpagina L.A. Molecular genetic
marcers of cardiovascular pathology in patients with a
new coronavirus infection COVID-19. Modern Probl. Sci. Educаt., 2023; 3: 98. (In Russ). doi: 10.17513/
spno.32563 [Козик В.А., Шпагин И.С., Золотухи-
на Е.В., Паначева Л.А., Шпагина Л.А. Молеку-
лярно-генетические маркеры кардиоваскулярной
патологии у пациентов, перенесших новую коро-
навирусную инфекцию COVID-19. Соврем. пробл. науки и образования, 2023; 3: 98. doi: 10.17513/
spno.32563]. 6. Заключение ВНП rs12329760 гена TMPRSS2 не ассоци-
ирован с ОКС у пациентов, перенесших НКИ. 4. 4. Yaghoobi A., Lord J.S., Rezaiezadeh J.S., Yekanine-
jad M.S., Amini M., Izadi P. TMPRSS2 polymor-
phism (rs12329760) and the severity of the COVID-19
in Iranian population. PLoS One, 2023; 18 (2):
e0281750. doi: 10.1371/journal.pone.0281750 Обсуждение ontgomery H., Openshaw P.J.M., Begg C., Walsh T., nesa A., Flores C., Riancho J.A., Rojas-Martinez A., Lapunzina P.; GenOMICC Investigators; SCOURGE
Consortium;
ISARICC
Investigators;
23andMe Vitart V., Abedalthagafi M., Luchessi A.D., Parra E.J., Cruz R., Carracedo A., Fawkes A., Murphy L.,
Rowan K., Pereira A.C., Law A., Fairfax B.,
Hendry S.C., Baillie J.K. GWAS and meta-analysis
identifies 49 genetic variants underlying critical
COVID-19. Nature. 2023; 617 (7962): 764–768. doi:
10.1038/s41586-023-06034-3 Обсуждение В украинской
популяции изучался ген-кандидат rs12329760
гена TMPRSS2 и его ассоциация с повышен-
ным риском развития НКИ, а также смертно-
стью [8]. Всего исследовали три группы боль-
ных: 62 человека без кислородной терапии,
92 с неинвазивной и 35 с инвазивной кисло- казали, что выживаемость носителей генотипа
GG была меньше по сравнению с больными с
другими генотипами (p = 0,009). При много-
факторном анализе генотип GG был фактором,
независимо связанным с риском смерти у по-
жилых людей (относительный риск 4,03, 95 %
ДИ 1,49–10,84). В исследовании иранских уче-
ных, включавшем 251 пациента с НКИ, пока-
зано, что аллель Т rs12329760 в гене TMPRSS2
представляет собой аллель риска тяжелой фор-
мы заболевания [4], в отличие от большинства 39 Атеросклероз. Т. 20. № 1. 2024 / Ateroscleroz. Vol. 20. N 1. 2024 родной терапией. Обращает на себя внимание
преобладание носительства гомозигот (СС и
ТТ) над гетерозиготой (СТ), что сопоставимо
с результатами нашего исследования. В работе
[9] доказана связь rs12329760 гена-кандидата
TMPRSS2 с повышением риска развития более
тяжелых форм НКИ: при исследовании 693
пациентов с положительным исходом болезни
(выздоровление) и 592 человек с негативным
исходом (смерть) выявлено, что генотип CC
rs12329760 TMPRSS2 связан с повышенным
уровнем смертности от COVID-19, т. е. пред-
ставляет собой полиморфизм, связанный со
значительно более высокой заболеваемостью и
тяжелым течением НКИ. родной терапией. Обращает на себя внимание
преобладание носительства гомозигот (СС и
ТТ) над гетерозиготой (СТ), что сопоставимо
с результатами нашего исследования. В работе
[9] доказана связь rs12329760 гена-кандидата
TMPRSS2 с повышением риска развития более
тяжелых форм НКИ: при исследовании 693
пациентов с положительным исходом болезни
(выздоровление) и 592 человек с негативным
исходом (смерть) выявлено, что генотип CC
rs12329760 TMPRSS2 связан с повышенным
уровнем смертности от COVID-19, т. е. пред-
ставляет собой полиморфизм, связанный со
значительно более высокой заболеваемостью и
тяжелым течением НКИ. шин С.С. Острый коронарный синдром без
подъема
сегмента
ST
электрокардиограммы. Клинические рекомендации 2020. Рос. кардиол. журн., 2021; 26, (4): 149–202. doi: 10.15829/1560-
4071-2021-4449]. шин С.С. Острый коронарный синдром без
подъема
сегмента
ST
электрокардиограммы. Клинические рекомендации 2020. Рос. кардиол. журн., 2021; 26, (4): 149–202. doi: 10.15829/1560-
4071-2021-4449]. родной терапией. Обращает на себя внимание
преобладание носительства гомозигот (СС и
ТТ) над гетерозиготой (СТ), что сопоставимо
с результатами нашего исследования. Обсуждение В работе
[9] доказана связь rs12329760 гена-кандидата
TMPRSS2 с повышением риска развития более
тяжелых форм НКИ: при исследовании 693
пациентов с положительным исходом болезни
(выздоровление) и 592 человек с негативным
исходом (смерть) выявлено, что генотип CC
rs12329760 TMPRSS2 связан с повышенным
уровнем смертности от COVID-19, т. е. пред-
ставляет собой полиморфизм, связанный со
значительно более высокой заболеваемостью и
тяжелым течением НКИ. 2. 2. Kozik V.A., Shpagin I.S., Shpagina L.A., Loktin E.M. Damage of the cardiovascular system in patients with
a new coronavirus infection: current state of the
problem. Modern Probl. Sci. Educt., 2023; 4: 135. (In
Russ). doi: 10.17513/spno.32651 [Козик В.А., Шпа-
гин И.С., Шпагина Л.А., Локтин Е.М. Пораже-
ние сердечно-сосудистой системы у пациентов,
перенесших новую коронавирусную инфекцию:
современное состояние проблемы. Соврем. пробл. науки и образования, 2023; 4: 135. doi: 10.17513/
spno.32651]. 3. 3. Pairo-Castineira E., Rawlik K., Bretherick A.D., Qi T.,
Wu Y., Nassiri I., McConkey G.A., Zechner M.,
Klaric L., Griffiths F., Oosthuyzen W., Kousathanas A.,
Richmond A., Millar J., Russell C.D., Malinauskas T.,
Thwaites R., Morrice K., Keating S., Maslove D.,
Nichol A., Semple M.G., Knight J., Shankar-Hari M.,
Summers C., Hinds C., Horby P., Ling L., McAuley D.,
Montgomery H., Openshaw P.J.M., Begg C., Walsh T.,
Tenesa A., Flores C., Riancho J.A., Rojas-Martinez A.,
Lapunzina P.; GenOMICC Investigators; SCOURGE
Consortium;
ISARICC
Investigators;
23andMe
COVID-19 Team; Yang J., Ponting C.P., Wilson J.F.,
Vitart V., Abedalthagafi M., Luchessi A.D., Parra E.J.,
Cruz R., Carracedo A., Fawkes A., Murphy L.,
Rowan K., Pereira A.C., Law A., Fairfax B.,
Hendry S.C., Baillie J.K. GWAS and meta-analysis
identifies 49 genetic variants underlying critical
COVID-19. Nature. 2023; 617 (7962): 764–768. doi:
10.1038/s41586-023-06034-3 Неоднородные результаты научных иссле-
дований еще раз доказывают, что влияние
на тяжесть развития заболеваний оказывает
не один полиморфизм гена, а множество. Существенно
ограничивает
интерпретацию
результатов тот факт, что в подавляющем
большинстве работ используется подход подбора
отдельных
генов-кандидатов. Возможно,
в
будущих исследованиях, где будут изучаться
полиморфизмы всего генома (GWAS) у боль-
шей выборки людей, разных мест проживания,
рас, возрастов, результаты будут более показа-
тельными. Неоднородные результаты научных иссле-
дований еще раз доказывают, что влияние
на тяжесть развития заболеваний оказывает
не один полиморфизм гена, а множество. Существенно
ограничивает
интерпретацию
результатов тот факт, что в подавляющем
большинстве работ используется подход подбора
отдельных
генов-кандидатов. Возможно,
в
будущих исследованиях, где будут изучаться
полиморфизмы всего генома (GWAS) у боль-
шей выборки людей, разных мест проживания,
рас, возрастов, результаты будут более показа-
тельными. Сведения об авторах: Валентина Александровна Козик, канд. мед. наук, ассистент кафедры госпитальной терапии и медицинской
реабилитации, Новосибирск, Россия, ORCID: 0000-0001-7128-7887, e-mail: valiyta90@mail.ru р
,
р
,
,
,
y
Любовь Анатольевна Шпагина, д-р мед. наук, проф., зав. кафедрой госпитальной терапии и медицинской
реабилитации, Новосибирск, Россия, ORCID: 0000-0003-3446-8018, e-mail: mkb-2@yandex.ru Любовь Анатольевна Шпагина, д-р мед. наук, проф., зав. кафедрой госпитальной терапии
реабилитации, Новосибирск, Россия, ORCID: 0000-0003-3446-8018, e-mail: mkb-2@yandex.ru Любовь Анатольевна Шпагина, д-р мед. наук, проф., зав. кафедрой госпитальной терапии и медицинской
реабилитации, Новосибирск, Россия, ORCID: 0000-0003-3446-8018, e-mail: mkb-2@yandex.ru Илья Семенович Шпагин, д-р мед. наук, проф. кафедры госпитальной терапии и медицинской реабилитации,
Новосибирск, Россия, ORCID: 0000-0002-3109-9811, e-mail: dr.ilya.shpagin@gmail.com Илья Семенович Шпагин, д-р мед. наук, проф. кафедры госпитальной терапии и медицинской реабилитации,
Новосибирск, Россия, ORCID: 0000-0002-3109-9811, e-mail: dr.ilya.shpagin@gmail.com ,
,
y
p g
g
димировна Максимова, ординатор кафедры медицинской генетики и биологии, младший научны
восибирск, Россия, ORCID: 0000-0002-2472-181X, e-mail: 99naruto@mail.ru р
,
,
,
y
p g
g
Софья Владимировна Максимова, ординатор кафедры медицинской генетики и биологии, м
сотрудник, Новосибирск, Россия, ORCID: 0000-0002-2472-181X, e-mail: 99naruto@mail.ru ру
р
Наталья Геннадьевна Ложкина, д-р мед. наук, проф., рук. группы «Клиническая и экспериментальная
кардиология», Новосибирск, Россия, ORCID: 0000-0002-4832-3197, e-mail: lozhkina.n@mail.ru Наталья Геннадьевна Ложкина, д-р мед. наук, проф., рук. группы «Клиническая и экспериментальная
кардиология», Новосибирск, Россия, ORCID: 0000-0002-4832-3197, e-mail: lozhkina.n@mail.ru Владимир Николаевич Максимов, д-р мед. наук, проф., зав. лабораторией молекулярно-генетических
исследований терапевтических заболеваний, проф. кафедры медицинской генетики и биологии, Новосибирск,
Россия, ORCID: 0000-0002-7165-4496, e-mail: medik11@mail.ru Список литературы/ References Andrade C.C., Silva A.T.P., Vasconcelos L.R.S.,
Oliveira
P.R.S.,
de
Souza
C.D.F.,
da
Costa
Armstrong A., do Carmo R.F. A Polymorphism in the
TMPRSS2 gene increases the risk of death in older
patients hospitalized with COVID-19. Viruses, 2022;
14 (11): 2557. doi: 10.3390/v14112557 (
)
/
7. Sekiya T., Ogura Y., Kai H., Kawaguchi A., Oka-
wa S., Hirohama M., Kuroki T., Morii W., Hara A.,
Hiramatsu Y., Hitomi S., Kawakami Y., Arakawa Y.,
Maruo K., Chiba S., Suzuki H., Kojima H., Tachika-
wa H., Yamagata K. TMPRSS2 gene polymorphism
common in East Asians confers decreased COVID-19 40 В.А. Козик, Л.А. Шпагина, И.С. Шпагин и др. Ukrainian population. Acta Biomed., 2023; 94 (1):
e2023030. doi: 10.23750/abm.v94i1.13543 Ukrainian population. Acta Biomed., 2023; 94 (1):
e2023030. doi: 10.23750/abm.v94i1.13543 /
8. Аidashev I., Izmailova O., Shlykova O., Kabaliei A.,
Vatsenko A., Ivashchenko D., Dudchenko M., Vo-
lianskyi A., Zelinskyy G., Koval T., Dittmer U. Poly-
morphism of tmprss2 (rs12329760) but not ace2
(rs4240157), tmprss11a (rs353163) and cd147 (rs8259)
is associated with the severity of COVID-19 in the /
9. Beheshti
Shirazi
S.S.,
Sakhaee
F.,
Sotoodeh-
nejadnematalahi F., Zamani M.S., Ahmadi I., An-
vari E., Fateh A. rs12329760 Polymorphism in
transmembrane serine protease 2 gene and risk of
coronavirus disease 2019 mortality. Biomed. Res. Int.,
2022; 2022: 7841969. doi: 10.1155/2022/7841969 Ukrainian population. Acta Biomed., 2023; 94 (1):
e2023030. doi: 10.23750/abm.v94i1.13543
9. Beheshti
Shirazi
S.S.,
Sakhaee
F.,
Sotoodeh-
nejadnematalahi F., Zamani M.S., Ahmadi I., An-
vari E., Fateh A. rs12329760 Polymorphism in
transmembrane serine protease 2 gene and risk of
coronavirus disease 2019 mortality. Biomed. Res. Int.,
2022; 2022: 7841969. doi: 10.1155/2022/7841969 Статья поступила 03.12.2023
После доработки 03.01.2024
Принята к печати 14.01.2024 susceptibility. Front. Microbiol., 2022; 13: 943877. doi:
10.3389/fmicb.2022.943877
8. Аidashev I., Izmailova O., Shlykova O., Kabaliei A.,
Vatsenko A., Ivashchenko D., Dudchenko M., Vo-
lianskyi A., Zelinskyy G., Koval T., Dittmer U. Poly-
morphism of tmprss2 (rs12329760) but not ace2
(rs4240157), tmprss11a (rs353163) and cd147 (rs8259)
is associated with the severity of COVID-19 in the susceptibility. Front. Microbiol., 2022; 13: 943877. doi:
10.3389/fmicb.2022.943877
8. Аidashev I., Izmailova O., Shlykova O., Kabaliei A.,
Vatsenko A., Ivashchenko D., Dudchenko M., Vo-
lianskyi A., Zelinskyy G., Koval T., Dittmer U. Poly-
morphism of tmprss2 (rs12329760) but not ace2
(rs4240157), tmprss11a (rs353163) and cd147 (rs8259)
is associated with the severity of COVID-19 in the
Ukrainian population. Acta Biomed., 20
e2023030. doi: 10.23750/abm.v94i1.13543
9. Beheshti
Shirazi
S.S.,
Sakhaee
F.,
nejadnematalahi F., Zamani M.S., Ahm
vari E., Fateh A. rs12329760 Polym
transmembrane serine protease 2 gene
coronavirus disease 2019 mortality. Biom
2022; 2022: 7841969. doi: 10.1155/2022/7 Ukrainian population. Acta Biomed., 2023; 94 (1):
e2023030. doi: 10.23750/abm.v94i1.13543 susceptibility. Front. Microbiol., 2022; 13: 943877. doi:
10.3389/fmicb.2022.943877 Information about the authors: Valentina A. Kozik, candidate of medical sciences, assistant at the department of hospital therapy and medical
rehabilitation, Novosibirsk, Russia, ORCID: 0000-0001-7128-7887, e-mail: valiyta90@mail.ru Lyubov A. Shpagina, doctor of medical sciences, professor, head of the department of hospital therapy and medical
rehabilitation, Novosibirsk, Russia, ORCID: 0000-0003-3446-8018, e-mail: mkb-2@yandex.ru pagin, doctor of medical sciences, professor of the department of hospital therapy and medic
Novosibirsk, Russia, ORCID: 0000-0002-3109-9811, e-mail: dr.ilya.shpagin@gmail.com Ilya S. Shpagin, doctor of medical sciences, professor of the department of hospital the
rehabilitation, Novosibirsk, Russia, ORCID: 0000-0002-3109-9811, e-mail: dr.ilya.shpagin@gmail.com Sofya V. Maksimova, resident of the department of medical genetics and biology, junior researcher, Novosibirsk,
Russia, ORCID: 0000-0002-2472-181X, e-mail: 99naruto@mail.ru Sofya V. Maksimova, resident of the department of medical genetics and biology, junior researcher, Novosibirsk,
Russia, ORCID: 0000-0002-2472-181X, e-mail: 99naruto@mail.ru Natalya G. Lozhkina, doctor of medical sciences, professor, head of the clinical and experimental cardiology group,
Novosibirsk, Russia, ORCID: 0000-0002-4832-3197, e-mail: lozhkina.n@mail.ru Vladimir N. Maksimov, doctor of medical sciences, professor, head laboratory of molecular genetic research of
therapeutic diseases, professor of the department of medical genetics and biology, Novosibirsk, Russia, ORCID: 0000-
0002-3157-7019, e-mail: medik11@mail.ru Received 03.12.2023
Revision received 03.01.2024
Accepted 14.01.2024 Статья поступила 03.12.2023
После доработки 03.01.2024
Принята к печати 14.01.2024 Received 03.12.2023
Revision received 03.01.2024
Accepted 14.01.2024 Статья поступила 03.12.2023
После доработки 03.01.2024
Принята к печати 14.01.2024 41
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https://revistas.ucm.es/index.php/CIYC/article/download/60692/4564456547542
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es
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"Teoría e historia de la propaganda" (2017), Adrián Huici Módenes, Madrid, Editorial Síntesis, 2017, pp. 278.
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Cuadernos de información y comunicación
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cc-by
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RESEÑAS
CIC. Cuadernos de Información y Comunicación
ISSN: 1135-7791
http://dx.doi.org/10.5209/CIYC.60692
Teoría e historia de la propaganda (2017), Adrián Huici Módenes, Madrid, Editorial
Síntesis, 2017, pp. 278.
Adrián Huici Módenes es profesor titular en la Facultad de Comunicación de la Universidad de Sevilla. Durante su carrera académica se ha especializado en el campo
de la comunicación pública y la propaganda, investigando en torno a la relación de
ésta última con el mito y la cultura popular. En su último libro nos presenta amplio
repaso por la teoría y el desarrollo histórico de los fenómenos propagandísticos,
desde sus primeros antecedentes en las culturas antiguas, hasta sus más modernas
expresiones en torno a las nuevas herramientas que nos ofrece internet.
A lo largo de los dos primeros capítulos, en los cuales se presenta la propaganda
en términos teóricos, el autor conecta sus posiciones con una serie de obras fundamentales tanto dentro de la academia española como en las bases de la doctrina en
términos científicos a principios del siglo XX. Algunas de las mencionadas en este
sentido son Alejandro Pizarroso, Mario Herreros, Maria V. Reyzábal, Juan Rey y
Antonio Pineda. Respecto de las principales contribuciones desde el ámbito internacional, el autor menciona a numerosos autores entre los que se encuentran pioneros
como Edward Bernays o Harold Lasswell y otros autores algo posteriores como
Jacques Ellul, Jean-Marie Domenach, Leonard Doob, Paul Watzlawick, Elliot Aronson y Anthony Pratkanis. Intercalando su saber con las aportaciones de éstos, el autor
expone en el segundo capítulo, el más extenso, el contenido propagandístico sustancial, enfatizando su naturaleza práctica. De ese modo se introduce la relación entre
ésta y otros modelos de enunciados persuasivos, subrayando la importancia de la
imagen como elemento fundacional de la misma y su relación con el símbolo y el
mito. De igual modo se presentan una serie de reglas básicas para la articulación de
discursos propagandísticos, tanto en un contexto de gestión de la opinión pública en
tiempo de paz como durante la guerra. Se pone fin al desarrollo de la teoría de la
propaganda teniendo en cuenta de que modos se articulan cada uno de los lenguajes
de los cuales esta se sirve y sus diferentes códigos y canales.
Resulta muy reseñable el esfuerzo llevado a cabo por el autor en su intento de
abarcar, aunque de una manera esquemática, la casi totalidad de la historia de Occidente. De ese modo, el autor nos invita en el capítulo tres a ver nuestra historia,
desde los sumerios hasta la protopropaganda de los imperios napoleónicos, a través
de los ojos del experto en propaganda. Éste último término, “protopropaganda”, es
utilizado como forma de conceptualizar todos aquellos elementos propagandísticos
propios de la política a lo largo de toda la historia previa al establecimiento de los
modernos medios de comunicación de masas: “…esto no significa que antes del
siglo XVIII no existieran formas persuasivas vinculadas con el poder y la ideología,
solo que carecían de los medios y de la sofisticación que aportan las nuevas tecnologías de la comunicación.” (p. 81) El extensísimo recorrido histórico al que se nos
invita puede inducirnos a ser conscientes de dos elementos problemáticos, que sin
CIC. Cuad. inf. com. 23, 2018: 235-238
235
236
Reseñas. CIC. Cuad. inf. comu. 23, 2018: 235-238
embargo no restan valor a la propuesta del autor. En primer lugar, debemos ser
conscientes de que no podemos abordar el capítulo tres ni como una historia propiamente dicha, ya que cada uno de los asuntos esquemáticamente tratados merecería
un abordaje mucho más extenso y detallado, ni como una historia de las ideas políticas. En segundo lugar, la obra parece en ocasiones conducirnos hacía una visión
de la política como mera canalización de mensajes propagandísticos. Ejemplos de
ello es la posibilidad de entender cualquier ámbito político como un terreno para la
expresión de la manipulación, desde la arquitectura monumental egipcia (p. 82) o la
propagación del modelo democrático hecho por los atenienses (p. 85) hasta el uso
de la guillotina por parte de los revolucionarios franceses (p. 113). Otro ejemplo se
encuentra en el tratamiento del nacionalismo, enfocado desde la perspectiva de las
Comunidades imaginadas de Anderson, y por tanto enfatizando su carácter contingente: “debemos situar a la propaganda en la génesis misma del nacionalismo”
(p. 126)
Uno de los puntos fuertes de la obra es la posibilidad de dar cuenta de algunos de
los elementos de los que se está nutriendo la propaganda en nuestros días. El libro
trata por ello de tener en cuenta, aunque de una manera provisoria, algunos fenómenos experimentados en los últimos cinco o diez años entre los que podemos citar la
irrupción de las redes sociales como un nuevo agente dentro de la comunicación
política o la maquinaria propagandística de la que se sirve el ISIS. Resulta evidente
que respecto de los fenómenos novedosos la obra no puede profundizar, de tal modo
que sólo el futuro nos dirá si las intuiciones plasmadas por el autor se cumplen o no.
En cualquier caso, algunas de éstas podrían ser matizadas: “…YouTube como un
nuevo ágora político, que en cierto sentido, superaba a la idealizada democracia ateniense.” (p. 270) También resulta adecuado señalar cómo la importancia de la imagen dentro de las técnicas propagandísticas, lo cual es enfatizado en diversas ocasiones, encuentra su paralelismo con la importancia que dentro del recorrido
argumentativo del libro tienen las diversas ilustraciones que acompañan cada una de
los puntos elaborados. De este modo podemos encontrar imágenes que da cuenta
desde el poder simbólico de los cuadros de Napoleón a la relevancia del debate televisado entre Nixon y Kennedy en el año 1960.
Por otro lado, la obra contiene un evidente tono crítico hacia la propaganda
como técnica de condicionamiento social. Ciertamente, como es de esperar en alguien que se ha especializado en este campo, escapa de las visiones maniqueas que
se refieren a la propaganda como un elemento propio de los regímenes totalitarios
en tanto que se define por el uso de la desinformación, manipulación y mentira.
Frente a esto, el autor nos advierte desde un inicio en que los regímenes democráticos hacen un uso constante de la propaganda ya que ésta es indisociable de la
competición política, de tal modo que, si bien por medio de eufemismos como
“marketing político, publicidad política, marketing electoral o relaciones públicas…la propaganda está tan vigente hoy como en os oscuros tiempos de Goebbels
o Stalin…” (12). Este fragmento nos permite de hecho dar cuenta de que, si bien el
autor es crítico con la relación entre democracia y propaganda, el verdadero blanco
de sus críticas parecen ser los nefastos usos de la propaganda por parte de modelos
políticos monstruosos surgidos en el periodo de entreguerras. La propaganda se
convierte así en un elemento técnico al servicio de regímenes políticos buenos y
malos. Si los malos son los totalitarios, en especial el nazismo y el comunismo, los
cuáles parecen juzgarse en los mismos términos, con una continua apelación a las
Reseñas. CIC. Cuad. inf. comu. 23, 2018: 235-238
237
similitudes entre Hitler y Stalin, los buenos, aunque con matices, son las democracias liberales del mundo occidental. De este modo el libro se posiciona claramente
a favor de una opción, simplificando ciertos elementos, lo cual le da precisamente
un leve tiente propagandístico a su defensa de los valores hegemónicos (“En este
caso, el proceso de demonización parece un tanto gratuito, puesto que el carácter
intrínsecamente demoniaco de Hitler no necesitaba de mayores explicaciones: el
hombre se retrataba solo”, p. 51. “…procuraban identificar la figura poco agraciada de Franco con el mismísimo Campeador, algo que hoy resulta irrisorio, tanto
como los increíbles calificativos que la literatura del régimen prodigaba a Stalin:
los extremos siempre acaban tocándose.”, p. 59. “Afortunadamente, existen ejemplos en positivo y, sin duda, el más espectacular es el caso del rugby en Sudáfrica.
Recordemos que Nelson Mandela utilizó…” p. 70. “Naturalmente, cuando Lenin
dice “educación” o “ideología”, en lo que está pensando es en la propaganda.”
p. 164.)
Por tanto, el desarrollo argumentativo de la obra nos induce a planear lo siguiente: si aceptamos, tal y como se sostiene en la obra, que habitualmente se pretende
convencer de que propaganda es “lo de los otros”, mientras que “nosotros propagamos la verdad”, ¿por qué el autor, que debiera enfatizar su visión crítica del fenómeno, acaba limitándose a considerar que existen propagandas “buenas” y propagandas
“malas”? En este sentido parece que se refiere implícita y frecuentemente a propagandas que se justificarían por sus beneficiosos resultados y otras que, tal y como
podemos observar desde el prisma histórico, no solo no podemos justificar sino que
debemos contrarrestar en tanto que partícipes de un proyecto aberrante. A pesar de
esto, incluso en algunos fragmentos el autor parece inclinarse finalmente al argumento central de aquellos que “luchan contra la propaganda en favor de la democracia”, sosteniendo, a colación de la habitual frivolidad con la que se hace uso del
marketing político y la campañas negativas, que “todo ello, sin duda, va en desmedro
de la calidad de los mensajes, que no pueden profundizar sobre ningún tema, y de la
democracia misma, cuyo combustible fundamental es la información amplia y de
calidad” (p. 265)
Frente a los ejemplos anteriores, en los cuales el autor parece seguir a pies juntillas la ortodoxia cultural occidental, resulta interesante el modo en que reivindica
una lectura crítica en torno al modo en que el cristianismo se ha comportado históricamente, tratando a su vez de señalar cómo el islam ha sido y sigue siendo atacado
desde unos planteamientos que no se aplican en el caso de los seguidores de Jesucristo. En este sentido resulta muy reseñable el modo en que argumenta en torno al modo
en que el aparato mediático estadounidense y europeo se ha esforzado sistemáticamente, tras la caída del muro de Berlín, por construir un nuevo monstruo, en este
caso el fanático islamista. En esta empresa el imperialismo cultural estadounidense,
encabezado por la industria cinematográfica, ha sido capaz de construir una imagen
del musulmán a través de películas citadas como Aladín, Mentiras arriesgadas o
Delta Force, de las cuales se desprende el estereotipo del “hombre barbudo, con un
turbante, un cinturón de explosivos y un Corán y un fusil de asalto en cada
mano.”(p. 273)
A modo de valoración general podemos considerar que la obra nos ofrece una
interesante recopilación de un material amplísimo tanto en términos teóricos como
históricos. Por tanto, podríamos considerarlo como un buen punto de partida para
llevar a cabo una investigación sistemática, teniendo en cuenta que introduce gran
238
Reseñas. CIC. Cuad. inf. comu. 23, 2018: 235-238
cantidad de temas y da cuenta de muchos de los más significativos estudiosos en
torno a la propaganda.
Miguel Fernández de la Peña
Universitat de Barcelona
miguelmixel@hotmail.com
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https://openalex.org/W4311505148
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https://www.frontiersin.org/articles/10.3389/fragi.2022.1063760/pdf
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English
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Living alone reduces the decline of calf circumference among Chinese older adults: A 4-year longitudinal study
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Frontiers in aging
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cc-by
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TYPE Original Research
PUBLISHED 15 December 2022
DOI 10.3389/fragi.2022.1063760 TYPE Original Research
PUBLISHED 15 December 2022
DOI 10.3389/fragi.2022.1063760 TYPE Original Research
PUBLISHED 15 December 2022
DOI 10.3389/fragi.2022.1063760 OPEN ACCESS OPEN ACCESS
EDITED BY
Alan Bruno Silva Vasconcelos,
University Center—AGES, Brazil
REVIEWED BY
Marcos Raphael Pereira Monteiro,
Federal University of Sergipe, Brazil
Leandro Henrique Albuquerque
Brandão,
Federal University of Minas Gerais, Brazil
Xiaolei Liu,
Sichuan University, China
*CORRESPONDENCE
Jun Zhang,
who626zj@126.com
SPECIALTY SECTION
This article was submitted to
Musculoskeletal Aging,
a section of the journal
Frontiers in Aging
RECEIVED 07 October 2022
ACCEPTED 06 December 2022
PUBLISHED 15 December 2022
CITATION
Wang D and Zhang J (2022), Living
alone reduces the decline of calf
circumference among Chinese older
adults: A 4-year longitudinal study. Front. Aging 3:1063760. doi: 10.3389/fragi.2022.1063760 OPEN ACCESS
EDITED BY
Alan Bruno Silva Vasconcelos,
University Center—AGES, Brazil
REVIEWED BY
Marcos Raphael Pereira Monteiro,
Federal University of Sergipe, Brazil
Leandro Henrique Albuquerque
Brandão,
Federal University of Minas Gerais, Brazil
Xiaolei Liu,
Sichuan University, China
*CORRESPONDENCE
Jun Zhang,
who626zj@126.com
SPECIALTY SECTION
This article was submitted to
Musculoskeletal Aging,
a section of the journal
Frontiers in Aging
RECEIVED 07 October 2022
ACCEPTED 06 December 2022
PUBLISHED 15 December 2022
CITATION
Wang D and Zhang J (2022), Living
alone reduces the decline of calf
circumference among Chinese older
adults: A 4-year longitudinal study. Front. Aging 3:1063760. doi: 10.3389/fragi.2022.1063760 Dong Wang and Jun Zhang*
Department of Neurology, Peking University People’s Hospital, Beijing, China Dong Wang and Jun Zhang* Department of Neurology, Peking University People’s Hospital, Beijing, China Background: Calf circumference (CC) is regarded as a surrogate marker of
skeletal muscle mass with high sensitivity and specificity for predicting
sarcopenia. A cross-sectional study reported older adults living alone were
at high risk of developing sarcopenia. Whether living alone affects the change of
calf circumference is unknown and there is no evidence from longitudinal
study. The purpose of this study was to investigate the relationship between
living arrangements and the change of calf circumferences among older adults
in China. CITATION
Wang D and Zhang J (2022), Living
alone reduces the decline of calf
circumference among Chinese older
adults: A 4-year longitudinal study. Front. Aging 3:1063760. doi: 10.3389/fragi.2022.1063760 Methods: The data were from the Chinese Longitudinal Healthy Longevity
Survey. A total of 2,203 older adults (age ≥65 years, mean age: 80.61 ±
8.30 years,
50.0%
female)
who
were
interviewed
in
2014
and
then
2018 follow-up survey were finally included for analysis. Living arrangements
and other information were collected in 2014. living alone, calf circumference, older adults, sarcopenia, longitudinal study 1 Introduction FIGURE 1
Participant flow in this study. Sarcopenia is defined as age-related loss of skeletal muscle
mass, accompanied by decline in muscle strength and/or reduced
physical function (Chen et al., 2020). Sarcopenia increases the
risks of falls, fractures, disability, and mortality among older
adults. Globally, the prevalence of sarcopenia in adults over the
age of 60 is 10% (Shafiee et al., 2017), and the prevalence of
sarcopenia among older adults over the age of 80 is about 50%
(Meng et al., 2014). With an increasingly ageing population,
sarcopenia has become a serious public health issue in modern
society. Skeletal muscle mass is one of the core dimensions for
assessing sarcopenia. Calf circumference (CC) can be used as
a surrogate marker of skeletal muscle mass, which has high
sensitivity and specificity for predicting sarcopenia (Kawakami
et al., 2015). Measures of CC may be used as a diagnostic proxy in
settings where no other muscle mass diagnostic methods are
available, and CC < 34 cm for men and <33 cm for women for
screening sarcopenia is recommended in the Asian Working
Group for Sarcopenia (AWGS) 2019 consensus (Cruz-Jentoft
et al., 2019; Chen et al., 2020). In addition, CC is also an
important variable in predictive models of other disorders,
such as cardiovascular disease (Wu et al., 2018). The CC
decline is often regarded as a risk factor affecting health,
especially among older adults. Therefore, it is of great
significance to explore the factors influencing the change of
the CCs among older adults. cross-sectional studies suggested that living alone was a risk
factor for frailty in older adults, especially in men (Yamanashi
et al., 2015; Kojima et al., 2020); however, meta-analysis of cohort
studies did not yield statistically significant results (OR = 0.88, 95
%CI = 0.76–1.03) (Kojima et al., 2020). As for sarcopenia,
previous cross-sectional study suggested that older adults
living alone are at higher risk for sarcopenia (Cheng et al.,
2021), while evidence from cohort studies is still lacking. CC
decline among older adults means reduction of skeletal muscle
mass and increased risk of developing sarcopenia. There is no
report about the associations of some social factors with CC
decline among older adults. OPEN ACCESS Calf circumferences were
measured and recorded in the questionnaires of two waves and the
differences were calculated. Logistic regression analyses were conducted to
evaluate the association of living arrangements (living alone or not living alone)
with the change of calf circumferences (decline or no decline). COPYRIGHT
© 2022 Wang and Zhang. This is an
open-access article distributed under
the terms of the Creative Commons
Attribution License (CC BY). The use,
distribution or reproduction in other
forums is permitted, provided the
original author(s) and the copyright
owner(s) are credited and that the
original publication in this journal is
cited, in accordance with accepted
academic practice. No use, distribution
or reproduction is permitted which does
not comply with these terms. Results: There were 446 (20.2%) participants living alone and 1,757 (79.8%)
participants not living alone. After about 4 years, calf circumferences of 866
(39.3%) older adults declined. Compared to not living alone, living alone was
negatively associated with calf circumference decline, even after adjustment for
covariates (OR: 0.702, 95% CI: 0.547–0.901, p = 0.005). This significant
association was found in 65–79 years old (OR: 0.619, 95% CI: 0.407–0.942,
p = 0.025), female (OR: 0.567, 95% CI: 0.408–0.790, p = 0.001) and city/town
(OR: 0.461, 95% CI: 0.310–0.685, p < 0.001) subgroups but not in advanced age
(≥80 years old), male and rural subgroups. Conclusion: This study revealed that living alone was associated with a
decreased risk of calf circumference decline among older adults, and might
be a protective factor for sarcopenia. KEYWORDS living alone, calf circumference, older adults, sarcopenia, longitudinal study Frontiers in Aging 01 frontiersin.org Wang and Zhang 10.3389/fragi.2022.1063760 1 Introduction Therefore, we used the cohort data
from the Chinese Longitudinal Healthy Longevity Survey
(CLHLS)
to
explore
the
relationship
between
living
arrangements and the change of CC among older adults, and
analyze the effect of living alone on the changes in skeletal muscle
mass and the development of sarcopenia. Living arrangement is an important observational variable in
many older adults-related studies about mental health, cognitive
impairment and other diseases (Beghi et al., 2021; Rosenwohl-
Mack et al., 2021). Living arrangements are influenced by
regional culture and the economic development of different
times. Under the influence of Confucianism, the traditional
Chinese
family
is
of
large
size
and
multigenerational. However, in the new era, the influence of the traditional
family concept has declined. Due to the needs of work and
the development of urbanization, many young people live far
away from their parents, and the traditional family model is
gradually deconstructed (Phillips and Feng, 2015). Among older
adults, the phenomenon of living alone due to the death of one’s
spouses is becoming more and more common. With an
increasingly aging population, the older adults living alone has
received more and more attention from the society. 2.3 Living arrangements Living arrangements were assessed by the item “co-residence
of interviewee” in the questionnaire. The options were “with
household member(s),” “alone,” and “in an institution,” with
1,735 (78.8%), 446 (20.2%), 22 (1.0%) respondents respectively. The living arrangements were dichotomized as living alone (LA)
and
not
living
alone
[NLA,
including
“with
household
member(s)” and “in an institution”] for analysis. 2.2 Assessment of CC In this survey, CCs were recorded and rounded up to the
nearest integer in centimeters. The CC difference was defined as
CC at 2018 wave minus CC at 2014 wave. Given the possible
measurement errors and the round-off recording principles, the
decline of CC in this survey was defined as CC difference <−1 cm,
and CC difference ≥−1 cm means no decline. Finally, there were
866 (39.3%) subjects who had declined CCs. 2.1 Study population A modified Mini Mental State Examination
(mMMSE) score was calculated by 23 items in original
questionnaire to assess the cognitive function. A detailed
introduction to this mMMSE has been published elsewhere
(Guo
et
al.,
2022). Every
interviewee
was
asked
about
diagnoses
(hypertension,
diabetes,
heart
disease,
stroke,
respiratory diseases, tuberculosis, cancer, Parkinson’s disease,
arthritis) by hospital. to answer questions, the interview was finished by a proxy
interviewer, often a spouse or other close relative. At first
wave, 7,192 respondents were interviewed in 2014, out of
which 1,525 respondents were lost to follow-up at second
wave in the 2018 survey and 2,226 died before 2018. Subjects
under the age of 65 were excluded. We checked and cleaned the
missing data and registration errors of important variables. Finally, 2,203 cases were included for analysis (Figure 1). 2.5 Statistical analysis IBM SPSS statistics 26.0 (IBM Corporation, Armonk, NY,
United States) was used for statistical analysis. The mean,
standard deviation, median, and ratio were calculated for
statistical descriptions according to different types of variables. When data was normally distributed, the differences of
continuous
variables
between
LA
and
NLA
group
at
2014 wave were determined by the independent-sample t test,
and the homogeneity of variance was tested by Levene’s test. The
Mann-Whitney U test was used when data was not normally
distributed. The statistical analyses of categorical variables were
performed by Pearson χ2 test. Both univariate and multivariate
logistic regression analyses were used to examine the associations
of living arrangements with CC difference. Odds ratios (ORs)
and 95% confidence intervals (CIs) were calculated. The
statistical significance was defined as p < .05, and all were
two-tailed tests. 2.4 Covariates The sociodemographic variables including age, sex, weight
(kg), height (cm), education (uneducated or educated), marital
status, residence (rural or city/town) and financial support
(sufficient or insufficient) were selected from the dataset. The
options of current marital status were “currently married and
living with spouse,” “married but not living with spouse,”
“divorced,”
“widowed,” and
“never
married.” They
were
dichotomized as “separated/divorced/widowed/single” (SDWS)
or not. Body mass index (BMI) was calculated as weight in
kilograms divided by the square of height in meters. The sleep
quality was categorized as “very good,” “good,” “so,” “bad,” and
“very bad” in primitive questionnaire and was dichotomized as
bad and not bad for analysis. Sleep duration were recorded in this
survey as short (<5 h), medium (5–9 h) and long (>9 h)
according to the recommendation (Hirshkowitz et al., 2015). For smoking and drinking, respondents were categorized as
current, ex-smoker/drinker, or never. By asking questions:
“Exercise or not at present?” and “Have you done physical
labor regularly?,” data about exercise/physical labor or not 2.1 Study population The physical and mental health of older adults living alone
may be affected when the level of support from family or society
changes (Lou and Ng, 2012). It is generally believed that lack of
family companionship is harmful to the physical and mental
health, but the effects of living alone on the health of older adults
are currently inconsistent in different literatures (Holt-Lunstad
et al., 2015; Zhou et al., 2018; Gu et al., 2019). Both sarcopenia
and frailty are age-related syndromes with some overlap in
clinical manifestations (Roberts et al., 2021). Meta-analysis of The data were from CLHLS 2014–2018 longitudinal dataset,
which is a nationally representative study of Chinese older adults
and covers 23 out of 31 provinces in Mainland China. The
CLHLS study was approved by the Research Ethics Committee of
Peking University (IRB00001052-13074). The interview was
conducted at participants’ homes by well-trained investigators
using a structured questionnaire. When a participant was unable Frontiers in Aging frontiersin.org 02 Wang and Zhang 10.3389/fragi.2022.1063760 10.3389/fragi.2022.1063760 were recorded. Depression was measured using two questions:
“Have you felt sad, blue, or depressed for 2 weeks or more in last
12 months?” and “Have you lost interest in most things like
hobbies, work, or similar activities?.” Answer of “yes” to any
question is considered a representation of depression (Su et al.,
2021). Activities of daily living (ADL) disability was defined as
needs for support in one or more of the five activities (bathing,
dressing, toileting, indoor transferring, and feeding) or being
incontinent. A modified Mini Mental State Examination
(mMMSE) score was calculated by 23 items in original
questionnaire to assess the cognitive function. A detailed
introduction to this mMMSE has been published elsewhere
(Guo
et
al.,
2022). Every
interviewee
was
asked
about
diagnoses
(hypertension,
diabetes,
heart
disease,
stroke,
respiratory diseases, tuberculosis, cancer, Parkinson’s disease,
arthritis) by hospital. were recorded. Depression was measured using two questions:
“Have you felt sad, blue, or depressed for 2 weeks or more in last
12 months?” and “Have you lost interest in most things like
hobbies, work, or similar activities?.” Answer of “yes” to any
question is considered a representation of depression (Su et al.,
2021). Activities of daily living (ADL) disability was defined as
needs for support in one or more of the five activities (bathing,
dressing, toileting, indoor transferring, and feeding) or being
incontinent. 3 Results At 2014 wave, there were 446 (20.2%) participants living alone
and 1,757 (79.8%) participants not living alone. Table 1 presents the
baseline characteristics by two types of living arrangements. No
significant differences were found among BMI, sleep quality,
exercise,
physical
labor,
depression,
ADL
disability,
and
comorbidities. Compared to subjects not living alone, older,
women, uneducated, SDWS, rural, insufficient financial support,
short-sleeper, non-smoker, non-drinker, and lower mMMSE scorer
were more common in subjects living alone. The outcomes of logistic regression models are illustrated in
Table 2. Compared to NLA group, the OR of the LA group for
crude model was 0.728 (95% CI: 0.585–0.905, p = .004). After
adjustments for covariates, the results were also statistically Frontiers in Aging frontiersin.org 03 10.3389/fragi.2022.1063760 Wang and Zhang TABLE 1 Baseline characteristics by living arrangements at 2014 wave. TABLE 1 Baseline characteristics by living arrangements at 2014 wave. DWS, separated/divorced/widowed/single; ADL, activities of daily living; mMMSE, modified Mini Mental state examination. *median and 25th–75th percentile.
tistical difference. 3 Results Living alone (n = 446)
Not living alone (n = 1,757)
p
Age (year)
82.19 ± 7.77
80.21 ± 8.38
<.001
Advanced age (≥80 years), n (%)
267 (59.9%)
809 (46.0%)
<.001
Female, n (%)
267 (59.9%)
834 (47.5%)
<.001
BMI (kg/m2)
22.37 ± 3.81
22.54 ± 3.98
.410
Uneducated, n (%)
277 (62.1%)
786 (44.7%)
<.001
Marital status (SDWS), n (%)
422 (94.6%)
656 (37.3%)
<.001
Residence (Rural), n (%)
264 (59.2%)
939 (53.4%)
.029
Financial support (insufficient), n (%)
96 (21.5%)
269 (15.3%)
.002
Sleep quality (Bad), n (%)
52 (11.7%)
201 (11.4%)
.897
Sleep duration
.024
Short, n (%)
46 (10.3%)
115 (6.5%)
Long, n (%)
72 (16.1%)
290 (16.5%)
Medium, n (%)
328 (73.5%)
1,352 (76.9%)
Smoking
.020
Current, n (%)
78 (17.5%)
349 (19.9%)
Ex-smoker, n (%)
42 (9.4%)
237 (13.5%)
Never, n (%)
326 (73.1%)
1,171 (66.6%)
Drinking
.034
Current, n (%)
78 (17.5%)
346 (17.9%)
Ex-drinker, n (%)
33 (7.4%)
191 (10.9%)
Never, n (%)
335 (75.1%)
1,220 (69.4%)
Exercise, n (%)
145 (32.5%)
619 (35.2%)
.281
Physical labor, n (%)
382 (85.7%)
1,481 (84.3%)
.478
Depression, n (%)
50 (11.2%)
195 (11.1%)
.946
ADL disability, n (%)
27 (6.1%)
133 (7.6%)
.271
mMMSE
22.00 (19.00–22.25) p
22 (20.00–23.00) p
.006
Disease
Hypertension, n (%)
184 (41.3%)
730 (41.5%)
.911
Diabetes, n (%)
75 (16.8%)
282 (16.1%)
.695
Heart disease, n (%)
98 (22.0%)
348 (19.8%)
.309
Stroke, n (%)
67 (15.0%)
257 (14.6%)
.833
Respiratory diseases, n (%)
66 (14.8%)
281 (16.0%)
.536
Tuberculosis, n (%)
47 (10.5%)
135 (7.7%)
.051
Cancer, n (%)
23 (5.2%)
114 (6.5%)
.298
Parkinson’s disease, n (%)
22 (4.9%)
92 (5.2%)
.796
Arthritis, n (%)
60 (13.5%)
235 (13.4%)
.966
BMI, body mass index; SDWS, separated/divorced/widowed/single; ADL, activities of daily living; mMMSE, modified Mini Mental state examination. *median and 25th–75th percentile. Frontiers in Aging 04 frontiersin.org 10.3389/fragi.2022.1063760 Wang and Zhang significant. Living alone was independently and negatively
associated with CC decline in adjusted model 2: the adjusted
OR of LA group was 0.702 (95% CI: 0.547–0.901, p = .005). In
adjusted model 2, advanced age (≥80 years) and stroke were two
covariates that were significantly associated with CC decline
(Table 2). TABLE 2 Associations between CC decline and living arrangements by logistic regression models. 3 Results Crude model
Adjusted model 1
Adjusted model 2
OR (95% CI)
p
OR (95% CI)
p
OR (95% CI)
p
Living alone
0.728 (0.585–0.905)
0.004
0.711 (0.570–0.887)
0.002
0.702 (0.547–0.901)
0.005
Age (≥80 years)
1.246 (1.048–1.483)
0.013
1.230 (1.013–1.494)
0.037
Sex (female)
0.938 (0.789–1.116)
0.471
0.869 (0.693–1.091)
0.226
Uneducated
0.893 (0.727–1.098)
0.283
Marital status (SDWS)
1.096 (0.883–1.362)
0.405
Residence (Rural)
0.992 (0.826–1.191)
0.928
Financial support (insufficient)
1.026 (0.806–1.306)
0.835
Sleep quality (bad)
1.097 (0.798–1.506)
0.570
Sleep duration
Short
0.719 (0.485–1.068)
0.102
Long
1.046 (0.824–1.328)
0.710
Medium
Ref. Smoking
Current, n (%)
0.832 (0.641–1.079)
0.165
Ex-smoker, n (%)
0.873 (0.643–1.184)
0.383
Never, n (%)
Ref. Drinking
Current
0.983 (0.767–1.261)
0.894
Ex-drinker
0.941 (0.682–1.298)
0.710
Never
Ref. Exercise
0.969 (0.799–1.177)
0.753
Physical labor
0.860 (0.673–1.099)
0.228
Depression
0.829 (0.621–1.106)
0.202
ADL disability
1.003 (0.713–1.412)
0.985
mMMSE
0.992 (0.970–1.015)
0.490
Hypertension
1.122 (0.918–1.372)
0.261
Diabetes
0.847 (0.608–1.179)
0.324
Heart disease
1.126 (0.852–1.487)
0.405
Stroke
1.486 (1.085–2.035)
0.014
Respiratory diseases
1.122 (0.840–1.498)
0.435
tuberculosis
0.607 (0.342–1.079)
0.089
Cancer
1.050 (0.527–2.093)
0.889
Parkinson’s disease
1.517 (0.710–3.241)
0.281
Arthritis
0.812 (0.588–1.121)
0.206
OR, odds ratio; CI, confidence interval; SDWS, separated/divorced/widowed/single; ADL, activities of daily living; mMMSE, modified Mini Mental state examination. Bold values represent statistical difference. TABLE 2 Associations between CC decline and living arrangements by logistic regression models. adjusted model 2, advanced age (≥80 years) and stroke were two
covariates that were significantly associated with CC decline
(Table 2). adjusted model 2, advanced age (≥80 years) and stroke were two
covariates that were significantly associated with CC decline
(Table 2). significant. Living alone was independently and negatively
associated with CC decline in adjusted model 2: the adjusted
OR of LA group was 0.702 (95% CI: 0.547–0.901, p = .005). In significant. Living alone was independently and negatively
associated with CC decline in adjusted model 2: the adjusted
OR of LA group was 0.702 (95% CI: 0.547–0.901, p = .005). In 05 Frontiers in Aging 05 frontiersin.org 10.3389/fragi.2022.1063760 Wang and Zhang TABLE 3 Associations between CC decline and LA by subgroup analysis. Table 3 Table 3 shows the results of subgroup analysis. LA was
significantly associated with CC decline in 65–79 years old,
female and city/town subgroups: the adjusted ORs of LA
group were 0.619 (95% CI: 0.407–0.942, p = .025), 0.567 (95%
CI: 0.408–0.790, p = .001) and 0.461 (95% CI: 0.310–0.685, p <
.001) respectively. LA was not significantly associated with CC
decline in advanced age (≥80 years old), male and rural
subgroups. Gu et al. presented a theoretical framework of reciprocal
causality between LA and health. That is, the solitary living
arrangement needs to be conceptualized as the cause of
subsequent health outcomes as well as the outcome of the
prior health status (Gu et al., 2019). Health status of older
adults affected their preference for living arrangements and
both physical and mental health conditions play a role in the
transitions of living arrangements (Brown et al., 2002). A
disabled
older
adult
who
loses
the
capability
to
live
independently would be more inclined to live with others. In
addition, older adults living alone need to cope with more jobs of
daily life by themselves, helping them get rid of their bad
sedentary habits; and a better cognitive or physical function is
needed to support them in handling the daily jobs. Physical
activity, especially resistance exercise, which can improve skeletal
muscle mass and strength, is the primary method for sarcopenia
prevention and treatment (Cruz-Jentoft and Sayer, 2019). However,
the
BMI,
exercise
and
physical
labor,
ADL
disability,
depression
status,
and
comorbidities
were
all
comparable at baseline and didn’t change the result in the
adjusted model. And the mMMSE score was lower in the LA
group probably due to lower education level. It suggests that the
reciprocal causality alone cannot explain the findings. 3 Results y
g
p
y
Crude model
Adjusted model
OR (95% CI)
p
OR (95% CI)
p
65–79 years old
0.628 (0.443–0.891)
0.009
0.619 (0.407–0.942)
0.025
≥80 years old
0.764 (0.575–1.016)
0.065
0.760 (0.553–1.043)
0.090
Female
0.556 (0.412–0.750)
<0.001
0.567 (0.408–0.790)
0.001
Male
1.034 (0.746–1.432)
0.841
0.960 (0.646–1.428)
0.841
Rural
0.875 (0.659–1.317)
0.354
0.943 (0.676–1.317)
0.731
City/town
0.560 (0.396–0.794)
0.001
0.461 (0.310–0.685)
<0.001
Adjusted model: adjustment for age, sex, education, marital status, residence, financial, sleep quality, sleep duration, smoking, drinking, exercise, physical labor, depression, ADL disability,
mMMSE and comorbidities, the grouping variable were excluded in each subgroup analysis. Bold values represent statistical difference. Adjusted model: adjustment for age, sex, education, marital status, residence, financial, sleep quality, sleep duration, smoking, drinking, exercise, physical labor, depression, ADL disability,
mMMSE and comorbidities, the grouping variable were excluded in each subgroup analysis. Bold values represent statistical difference. 2020). In addition, LA was often accompanied by decreased
family and social support and social isolation for older adults
(Lou and Ng, 2012; Teerawichitchainan et al., 2015), and thus
modified the associations of loneliness with adverse health
outcomes in previous study (Wei et al., 2022). Maybe less
tobacco use could be protective (Locquet et al., 2021). However, the results were sustained in multivariate logistic
analyses adjusted for these covariates. There must be some
other mediators that make LA protective for CC decline. 4 Discussion To the best of our knowledge, this is the first longitudinal
study to evaluate the association of living arrangements with CC
decline among older adults. Both univariate and multivariate
logistic regression results of our study showed that LA was a
protective factor for CC decline, which might help preserve
skeletal muscle mass and reduce the risk of sarcopenia. The
results of cross-sectional and longitudinal study about living
arrangements and frailty were different (Woods et al., 2005;
Yamanashi et al., 2015), and the result of our longitudinal study
was also inconsistent with the result of cross-sectional study
(Cheng et al., 2021). The association of LA with CC decline seems
to be specific for older adults in 65–79 years old, female and city/
town subgroups when conducting the subgroup analysis. The reason why LA was protective was not clear yet. In our
results, the age structure, gender ratio, education level, marital
status, urban-rural distribution, economic status, sleep duration,
smoking and drinking status, and mMMSE score were different
between LA and NLA groups; older, women, uneducated, SDWS,
rural, insufficient financial support, short-sleeper, non-smoker,
non-drinker, and lower mMMSE scorer were more common in
subjects living alone. These variables with statistical difference
above, such as lower economic status and educational level in LA
group, didn’t seem to be beneficial for skeletal muscle mass
maintenance and sarcopenia prevention (Brennan-Olsen et al., LA does not always have adverse effects on physical and
mental health, depending on the different context (Yeung and
Cheung, 2015). Previous studies suggested that LA in older adults
was not always a risk factor for health, and adverse health Frontiers in Aging frontiersin.org 06 Wang and Zhang 10.3389/fragi.2022.1063760 outcomes among older adults living alone might be confounded
by poor social network (Sakurai et al., 2019). Women live longer
than men and are more likely to be widowed. Compared to men,
women tend to have larger social networks (Cornwell, 2011),
which may explain the protective effect specific in women in our
result. Another advantage of LA is that older adults who live
alone can be relieved of family obligations and have more free
time (Eshbaugh, 2008; Gu et al., 2019), which may offset the
adverse effects of LA for physical and mental health. Actually,
other study also found that women living alone have better
psychologic function or lower frailty risk than women living
with a spouse (Michael et al., 2001; Trevisan et al., 2016). 4 Discussion Compared to very old adults over the age of 80 or rural
residents, people aged 65 to 79 or urban residents in China
have more energy or better conditions to do the things they love
or to be socially active (Liu et al., 2019). This may explain the
protective effect specific for older adults in 65–79 years old and
city/town subgroups in our result. It reminds us that the public
health policy should be specific for different area groups,
different
age
groups,
and
different
gender
groups. The
implementation of the newly proposed rural revitalization
strategy of China will bridge the urban-rural gap and might
reduce the risk of sarcopenia among rural older adults. And
future research needs better quantification of social factors to
differentiate between different context. specific for different area groups, different age groups, and
different gender groups. The results of this study will also
provide a theoretical basis for the rural revitalization strategy
of China to improve the health of rural older adults. Further
studies are required to confirm these findings and identify the
specific mechanisms that can be applied to improve the public
health of older adults. Conflict of interest The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could
be construed as a potential conflict of interest. Acknowledgments The authors wish to thank the participants of the CLHLS. Data availability statement The datasets presented in this study can be found in online
repositories. The names of the repository/repositories and
accession number(s) can be found below: https://opendata. pku.edu.cn/dataverse/CHADS. Author contributions The strength of this study was that it was the first longitudinal
study to investigate the relationship between living arrangements
and CC decline, revealing the causality better. However, there
were also limitations. First, the measurement of the CCs was not
accurate enough, which were rounded up to the nearest integer in
centimeters. And CC is recommended as one of the methods for
sarcopenia screening in AWGS 2019 but not a gold standard for
diagnosis. It could reflect but not substitute for skeletal muscle
mass when it comes to sarcopenia. Second, we did not consider
the change of living arrangements between two waves. The
advantage, however, is that compared to other variables, such
as sleep quality or sleep duration at different time, living
arrangement is a more stable exposure in a 4-year time span
(Nakakubo et al., 2021). Third, several potential confounders,
such as social networks, were not included in our study. The
better methods of social factors measurement are needed in
future studies. DW contributed to design of the study, information
extraction, statistical analysis and drafting the manuscript. JZ
supervised all aspects of the study and revised the manuscript. Ethics statement The studies involving human participants were reviewed and
approved by Research Ethics Committee of Peking University. The patients/participants provided their written informed
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Chinese community-dwelling older adults living alone. Sci. 5 Conclusion In summary, this study revealed that living alone was
associated with a reduced risk of calf circumference decline
among older adults, especially for older adults of 65–79 years
old, women and city/town dwellers. It suggests that living alone
may be a protective factor for skeletal muscle mass maintenance
and sarcopenia prevention. Public health policy should be 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. Frontiers in Aging frontiersin.org 07 Wang and Zhang Wang and Zhang 10.3389/fragi.2022.1063760 References National sleep foundation’s sleep time duration recommendations:
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of
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outcomes in older adults: evidence from the CLHLS. BMC Geriatr. 22 (1), 59. doi:10.1186/s12877-021-02742-5 Holt-Lunstad, J., Smith, T. B., Baker, M., Harris, T., and Stephenson, D. (2015). Loneliness and social isolation as risk factors for mortality: a meta-analytic review. Perspect. Psychol. Sci. 10 (2), 227–237. doi:10.1177/1745691614568352 Woods, N. F., LaCroix, A. Z., Gray, S. L., Aragaki, A., Cochrane, B. B., Brunner, R. L., et al. (2005). Frailty: emergence and consequences in women aged 65 and older
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in asia. Demogr. Res. 32, 1099–1112. doi:10.4054/demres.2015.32.40 Locquet, M., Bruyere, O., Lengele, L., Reginster, J. Y., and Beaudart, C. (2021). Relationship between smoking and the incidence of sarcopenia: The SarcoPhAge
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Presence of Spodoptera frugiperda Multiple Nucleopolyhedrovirus (SfMNPV) Occlusion Bodies in Maize Field Soils of Mesoamerica
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Insects
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Presence of Spodoptera frugiperda Multiple
Nucleopolyhedrovirus (SfMNPV) Occlusion Bodies in Maize
Field Soils of Mesoamerica Trevor Williams 1,*
, Guadalupe del Carmen Melo-Molina 2, Jaime A. Jiménez-Fernández 2,
Holger Weissenberger 3, Juan S. Gómez-Díaz 1, Laura Navarro-de-la-Fuente 1 and Andrew R. R 1
Instituto de Ecología AC (INECOL), Xalapa 91073, Veracruz, Mexico
2
El Colegio de la Frontera Sur (ECOSUR), Tapachula 30700, Chiapas, Mexico
3
El Colegio de la Frontera Sur (ECOSUR), Chetumal 77014, Quintana Roo, Mexico
4
CSIRO Division of Entomology, Canberra, ACT 2601, Australia
*
Correspondence: trevor.williams@inecol.mx Simple Summary: Nucleopolyhedroviruses of caterpillars (Lepidoptera) produce occlusion bodies
(OBs) that protect the virions within a protein matrix. This allows the virus to persist in the envi-
ronment until ingested by a susceptible insect. The soil is an important reservoir of OBs that can be
transported to plants for transmission to the host caterpillar. The Spodoptera frugiperda multiple
nucleopolyhedrovirus is a lethal pathogen of the fall armyworm, a major pest of maize, rice, and
sorghum. Analysis of 186 soil samples collected from maize fields in southern Mexico, Belize, and
Guatemala revealed that almost 19% of samples had OBs present at low concentrations. Genetic
analysis revealed the presence of genetic diversity in the soil OB populations. The presence of OBs
was higher in maize fields with living crops and in specific types of soils. These findings suggest that
the soil could be a valuable source of genetic diversity for the design of virus-based insecticides to
control this important pest. insects insects insects insects insects Citation: Williams, T.; Melo-Molina,
G.d.C.; Jiménez-Fernández, J.A.;
Weissenberger, H.; Gómez-Díaz, J.S.;
Navarro-de-la-Fuente, L.; Richards,
A.R. Presence of Spodoptera
frugiperda Multiple
Nucleopolyhedrovirus (SfMNPV)
Occlusion Bodies in Maize Field Soils
of Mesoamerica. Insects 2023, 14, 80. https://doi.org/10.3390/
insects14010080 Abstract: The occlusion bodies (OBs) of lepidopteran nucleopolyhedroviruses can persist in soil
for extended periods before being transported back on to the foliage for transmission to the host
insect. A sensitive insect bioassay technique was used to detect OBs of Spodoptera frugiperda
multiple nucleopolyhedrovirus (SfMNPV) in 186 soil samples collected from maize fields in the
southern Mexican states of Chiapas, Tabasco, Campeche, Yucatán, and Quintana Roo, as well Belize
and Guatemala. Overall, 35 (18.8%) samples proved positive for SfMNPV OBs. The frequency of
OB-positive samples varied significantly among Mexican states and countries (p < 0.05). Between
1.7 and 4.4% of S. frugiperda larvae that consumed OB-positive samples died from polyhedrosis
disease. Restriction endonuclease analysis using PstI and HindIII confirmed that the soil-derived
isolates were strains of SfMNPV and that genetic diversity was evident among the isolates. The
prevalence of OB-positive soil samples did not differ with altitude or extension (area) of the maize
field, but it was significantly higher in fields with the presence of living maize plants compared
to those containing dead plants or crop residues (p < 0.05). Georeferenced soil samples were used
to identify soil types on digitized soil maps. Lithosol and Luvisol soils had a higher than average
prevalence of OB-positive samples (42–45% positive) (p = 0.006), as did Andosol, Gleysol, and Vertisol
soils (33–60% OB-positive), although the sample sizes were small (<5 samples) for the latter three
soils. In contrast, Cambisol soils had a lower than average prevalence of OB-positive samples (5%
positive). Bioassays on Acrisol, Fluvisol, Phaeozem, and Rendzina soils resulted in intermediate
levels of OB-positive samples. We conclude that certain soil types may favor OB persistence and
virus-mediated biological pest control. The soil is also likely to provide a valuable source of genetic
diversity for the design of virus-based insecticides against this pest. Academic Editors: Wataru
Mitsuhashi and Kotaro Konno Received: 30 December 2022
Accepted: 11 January 2023
Published: 13 January 2023 Received: 30 December 2022
Accepted: 11 January 2023
Published: 13 January 2023 1. Introduction Nucleopolyhedrovirus occlusion bodies (OBs) that are released from virus-killed
insects are washed off the plant by the action of rainfall or by the senescence and shedding
of OB-contaminated leaves and subsequently enter the soil [1,2]. As a result, the soil
represents an important environmental reservoir of nucleopolyhedrovirus OBs [3], although
the persistence of OB populations in soil is poorly understood [4]. The OB structure allows
the occlusion-derived virions (ODVs) to remain viable for months or years in soil, given
favorable conditions of pH and temperature [5]. OBs in soil are also protected from the
adverse effects of solar ultraviolet (UV) radiation that can rapidly inactivate these viruses [6]. OBs in the soil are transported back on to host plants during plant growth through the soil,
through the action of wind-blown dust and rain-splash [7–9], or they are carried on the
bodies of foraging invertebrates [10]. As OBs adhere strongly to soil particles, it is difficult to separate them by conventional
techniques [11]. Techniques have been developed to detect the presence of OBs in soil by
polymerase chain reaction (PCR), although amplification can be hindered by a diversity
of soil components [12–14]. A sensitive bioassay technique has been developed involving
feeding early instar larvae with mixtures of soil and semi-synthetic diet [15]. This technique
has been used to obtain novel occluded virus isolates [16] and to study the abundance and
genetic composition of soil OB populations [17,18] and their interaction with soil-dwelling
invertebrates [19,20]. The Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV) (family Bac-
uloviridae; genus Alphabaculovirus) is a common pathogen in natural populations of the
fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae) in the Americas, and it
has now been reported in China [21], India [22], and Indonesia [23], where the pest has
recently invaded. The virus usually causes mortality of between 1% and 5% in S. frugiperda
larvae [24–26], although some populations can experience seasonal epizootics [27,28]. This
virus has attracted attention as the active ingredient in the development of biological
insecticides against this pest, and several commercial products have been developed [29]. As part of a long-term study on the biology, ecology, and bioinsecticide potential of
the S. frugiperda—SfMNPV pathosystem, we examined the prevalence of SfMNPV OBs in
maize field soils in southern Mexico and part of Central America, over an area of >2000 km2. Copyright:
© 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article
distributed
under
the
terms
and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/). Keywords: Baculoviridae; Alphabaculovirus; virus persistence; insect bioassay; genetic diversity; soil
type; fall armyworm https://www.mdpi.com/journal/insects Insects 2023, 14, 80. https://doi.org/10.3390/insects14010080 2 of 15 Insects 2023, 14, 80 1. Introduction The sampling program generated multiple isolates, some of which were gifted to other
researchers who examined their insecticidal characteristics [30]. As digitalized maps of soil
types in this region recently became available, we used our data, collected two decades ago,
to analyze the relationship between soil type and the presence of SfMNPV OBs in maize
fields in the region. 2. Materials and Methods 2.1. Insect Colonies and Reference Virus Isolate 2.2. Soil Sampling Between 30 June and 20 October 2000, a total of 186 soil samples were collected from
maize fields in the southern Mexican states of Chiapas (120 samples), Tabasco (1 sample),
Campeche (8 samples), Yucatán (16 samples), and Quintana Roo (10 samples), as well
as parts of Belize (4 samples) and Guatemala (27 samples). Only a single sample was
taken in Tabasco as the road to Campeche passed through this state for only a very short
distance (~5 km). Sampling was non-systematic and consisted of driving along roads and selecting
accessible fields with evidence of maize cultivation (planted crop or crop residues). In
areas of intense maize production, fields were sampled at intervals of hundreds of meters
(shortest distance 100 m) or a few kilometers, whereas in other areas, such as Belize where
the dominant crop is sugarcane, maize fields were sampled at intervals of tens of kilometers
(longest distance ~80 km). The aim was to obtain a range of samples in each area that
we passed through. A soil sample was taken by scraping away the upper 5 cm of soil
and collecting ~100 g of soil at a depth of 5–10 cm at five random points in each field to
produce a sample of ~500 g that was placed in a black plastic bag, mixed by shaking, sealed,
and labeled with a site code. Following the procedures of Richards & Christian [15], the
uppermost 5 cm of soil was not sampled as OBs in this layer would be exposed to extremes
of solar UV radiation and high temperatures that may have affected their viability. The geographical coordinates and altitude of each field were recorded using a GPS
locator (Garmin III Plus, Garmin International Inc., Olathe, KS, USA); the area of the field
was estimated (based on the approximate dimensions and shape of the field); the state of
the crop was classified in terms of growth status (living or dead), as maize is commonly
folded over and allowed to dry prior to harvesting in this region, or the presence of plant
residues from a previously harvested crop (Supplemental File S1). Soil samples were taken
to the laboratory in ECOSUR, Tapachula, Chiapas, spread on sheets of paper, and allowed
to air dry for 2–5 days in a dark room at 26–28 ◦C and then prepared for insect bioassay. 2.1. Insect Colonies and Reference Virus Isolate A laboratory colony was started in 1996 using larvae of S. frugiperda collected from
maize fields close to the city of Tapachula, Chiapas, Mexico. Larvae were reared individ-
ually in 30 mL plastic cups with a piece of semi-synthetic diet based on soya, yeast, and
maize flour without formaldehyde [31]. For oviposition, groups of adults were placed in
paper bags with a plastic dish containing a cotton pad soaked in 10% honey solution and
maintained at 25–28 ◦C. This colony was used for the isolation of SfMNPV OBs from soil
samples. In the year 2000, a sample from this colony was sent to the Universidad Pública
de Navarra, Spain, reared, and analyzed by reverse transcription PCR using primers tar-
geting the ie-0 and polh genes and was found to be free of sublethal nucleopolyhedrovirus
infection [32]. A second colony of S. frugiperda was started in 2021 using larvae collected from maize
plants in a field close to Cempoala, Veracruz. This colony was treated identically to that
of the first colony except that it was maintained at a constant 26 ± 1 ◦C in a temperature-
controlled room. This colony was used to amplify isolates prior to restriction endonuclease
analysis. PCR analysis using primers targeted at the sf58 gene [33] or the polh gene [34] did Insects 2023, 14, 80 3 of 15 3 of 15 not result in amplification of DNA samples taken from larvae, pupae, or adults, suggesting
that this colony did not harbor sublethal nucleopolyhedrovirus disease. y
p
y
The Nicaraguan isolate of SfMNPV (SfMNPV-NIC) was used as a reference isolate
that has been characterized in detail in previous studies [35–37]. This isolate naturally
comprises nine genotypic variants that are present in varying proportions. 2.2. Soil Sampling Following the bioassay procedure, all soils were autoclaved prior to disposal to eliminate
soil-borne pathogens. 2.3. Bioassay of Soil Samples The soil bioassay procedure involves larvae that are fed mixtures of soil and semi-
synthetic diet [15]. Air-dried soil samples were passed through a 1 mm sieve to eliminate
stones and large pieces of organic material. Sieves were washed and decontaminated by
treatment with 0.5% sodium hypochlorite solution to avoid cross-contamination. A 10 g
sample of sieved soil was placed in a 250 mL plastic cup and mixed thoroughly with 90 g
of semi-synthetic diet using a disposable wooden spatula to produce a smooth uniform
paste. The soil–diet mixture was then distributed evenly among 30 plastic cups (30 mL
capacity), and two S. frugiperda first instars from the first laboratory colony were placed
in each cup. An additional group of 10 larvae per sample was reared individually with a
mixture of sterilized soil and diet as controls. Larvae were incubated at 25 ◦C and were
checked at 48 h intervals until death or pupation. A small portion of each dead larvae was
taken with a toothpick, smeared on a microscope slide, stained using Giemsa stain, and
examined for the presence of OBs under a phase-contrast microscope at ×1000 with oil
immersion [38]. The efficacy of the soil sterilization procedure was checked by autoclaving
samples of 10 g soil that had been spiked with 1.9 × 106 OB/g soil (equivalent to the
estimated LC50 concentration mentioned in Section 3.1). None of the larvae that were Insects 2023, 14, 80 4 of 15 treated with soil+diet mixtures of autoclaved soil succumbed to polyhedrosis disease
(3 replicates of 24 larvae each). OB-positive larva were macerated in 50 µL of distilled water, mixed with 10% sucrose
and 0.05% Fluorella blue, and used to inoculate fourth instar larvae that had been starved
overnight. Larvae that consumed the inoculum within 15 min were individualized in the
wells of a 24-well cell culture plate with a piece of diet. Larvae were incubated at 25 ± 1 ◦C
and were checked daily for signs of polyhedrosis disease until death. Virus-killed larvae
were placed in 1.5 mL centrifuge tubes and stored at −80 ◦C for 21 years. Some of the
samples were gifted to other researchers to perform comparative studies on the insecticidal
characteristics of the isolates. A selection of 32 isolates was subjected to PCR amplification using primers Sf58.1
(5′-GTCCTCGGTGCTGAATCAGG-3′) and Sf58.2 (5′-TTACGTAGGTGCTGGAGGAG-3′)
targeted at the sf58 gene of SfMNPV [33]. 2.3. Bioassay of Soil Samples For this, viral DNA was extracted from OBs as
follows. Each infected larva was homogenized, filtered through an 80 micron pore steel
mesh, and centrifuged at 2500 rpm for 5 min to pellet debris. The OB suspension was
placed on a cushion of 50% glycerol and centrifuged at 12,000 rpm for 10 min. The resulting
OB pellet was washed with distilled water and resuspended in 200 µL of distilled water. A 100 µL volume of 3xDAS buffer (0.3 M Na2CO3, 0.5 M NaCl, 0.03 M EDTA; pH 10.5)
was added to the OB suspension and incubated at 45 ◦C for 45 min to release occlusion-
derived virions (ODVs). The suspension was centrifuged at 3000 rpm for 5 min to remove
undissolved OBs, and the supernatant was centrifuged at 12000 rpm for 10 min. The
resulting pellet of ODVs was resuspended in 100 µL MilliQ water, 10 µL of 10% SDS, and
3 µL of proteinase K (20 mg/mL), followed by incubation at 45 ◦C for 45 min. Viral genomic
DNA was then extracted by treatment with phenol-chloroform and precipitated in absolute
ethanol with 1/10 vol 3 M sodium acetate at −20 ◦C overnight. The DNA pellet was
washed with 70% ethanol and resuspended in 50 µL MilliQ water, and the concentration
was measured in a UV-spectrometer at 260 nm (BioSpec-Nano, Shimadzu, Japan). p
p
p
A 1 µL sample of the resulting DNA was amplified at 95 ◦C for 3 min, followed by
35 cycles of 95 ◦C for 30 s, 59 ◦C for 30 s, 72 ◦C for 1 min, and final extension at 72 ◦C for
7 min. Electrophoresis was performed in 1.2% agarose in TBE buffer (100 mM Tris, 90 mM
boric acid, 1 mM EDTA; pH 8.3) containing 35 µL/liter of ethidium bromide (10 mg/mL)
and photographed on a transilluminator (UV ChemiDoc XRS+ System with Image Lab
Software; Bio-Rad, Hercules, CA, USA). A sample of SfMNPV-NIC (106 OBs) was included
as a positive control and water as a negative extraction control. The frequency of OB-positive soil samples was compared across the states of southern
Mexico and Belize and Guatemala by Fisher’s exact test, whereas the prevalence of virus-
induced disease in bioassay larvae exposed to soil samples was compared across states
and countries by fitting a generalized linear model with a binomial error structure in
GLIM 4 [39]. 2.3. Bioassay of Soil Samples To quantify the sensitivity of the soil bioassay, the procedure was performed using a
characterized clay soil (pH 5.2) from the grounds of the Instituto de Ecología AC, Xalapa,
Mexico, which was studied previously [20]. For this, 1 mL suspensions of SfMNPV-NIC
OBs were mixed with 10 g soil samples to produce concentrations of 2 × 104, 2 × 105,
2 × 106, 2 × 107 OBs/g soil. Control samples were treated with 1 mL distilled water. Each
OB-contaminated soil was mixed with 90 g semi-synthetic diet and distributed among
the wells of 24 well cell culture plates, and a single S. frugiperda first instar larva from the
Cempoala colony was placed in each well and incubated at 27 ± 1 ◦C in darkness. Larvae
that died from polyhedrosis disease (confirmed by examination of Giemsa-stained smears)
were counted and recorded. The procedure was replicated four times using different
batches of insects. The results were subjected to logit regression in GLIM 4 [39]. 3.1. Calibration of the Soil Bioassay Mortality in the soil bioassay ranged from 1% in the 2 × 104 OB/g soil concentration to
99% in the 2 × 107 OB/g soil concentration (χ2 = 310.2, df = 1, p < 0.001). No virus-induced
deaths were observed in the control. The slope (± SE) of the logit regression was 1.383 ± 0.153,
and the estimated LC50 was 1.9 × 106 OBs/g soil (range of 95% C.I.: 1.48 × 106–2.48 × 106). There was no evidence of overdispersion in the data (residual deviance/residual d.f. = 1.02). 2.5. Relationship between Soil OBs and Environmental Factors 2.5. Relationship between Soil OBs and Environmental Factors Data collected on the altitude (GPS data) and the estimated size of maize fields were
compared for OB-positive and OB-negative soil samples by Kruskal–Wallis test and were
reported as medians ± interquartile range (IQR). The stage of the crop cycle was classified
as (i) green living stages from seedling to maize cob production or (ii) dry, dead plants and
crop residues, as farmers twist and bend the maize stem to allow the cobs to dry in the field
before they are harvested. The frequency of positive and negative samples was compared
for each crop stage by contingency table analysis. The GPS-referenced location of each sample was plotted on digitized maps produced
using vector data on southern Mexico [40] or Belize and Guatemala produced by the Food
and Agriculture Organization of the United Nations [41]. Maps were drawn, and sample
points were plotted using ArcGIS 10.4 software [42]. Intensive sampling was performed
in the southernmost Soconusco region of Chiapas State, so this region was plotted on a
separate map for clarity. Mapped information was used to identify soil type and subtype
for each of the 186 samples analyzed. The prevalence of OB-positive samples was compared
among soil types by contingency table analysis. Soils with fewer than five samples were
excluded from this analysis. 2.4. Restriction Endonuclease Analysis of Soil Isolates To examine the presence of genetic diversity in soil isolates, eight isolates were selected
at random. These isolates were amplified in S. frugiperda fourth instars from the Cempoala Insects 2023, 14, 80 5 of 15 5 of 15 colony. Larvae were individualized, starved overnight, and then allowed to drink a
suspension of 108 OBs/mL in 10% sucrose solution with 0.05% Fluorella blue. Larvae that
consumed the inoculum within 15 min were individualized in the wells of a 24-well cell
culture plate with a piece of diet. Larvae were incubated at 27 ± 1 ◦C and were checked
daily for signs of polyhedrosis disease. Virus-killed larvae were placed in 1.5 mL centrifuge
tubes and stored at −20 ◦C. To obtain viral DNA, larvae were individually homogenized in 1 mL distilled water. OBs were then purified, and DNA was extracted, purified by phenol-chloroform treatment,
and resuspended in 100 µL of MilliQ water as described in Section 2.3. Samples of 1 µg DNA
of each isolate were digested with restriction endonucleases PstI or HindIII (New England
Biolabs, Ipswich, MA, USA) at 37 ◦C following the manufacturer’s recommendations. After
6 h, the reaction was stopped by addition of loading buffer, and samples were loaded in a
0.6% (PstI) or 0.8% (HindIII) agarose gel in TBE buffer containing 35 µL/liter of ethidium
bromide (10 mg/mL). Electrophoresis was performed overnight at 30 V. Each gel was
then photographed on a transilluminator (UV ChemiDoc XRS+ System with Image Lab
Software; Bio-Rad, Hercules, CA, USA). 3.2. Bioassay of Soil Samples from Maize Fields Of the 186 soil samples that were collected from maize fields, 35 (18.8%) proved posi-
tive for SfMNPV OBs by insect bioassay (Figure 1, Table 1). The prevalence of OB-positive
samples varied significantly by state and country (Fisher’s exact p = 0.003). A single neg-
ative sample from Tabasco state was not included in the analysis. The highest number
of OB-positive samples (N = 14) was obtained from Chiapas state, reflecting the inten-
sive sampling in this state. The remaining states in Mexico had intermediate numbers
of OB-positive samples (N = 3–6). Two positive samples were obtained from Belize, and
six positive samples were collected in Guatemala (Table 1). 6 of 15
the am-
emental Insects 2023, 14, 80 Figure 1. Distribution of soils samples that proved positive (orange points) or negative (black points)
for SfMNPV OBs in the soil-diet bioassay. Samples were distributed across fives states of southern
Mexico, Belize, and Guatemala. Intensive sampling was performed in the Soconusco region in the
south of Chiapas State, Mexico (shown enclosed in a black rectangle). Figure 1. Distribution of soils samples that proved positive (orange points) or negative (black points)
for SfMNPV OBs in the soil-diet bioassay. Samples were distributed across fives states of southern
Mexico, Belize, and Guatemala. Intensive sampling was performed in the Soconusco region in the
south of Chiapas State, Mexico (shown enclosed in a black rectangle). Figure 1. Distribution of soils samples that proved positive (orange points) or negative (black points)
for SfMNPV OBs in the soil-diet bioassay. Samples were distributed across fives states of southern
Mexico, Belize, and Guatemala. Intensive sampling was performed in the Soconusco region in the
south of Chiapas State, Mexico (shown enclosed in a black rectangle). Figure 1. Distribution of soils samples that proved positive (orange points) or negative (black points)
for SfMNPV OBs in the soil-diet bioassay. Samples were distributed across fives states of southern
Mexico, Belize, and Guatemala. Intensive sampling was performed in the Soconusco region in the
south of Chiapas State, Mexico (shown enclosed in a black rectangle). Table 1. Results of insect bioassays on soil samples according to Mexican State and country. Table 1. Results of insect bioassays on soil samples according to Mexican State and country. State/Country 1
No. of
Samples Tested
No. of
Positive Samples (%)
No. of Larvae Tested Against
OB-Positive Samples
No. 3.3. Restriction Endonuclease Analysis of Soil Isolates
3.3. Restriction Endonuclease Analysis of Soil Isolates 3.3. Restriction Endonuclease Analysis of Soil Isolates
3.3. Restriction Endonuclease Analysis of Soil Isolates Restriction endonuclease analysis of eight randomly selected isolates indicated con-
siderable levels of genetic diversity across these isolates. Compared to the SfMNPV-NIC
reference isolate, all the soil-derived isolates differed in the number and size of restriction
fragments and also in the presence of sub-molar fragments that suggest that individual
isolates likely comprise mixtures of genotypes (clear differences are marked with asterisks
in Figure 2A,B). Digestion with PstI and HindIII revealed polymorphism that was apparent
across the range of fragment lengths. The results of these analyses should be viewed with
caution as the largest fragments (>48 Kb) may be difficult to distinguish from undigested
genomic DNA, or partially digested DNA in the case of some sub-molar bands. The three
short fragments of between 0.5 and 1 Kb produced by HindIII treatment were similar across
all isolates, but they are not easily visualized in Figure 2B. Restriction endonuclease analysis of eight randomly selected isolates indicated con-
siderable levels of genetic diversity across these isolates. Compared to the SfMNPV-NIC
reference isolate, all the soil-derived isolates differed in the number and size of restriction
fragments and also in the presence of sub-molar fragments that suggest that individual
isolates likely comprise mixtures of genotypes (clear differences are marked with asterisks
in Figure 2A,B). Digestion with PstI and HindIII revealed polymorphism that was appar-
ent across the range of fragment lengths. The results of these analyses should be viewed
with caution as the largest fragments (>48 Kb) may be difficult to distinguish from undi-
gested genomic DNA, or partially digested DNA in the case of some sub-molar bands. The three short fragments of between 0.5 and 1 Kb produced by HindIII treatment were
similar across all isolates, but they are not easily visualized in Figure 2B. Figure 2. Restriction endonuclease analysis of a selection of soil-derived isolates of SfMNPV (lanes
A-H) compared to the SfMNPV-NIC reference isolate (Nic). Genomic DNA was digested with (A)
PstI or (B) HindIII and subjected to electrophoresis in 0.6% and 0.8% agarose, respectively. The
Figure 2. Restriction endonuclease analysis of a selection of soil-derived isolates of SfMNPV (lanes
A-H) compared to the SfMNPV-NIC reference isolate (Nic). Genomic DNA was digested with (A) PstI
or (B) HindIII and subjected to electrophoresis in 0.6% and 0.8% agarose, respectively. 3.3. Restriction Endonuclease Analysis of Soil Isolates
3.3. Restriction Endonuclease Analysis of Soil Isolates The isolates
used for this analysis were SfMNPV-NIC (labeled Nic) sample #121 (labeled A), #36 (B), #37 (C),
#19 (D), #152 (E), #92 (F), #177 (G), and #50 (H), following the sample numbers given in Supplemental
File S1. Restriction fragments and sub-molar bands that differed from those of SfMNPV-NIC are
labeled with an asterisk. Molecular markers were NEB 1 Kb ladder (M1) and NEB λ mono-cut
DNA (M2). Figure 2. Restriction endonuclease analysis of a selection of soil-derived isolates of SfMNPV (lanes
A-H) compared to the SfMNPV-NIC reference isolate (Nic). Genomic DNA was digested with (A)
PstI or (B) HindIII and subjected to electrophoresis in 0.6% and 0.8% agarose, respectively. The
Figure 2. Restriction endonuclease analysis of a selection of soil-derived isolates of SfMNPV (lanes
A-H) compared to the SfMNPV-NIC reference isolate (Nic). Genomic DNA was digested with (A) PstI
or (B) HindIII and subjected to electrophoresis in 0.6% and 0.8% agarose, respectively. The isolates
used for this analysis were SfMNPV-NIC (labeled Nic) sample #121 (labeled A), #36 (B), #37 (C),
#19 (D), #152 (E), #92 (F), #177 (G), and #50 (H), following the sample numbers given in Supplemental
File S1. Restriction fragments and sub-molar bands that differed from those of SfMNPV-NIC are
labeled with an asterisk. Molecular markers were NEB 1 Kb ladder (M1) and NEB λ mono-cut
DNA (M2). 3.2. Bioassay of Soil Samples from Maize Fields of Virus
Killed Larvae (%)
Chiapas
120
14 (11.7%)
765
22 (2.9%)
Tabasco
1
0 (0%)
-
-
Campeche
8
4 (50.0%)
240
8 (3.3%)
Yucatán
16
6 (37.5%)
360
15 (4.2%)
Quintana Roo
10
3 (30.0%)
180
8 (4.4%)
Belize
4
2 (50.0%)
120
2 (1.7%)
Guatemala
27
6 (22.2%)
360
6 (1.7%)
Totals:
186
35 (18.8%)
2025
61 (3.0%)
1 A single soil sample from Tabasco state was negative in the insect bioassay and was not included in the
statistical analyses. On average (±SE), 1.74 ± 0.18 larvae died from polyhedrosis disease in the OB-positive
samples (range 1–4 larvae/sample). Lethal polyhedrosis disease was confirmed by exam-
ination of Geimsa smears. The prevalence of infection (range 1.7–4.4%; Table 1) did not
differ significantly among samples from different states and countries (GLM χ2 = 6.245,
df = 5, p = 0.283). An average (±SE) of 2.14 ± 0.38 larvae (range 0–8) died from other causes
in OB-positive samples, mainly bacterial deaths, and these larvae were not considered
further. None of the control larvae that consumed sterilized soil samples succumbed to
polyhedrosis disease. p
y
All 32 isolates tested proved positive for PCR amplification of the sf58 gene, resulting in
the expected 306 bp product, although there appeared to be slight variation in the amplicon
size, suggesting the presence of genotypic variation among isolates (Supplemental Figure S3). 7 of 15
ncluded 7 of 15
ncluded Insects 2023, 14, 80
Totals: 3.4. Relationship between Soil OBs and Environmental Factors Percentage values shown in gray
rectangles are based on the indicated sample sizes (N). Figure 3. Prevalence of OB-positive and -negative samples taken from maize fields with (A) living
green crops and (B) dry crops or post-harvest crop residues. Percentage values shown in gray rec-
tangles are based on the indicated sample sizes (N). Figure 3. Prevalence of OB-positive and -negative samples taken from maize fields with (A) living
green crops and (B) dry crops or post-harvest crop residues. Percentage values shown in gray
rectangles are based on the indicated sample sizes (N). Georeferenced information was used to locate samples taken from twelve types of
soil, and 16 soil subtypes that are present in this region. The soil type of all but five sam-
ples could be identified by plotting sample points on a combined map of the region incor-
porating data from southern Mexico, Belize, and Guatemala (Figure 4A). The five samples
from unknown soil types were classified as “uncharacterized”. Intensive sampling per-
formed in the Soconusco region of Chiapas State, Mexico was plotted on a separate map
for clarity (Figure 4B). Georeferenced information was used to locate samples taken from twelve types of
soil, and 16 soil subtypes that are present in this region. The soil type of all but five
samples could be identified by plotting sample points on a combined map of the region
incorporating data from southern Mexico, Belize, and Guatemala (Figure 4A). The five
samples from unknown soil types were classified as “uncharacterized”. Intensive sampling
performed in the Soconusco region of Chiapas State, Mexico was plotted on a separate map
for clarity (Figure 4B). y
g
The prevalence of OB-positive samples varied significantly among soil types
(χ2 = 22.998, df = 9, p = 0.006; soil types with a sample size of <5 were not included
in the analysis, namely Andosol, Nitisol, and Regosol). Compared to the average of 18.8%
of OB-positive samples (shown in Table 1), Lithosol and Luvisol soils had a clearly higher
than average prevalence of OB-positive samples (Figure 5). This was also the case for
Andosol, Gleysol, and Vertisol soils (33–60% OB-positive), but the sample sizes were small
for those soils (4–6 samples each). In contrast, Cambisol soils were well represented (21 sam-
ples) but had a lower than average prevalence of OB-positive samples. 3.4. Relationship between Soil OBs and Environmental Factors Maize fields were sampled at altitudes between 8 and 2630 m above mean sea level, but
altitude did not differ significantly between OB-positive soil samples (median [IQR]: 57 m
[105]) and OB-negative samples (69 m [197]) (Kruskal–Wallis H = 1.039, df = 1, p = 0.308). Maize fields varied in estimated area from 0.04 to 15 ha, but the size the field did not differ
significantly between OB-positive soil samples (median [IQR]: 1 ha [2.5]) and OB-negative
samples (1 ha [1.25]) (Kruskal–Wallis H = 0.00, df = 1, p = 0.997). The presence of the crop in the green living stages (from seedling to cob production)
was associated with an increased probability of OB-positive soil samples (Figure 3A,B). Overall, 29 out of 120 (24%) maize fields with living crops had OB-positive soils, whereas Insects 2023, 14, 80 8 of 15
uction)
3A B) 8 of 15
uction)
3A B) only 6 out of 66 (9%) maize fields with dead, dry crops and crop residues proved OB-positive
in the insect bioassay (χ2 = 6.335, df = 1, p = 0.012). Looking at these results in another
way, 83% (29 out of 35) of OB-positive soils originated from fields with green living crops,
whereas only 17% (6 out of 35) of the OB-positive soils originated from fields with dry
crops and crop residues. In this region, farmers twist and fold the maize stem to allow the
cobs to dry before harvesting. only 6 out of 66 (9%) maize fields with dead, dry crops and crop residues proved OB-
positive in the insect bioassay (χ2 = 6.335, df = 1, p = 0.012). Looking at these results in
another way, 83% (29 out of 35) of OB-positive soils originated from fields with green
living crops, whereas only 17% (6 out of 35) of the OB-positive soils originated from fields
with dry crops and crop residues. In this region, farmers twist and fold the maize stem to
allow the cobs to dry before harvesting. Figure 3. Prevalence of OB-positive and -negative samples taken from maize fields with (A) living
green crops and (B) dry crops or post-harvest crop residues. Percentage values shown in gray rec-
tangles are based on the indicated sample sizes (N). Figure 3. Prevalence of OB-positive and -negative samples taken from maize fields with (A) living
green crops and (B) dry crops or post-harvest crop residues. 3.4. Relationship between Soil OBs and Environmental Factors The other soil types
(Acrisol, Fluvisol, Phaeozem, and Rendzina soils) had intermediate levels of OB-positive
samples that were similar to the overall average prevalence (Figure 5). The numbers of
OB-positive samples among the soil subtypes are listed in Supplemental Material Table S1,
and a brief description of soil characteristics and properties is provided in Table S2. Insects 2023, 14, 80 9 of 15 (A)
(B)
Figure 4. Soil type composite maps for (A) southern Mexico, Belize, and Guatem
Soconusco region of Chiapas State, Mexico. Georeferenced soil samples were plotted
samples (open black circles) and OB-negative samples (black circles with a cross). H
versions of these maps are available as Supplemental Material (Figures S1 and S2). Figure 4. Soil type composite maps for (A) southern Mexico, Belize, and Guatemala and (B) the
Soconusco region of Chiapas State, Mexico. Georeferenced soil samples were plotted as OB-positive
samples (open black circles) and OB-negative samples (black circles with a cross). High resolution
versions of these maps are available as Supplemental Material (Figures S1 and S2) (A) (A)
(B)
Figure 4. Soil type composite maps for (A) southern Mexico, Belize, and Guatemala
Soconusco region of Chiapas State, Mexico. Georeferenced soil samples were plotted as
samples (open black circles) and OB-negative samples (black circles with a cross). Hig
versions of these maps are available as Supplemental Material (Figures S1 and S2). Figure 4. Soil type composite maps for (A) southern Mexico, Belize, and Guatemala and (B) the
Soconusco region of Chiapas State, Mexico. Georeferenced soil samples were plotted as OB-positive
samples (open black circles) and OB-negative samples (black circles with a cross). High resolution
versions of these maps are available as Supplemental Material (Figures S1 and S2). (A) (B) (B) Figure 4. Soil type composite maps for (A) southern Mexico, Belize, and Guatemala
Soconusco region of Chiapas State, Mexico. Georeferenced soil samples were plotted as
samples (open black circles) and OB-negative samples (black circles with a cross). Hig
versions of these maps are available as Supplemental Material (Figures S1 and S2). Figure 4. Soil type composite maps for (A) southern Mexico, Belize, and Guatemala and (B) the
Soconusco region of Chiapas State, Mexico. Georeferenced soil samples were plotted as OB-positive
samples (open black circles) and OB-negative samples (black circles with a cross). High resolution
versions of these maps are available as Supplemental Material (Figures S1 and S2). Discussion
4. Discussion Discussion
A 2600 km-long soil sampling trip around southern Mexico, Belize, and Guatemala
was followed by intensive sampling in the Soconusco coastal region of the state of Chia-
as. Fall armyworm is a major pest of maize in this region, especially during the rainy
eason (May–October) when this study was conducted. In general, the ecology of OBs in
he soil reservoir is poorly understood, which prompted the present study on the preva-
f OB i
i
fi ld i
M
i
A 2600 km-long soil sampling trip around southern Mexico, Belize, and Guatemala
was followed by intensive sampling in the Soconusco coastal region of the state of Chiapas. Fall armyworm is a major pest of maize in this region, especially during the rainy season
(May–October) when this study was conducted. In general, the ecology of OBs in the soil
reservoir is poorly understood, which prompted the present study on the prevalence of
OBs in maize fields in Mesoamerica. ence of OBs in maize fields in Mesoamerica. Overall, almost one in five soil samples (35 out of 186 samples; 18.8%) proved positive
or SfMNPV OBs by insect bioassay, indicating that this could be a productive strategy for
btaining novel virus isolates. A selection of 32 isolates all proved positive for PCR am-
lification of the sf58 gene, which encodes a per os infection factor (PIF9) present in al-
habaculoviruses and betabaculoviruses [33,43] (Supplemental Figure S3). This provides
dditional evidence that the majority of isolates were SfMNPV, although we were unable
Overall, almost one in five soil samples (35 out of 186 samples; 18.8%) proved positive
for SfMNPV OBs by insect bioassay, indicating that this could be a productive strategy
for obtaining novel virus isolates. A selection of 32 isolates all proved positive for PCR
amplification of the sf58 gene, which encodes a per os infection factor (PIF9) present in
alphabaculoviruses and betabaculoviruses [33,43] (Supplemental Figure S3). This provides
additional evidence that the majority of isolates were SfMNPV, although we were unable
to test the isolates that had been gifted to other researchers. o test the isolates that had been gifted to other researchers. The soil bioassay contrasts with the conventional method of collecting large numbers
f S. frugiperda larvae from infested fields and rearing them in the laboratory to detect
atural SfMNPV infections [24–26,28]. 3.4. Relationship between Soil OBs and Environmental Factors 10 of 15
brief Insects 2023, 14, 80 Figure 5. Prevalence of samples from different types of soil that were positive for SfMNPV OBs. Values above columns indicate the number of samples tested (N). Figure 5. Prevalence of samples from different types of soil that were positive for SfMNPV OBs. Values above columns indicate the number of samples tested (N). gure 5. Prevalence of samples from different types of soil that were positive for SfMNPV OBs. alues above columns indicate the number of samples tested (N). Figure 5. Prevalence of samples from different types of soil that were positive for SfMNPV OBs. Values above columns indicate the number of samples tested (N). Discussion
4. Discussion The reason why the LC50 values differed
by approximately 100-fold between droplet feeding and soil-diet bioassay techniques is
likely due to two reasons: (i) the OB-contaminated soil was diluted ten-fold by the addition
of the diet prior to the soil-diet bioassay, and (ii) OBs adhere strongly to components of
the soil, particularly clay minerals [49], and so may be less readily solubilized in the insect
midgut than purified OBs in water. g
p
The soil + diet bioassay technique was successfully applied in a study of natural
OB populations in greenhouse crops attacked by the beet armyworm, Spodoptera exigua
(Lepidoptera: Noctuidae) in southern Spain. Of a total of 267 substrate samples from green-
houses, 34% proved positive for the homologous nucleopolyhedrovirus (SeMNPV) [18]. Following this, a slightly modified soil + diet bioassay technique was used on early instars
of S. frugiperda to detect OB-positive samples (8% positive) in soils taken from maize fields
in northern Mexico [16]. Subsequent analysis indicated that some of these isolates were
mixtures of SfMNPV and a granulovirus, suggesting that the insect colony may have
harbored a sublethal granulovirus infection [50]. An alternative explanation involving
simultaneous acquisition of both types of virus from a single soil sample is unlikely given
the low concentration of OBs in soil. Nevertheless, granuloviruses can also persist in soil
for extended periods [51,52]. p
A selection of the SfMNPV isolates from the present study was gifted to A.M. Martínez-
Castillo (Universidad Michoacana de San Nicolás de Hidalgo, Mexico), and two of them,
from Chiapas and Yucatán, were subjected to analysis of insecticidal characteristics in
different Mexican populations of S. frugiperda. The Yucatán isolate was also used for field
tests with promising results [30], underlining the value of soil OB populations as a source
of new and highly insecticidal isolates. Larvae that became infected after consuming soil samples probably only consumed a
single OB, as consumption of one OB was previously demonstrated to result in 2.7–5.4%
mortality in S. frugiperda second instars [53]. Nonetheless, restriction endonuclease analysis
indicated that genetic diversity was present in each of the isolates tested, as evidenced
by the presence of fragment size polymorphisms and sub-molar fragments (Figure 4A,B). This was likely due to the presence of genotypic diversity present within individual OBs
present in the soil. Discussion
4. Discussion The soil bioassay approach has the additional ben-
fit that it could be used for isolate prospection during periods when the pest is not
The soil bioassay contrasts with the conventional method of collecting large numbers
of S. frugiperda larvae from infested fields and rearing them in the laboratory to detect
natural SfMNPV infections [24–26,28]. The soil bioassay approach has the additional benefit
that it could be used for isolate prospection during periods when the pest is not present
in the crop, or to quantify soil OB persistence over extended periods of time, even when
non-host crop plants have been planted as part of a crop rotation cycle. The prevalence of OB-positive samples varied across the sampled region, but the
prevalence of infection in larvae that consumed soil samples was invariably low (average
3%). Calibration of the soil bioassay indicated that the concentrations of OBs in virus-
positive maize field samples were likely between 104 and 105 OBs/g soil. This is not an
insignificant amount as the 50% lethal concentration of SfMNPV OBs by droplet feeding
bioassay is approximately 2 × 104 OBs/mL in second instar larvae of S. frugiperda [44]. y
pp
y
f
g p
The estimated concentration of SfMNPV in soil samples in our study (104–105 OBs/g)
was very similar to the concentration of OBs reported in other pest–virus pathosystems
in Spain [18], Botswana [45], Canada [46], and Connecticut, USA [47], although much
higher concentrations (7 × 107 OBs/g) were reported from soil under trees infested by
Hyphantria cunea in Japan [48]. It is important to note that these studies all used different
methodologies to quantify OBs in soil samples. In the case of the soil-diet bioassay in first instars, the estimated LC50 was 1.9 × 106 OBs/g
soil, which compared favorably with values derived from soil-diet bioassays on second Insects 2023, 14, 80 11 of 15 11 of 15 instars of 1.4 × 106 OBs/g soil [20] and 2.7 × 106 OBs/g soil, albeit with shorter periods
(2–4 days) of feeding on the soil–diet mixture [19]. Discussion
4. Discussion Restriction endonucleases provide a rapid visual assessment of genetic
similarities between isolates but are of limited value for determining the presence of genetic
diversity as large fragments, and sub-molar bands may be the result of incomplete digestion
of genomic DNA. Nonetheless, we performed several digestions of viral DNA with different
enzymes such as BamHI and EcoRI (data not shown), as well as several digestions using
HindIII and PstI (shown in Figure 2A,B), and in all cases sub-molar bands were present,
which lends support to the idea that the isolates comprised mixtures of genotypic variants. pp
p
g
yp
The SfMNPV-NIC isolate comprises a mixture of nine genotypic variants in various
proportions that are enveloped together within ODVs and co-occluded in OBs [53,54]. Genotypic diversity has a marked influence on the transmission and persistence of nu-
cleopolyhedroviruses [54–57]. A study on OB populations in greenhouse soil substrate
revealed that the prevalence of different genotypic variants of SeMNPV varied seasonally
and across different parts of the study area, suggesting that certain variants may be better
adapted than others to persist outside the host [18]. However, the genotypic diversity of
OBs on plant surfaces, in infected insects, and in the soil has not yet been subjected to
systematic comparison. y
p
The viable OB population in the soil reflects the result of processes that introduce
OBs and those that eliminate OBs from the soil habitat. OBs released from virus-killed
insects that fall off the plant and from OB-contaminated foliage are washed onto the soil by
rainfall [1] and through the shedding of OB-contaminated leaves [2,48]. As a result, there is
a clear correlation between the presence of the pest on the crop and the abundance of OBs
present in the soil [18,46,51,58]. This was confirmed in the present study as we detected
a positive association between OBs in soil samples and the living stage of the crop cycle, Insects 2023, 14, 80 12 of 15 12 of 15 prior to harvest, that is the stage attacked by S. frugiperda larvae (Figure 3A,B). Contrarily,
processes that eliminate OBs include UV-irradiation and high temperatures at the soil
surface, loss through OB transportation onto plant surfaces by wind and rain-splash, and
chemical and microbial degradation of OBs in the soil [59–61]. Discussion
4. Discussion In addition, processes such
as water percolation, ploughing, the movement of livestock or the activity of the soil fauna
can be responsible for altering the vertical distribution of OBs in soil [20,58,61]. p
g
An important finding was that the prevalence of OB-positive samples varied signifi-
cantly among soil types with higher than average prevalence in Lithosol, Luvisol, Andosol,
Gleysol, and Vertisol soils, although sample sizes were reduced for the later three soil types. Soils are extraordinarily variable in their physico-chemical and biological properties. How-
ever, it is worth noting that Luvisols, Andosols, Gleysols, and Vertisols are characterized
by high clay content, which is known to strongly bind to OBs [49]. Surface charge and
hydrophobic surface attributes appear to be involved in the interaction of OBs with soil
minerals [62,63]. Lithosols are also known as Leptosols and are characterized by very shal-
low soils over rock, highly calcareous or stony material without clearly expressed horizons. They comprise a wide variety of soils with a diversity of chemical and physical properties. Interestingly, Cambisol soils had a lower than average prevalence of OB-positive samples. Cambisols are known as “brown soils”, that are in the beginning of soil formation and
have weak horizon differentiation. Cambisols are characterized by the absence of a layer of
accumulated clay, humus, soluble salts, and iron or aluminum oxides [64–67]. The cause of
the low prevalence of OBs in Cambisols may be related to the low clay content or some
other factor(s) (see Supplemental Table S2). Finally, although we did not measure soil pH,
it is known that pH affects the long-term persistence of OBs in soil both at low pH (acidic)
and high pH (alkaline) [51]. Soil pH may also affect surface charge and the tendency of
OBs to bind to soil particles [63], which is another field of research that merits systematic
examination using modern analytical chemistry techniques. These findings have intriguing implications in that they suggest that soil type could
have a direct effect on the persistence of lepidopteran nucleopolyhedroviruses in the envi-
ronment. However, on a note of caution, additional factors such as the local maize planting
practices, the phenological development of the crop and the density of the S. frugiperda
infestation could also affect the likelihood of detecting OBs in soil samples, although we
did not measure these variables in the present study. Discussion
4. Discussion It would be interesting to examine sys-
tematically the influence of soil type on the prevalence of virus-induced disease in similar
pest–crop combinations grown in different types of soil, to explicitly test the hypothesis that
soils that favor OB persistence can augment the biological control of lepidopteran pests. 5. Conclusions We conclude that the soil represents an enormous and largely untapped source of
novel nucleopolyhedrovirus isolates. Such isolates harbor genotypic diversity that could be
useful in the development of biological insecticides. The probability of isolating novel virus
strains increased during periods of vegetative growth when the pest was likely present
in the maize crop. Certain soil types appeared to be more amenable than others to the
persistence of soil OB populations. That soil type could manifestly influence biological pest
control by these viruses is a notion that requires systematic evaluation. Finally, the soil-diet
bioassay technique has clear potential for the study of the ecology of soil OB populations
and the factors that mediate OB persistence in soil, such as soil composition, agronomic
practices, and the role of soil invertebrates in OB dispersal. Supplementary Materials: The following supporting information can be downloaded at: https://
www.mdpi.com/article/10.3390/insects14010080/s1, (FileS1.xlxs); Figure S1: High-resolution version
of Figure 4A; Figure S2: High-resolution version of Figure 4B; Figure S3: PCR amplification of soil
isolates using primers targeted at the sf58 gene of SfMNPV; Table S1: Number of OB-positive samples
according to soil type and subtype; Table S2: Properties and characteristics of soil types that differed
in the prevalence of OB-positive samples. 13 of 15 13 of 15 Insects 2023, 14, 80 Author Contributions: Conceptualization, A.R.R. and T.W.; methodology, A.R.R., T.W., J.S.G.-D. and H.W.; formal analysis, T.W.; investigation, G.d.C.M.-M., J.A.J.-F., A.R.R., T.W., J.S.G.-D. and
L.N.-d.-l.-F.; resources, A.R.R., T.W. and J.S.G.-D.; data curation, T.W.; writing—original draft
preparation, G.d.C.M.-M., J.A.J.-F. and T.W.; writing—review and editing, T.W., visualization, H.W. and L.N.-d.-l.-F.; supervision, T.W., project administration, T.W.; funding acquisition, T.W. and
A.R.R. All authors have read and agreed to the published version of the manuscript. Funding: The soil collection and virus detection study was funded by CONACYT-SIBEJ 0501047 and
the Australian Academy of Science via a travel grant to A.R.R. In addition, L.N.-d.-l.-F. received an
Ayudante de Investigador III award from CONACYT, Mexico. Data Availability Statement: All the data presented in this study are available in the file Supplementary
data file FileS1.xlxs. Acknowledgments: We thank Juan Cisneros and Dora I. Penagos (ECOSUR) for assistance with soil
bioassays and Cindy S. Molina (INECOL) for sharing PCR amplification results on the insect colony. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. References 1. D’Amico, V.; Elkinton, J.S. Rainfall effects on transmission of gypsy moth (Lepidoptera: Lymantriidae) n
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(accessed on 20 October 2022). 67. FAO Soils Portal—Data Hub. Available online: https://www.fao.org/soils-portal/data-hub/en/ (acce Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual
author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to
people or property resulting from any ideas, methods, instructions or products referred to in the content. Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual
author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to
people or property resulting from any ideas, methods, instructions or products referred to in the content.
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Unsupervised feature selection with least-squares quadratic mutual information
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Indonesian journal of electrical engineering and computer science
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Corresponding Author: Janya Sainui
Division of Computational Science, Faculty of Science
Prince of Songkla University
Songkhla, Thailand
Email: janya.s@psu.ac.th Indonesian Journal of Electrical Engineering and Computer Science
Vol. 22, No. 3, June 2021, pp. 1619∼1628
ISSN: 2502-4752, DOI: 10.11591/ijeecs.v22i3.pp1619-1628 Indonesian Journal of Electrical Engineering and Computer Science
Vol. 22, No. 3, June 2021, pp. 1619∼1628
ISSN: 2502-4752, DOI: 10.11591/ijeecs.v22i3.pp1619-1628 1619 Ì ABSTRACT We propose the feature selection method based on the dependency between features
in an unsupervised manner. The underlying assumption is that the most important
feature should provide high dependency between itself and the rest of the features. Therefore, the top m features with maximum dependency scores should be selected,
but the redundant features should be ignored. To deal with this problem, the objective
function that is applied to evaluate the dependency between features plays a crucial
role. However, previous methods mainly used the mutual information (MI), where the
MI estimator based on the k-nearest neighbor graph, resulting in its estimation de-
pendent on the selection of parameter, k, without a systematic way to select it. This
implies that the MI estimator tends to be less reliable. Here, we introduce the least-
squares quadratic mutual information (LSQMI) that is more sensible because its tuning
parameters can be selected by cross-validation. We show through the experiments that
the use of LSQMI performed better than that of MI. In addition, we compared the pro-
posed method to the three counterpart methods using six UCI benchmark datasets. The
results demonstrated that the proposed method is useful for selecting the informative
features as well as discarding the redundant ones. Unsupervised feature selection with least-squares quadratic
mutual information Janya Sainui, Chouvanee Srivisal
Division of Computational Science, Faculty of Science, Prince of Songkla University, Songkhla, Thailand Article Info
Article history:
Received Dec 27, 2020
Revised May 10, 2021
Accepted May 24, 2021 Keywords: Least-squares quadratic
mutual information
Mutual information
Unsupervised feature
selection This is an open access article under the CC BY-SA license. This is an open access article under the CC BY-SA license. 1.
INTRODUCTION Feature selection aims to select the most informative subset of features to capture the structure of
the original data. It usually appears as a pre-processing step for various tasks such as classification [1], [2],
clustering [3]-[5], data mining [6], resulting in better results. In a supervised scenario, the idea is to select
the features that are the most relevant to the class labels [7], [8]. However, in this paper, we focus on an
unsupervised feature selection that is more difficult to achieve than the supervised feature selection due to the
absence of the class labels [9]-[14]. In unsupervised manner, feature selection is important as it helps improve
the performance as well as reduce the computational time of clustering [15]-[19]. Moreover, unsupervised
feature selections may be used for various purposes such as visualization and pre-processing step for supervised
learning [7], [15], [20]. Unsupervised feature selection can be divided into two categories, namely wrapper
method and filter method [10], [15]. Many existing methods of unsupervised feature selection are based on the
wrapper method such that the selected features are dependent on the specific clustering algorithm. The main
drawbacks of the wrapper method are its computational complexity and limited use with a particular clustering Journal homepage: http://ijeecs.iaescore.com 1620 Ì ISSN: 2502-4752 algorithm. In this paper, we focus on the filter method which evaluates the important features without using
any clustering algorithm [21]-[25]. Thus, the filter method is more general and useful than the wrapper method. We review some existing unsupervised feature selection methods relating to our work as follows. algorithm. In this paper, we focus on the filter method which evaluates the important features without using
any clustering algorithm [21]-[25]. Thus, the filter method is more general and useful than the wrapper method. We review some existing unsupervised feature selection methods relating to our work as follows. Laplacian score (LS) [10] is based on Laplacian Eigenmap and locality preserving projection. For
each feature, the Laplacian score is estimated using the nearest neighbor graph. If it is a good feature, its
LS tends to be small. The LS is based on the observation that, two data points are probably related to the
same cluster if they are close to each other. The drawbacks of this method are that there is no systematic
way to tune the number of nearest neighbor k or Gaussian width, and the redundant features are not observed. 1.
INTRODUCTION multi-cluster feature selection (MCFS) [11] selects the features while preserving the cluster structure of the
data. The authors proposed to use multiple eigenvectors of graph Laplacian, which are defined on the affinity
matrix of data points to capture the multi-cluster structure of data. The algorithm performs especially well
when the number of selected features is small (e.g., <= 50). However, it performs best when the number of
used eigenvectors is equal to the number of clusters, but in an unsupervised manner, the number of clusters
remains unknown. Moreover, we observed through the experiments that this method tends to be sensitive to
noise features. Unsupervised feature selection based on mutual information (UFSMI) [9] is derived from the
observation that good features share information in common, and noisy features are less correlated with the
other features. Mutual information (MI) is then used as the objective function to capture the shared information
between a feature and the rest of features, and they choose the first m features that achieve the higher mutual
information. The higher level of noise leads to a smaller average value of the score function because the
addition of noise reduces the mutual information between features. The desired property of the feature selection
algorithm aims at removing noisy features. However, the estimation of MI is less reliable as it is computed
using the k-nearest neighbor graph. Thus, its performance is based on the tuning parameter, k, resulting in that
a subset of the selected features may not be the best. All methods mentioned above including several existing methods such as [12]-[14] are based on the
k-nearest neighbor graph or other tuning parameters, and there is no systematic way to choose such parameters
(i.e., k). In addition, they did not observe the redundant features. Therefore, in this paper, we would like to deal
with these problems. Our idea is inspired by [9] as they proved that a good feature is expected to be dependent
on the rest of features. However, the method did not take the redundancy of features into account, and thus
may not produce an optimal feature subset. Moreover, in [9], they used the mutual information (MI) as their
dependent measure, and the MI is estimated based on the k-nearest neighbors which lacks of the systematic
way to choose the appropriate k. So that the estimation of MI tends to be less reliable. 2.
THE LEAST-SQUARE QUADRATIC MUTUAL INFORMATION In this section, we first review the least-square quadratic mutual information (LSQMI) [27] that can
be used to measure a statistical dependence among features. As LSQMI is ranged from 0 to ∞, we need to
normalize LSQMI to [0,1] range. We then review the normalization of LSQMI [28] that is finally used as the
objective function for our proposed unsupervised feature selection method. 1.
INTRODUCTION In order to solve these
problems, we propose to apply the L2-distance variant of MI called the quadratic mutual information (QMI)
[26], where the least-square method is used to approximate QMI [27]. Therefore, the used parameters can be
obtained automatically by cross-validation. The contribution of this paper is a novel method of unsupervised
feature selection based on the least-squares quadratic mutual information (LSQMI), which is powerful for
selecting the most important features as well as rejecting the redundant features. In the experimental results,
we show that the LSQMI is more reliable than MI. In addition, we demonstrate that the proposed method is
promising through the experimental results on six UCI machine learning repositories. The rest of paper is organized as follows. In section 2, we review the least-square quadratic mutual
information and its normalization. We describe the proposed method in section 3. The experimental setup and
results are shown in section 4. In section 5, we provide the conclusion. Indonesian J Elec Eng & Comp Sci, Vol. 22, No. 3, June 2021 : 1619 – 1628 2.1.
LSQMI estimation Suppose that we are given a set of {(xi, yi)}n
i=1 independently drawn from a joint probability dis-
tribution with density p(x, y). Here, x ∈Rn is a featrue vector and y ∈Rn×(d−1) is the rest of feature
vectors, where n denotes the number of samples and d denotes the number of features. The quadratic mutual
information (QMI) [26] between x and y is defined as donesian J Elec Eng & Comp Sci, Vol. 22, No. 3, June 2021 : 1619 – 1628 Indonesian J Elec Eng & Comp Sci
ISSN: 2502-4752
Ì 1621 Indonesian J Elec Eng & Comp Sci
ISSN: 2502-4752 Ì QMI :=
ZZ
f(x, y)
2
dxdy, QMI :=
ZZ
f(x, y)
2
dxdy, QMI :=
ZZ
f(x, y)
2
dxdy, where where where f(x, y) := p(x, y) −p(x)p(y), f(x, y) := p(x, y) −p(x)p(y), f(x, y) := p(x, y) −p(x)p(y), and p(x) and p(y) denote the marginal densities of x and y, respectively. In LSQMI [27], the density difference
f(x, y) is modeled as and p(x) and p(y) denote the marginal densities of x and y, respectively. In LSQMI [27], the density difference
f(x, y) is modeled as g(x, y) =
n
X
ℓ=1
θℓK(x, xℓ)L(y, yℓ), g(x, y) =
n
X
ℓ=1
θℓK(x, xℓ)L(y, yℓ), g(x, y) =
n
X
ℓ=1
θℓK(x, xℓ)L(y, yℓ), where K(x, xℓ) and L(y, yℓ) are kernel functions for x and y, respectively. Then, θ = (θ1, . . . , θn)⊤is
learned by least-squares as where K(x, xℓ) and L(y, yℓ) are kernel functions for x and y, respectively. Then, θ = (θ1, . . . , θn)⊤is
learned by least-squares as min
θ
ZZ
g(x, y) −f(x, y)
2
dxdy. min
θ
ZZ
g(x, y) −f(x, y)
2
dxdy. An empirical and regularized version of the above optimization problem is given as bθ := argmin
θ
h
θ⊤Hθ −2θ⊤bh + λθ⊤θ
i
, where λ ≥0 is the regularization parameter, and H and bh are defined as where λ ≥0 is the regularization parameter, and H and bh are defined as Hℓ,ℓ′ :=
Z
K(x, xℓ)K(x, xℓ′)dx
Z
L(y, yℓ)L(y, yℓ′)dy,
(1)
bhℓ:= 1
n
n
X
i=1
K(xi, xℓ)L(yi, yℓ) −1
n2
n
X
i,j=1
K(xi, xℓ)L(yj, yℓ). 2.2.
The normalisation of LSQMI 2.2. The normalisation of LSQMI
As LSQMI is ranged from 0 to ∞, we here normalize it as follow: As LSQMI is ranged from 0 to ∞, we here normalize it as follow: As LSQMI is ranged from 0 to ∞, we here normalize it as follow: \
NQMI :=
[
QMI(x, y)
max([
QMI(x, x), [
QMI(y, y))
. (2) (2) The higher \
NQMI indicates the more information shared between the feature x and the rest of features y, while
it is closed to zero if the feature x is a noise feature. We assume that the values of [
QMI(y, y) and [
QMI(x, x)
are larger than zero and that of [
QMI(x, y), because they are the dependency of themselves. Now, the range of
\
NQMI is [0, 1]. However, in practice, \
NQMI may be higher than 1. The higher \
NQMI indicates the more information shared between the feature x and the rest of features y, while
it is closed to zero if the feature x is a noise feature. We assume that the values of [
QMI(y, y) and [
QMI(x, x)
are larger than zero and that of [
QMI(x, y), because they are the dependency of themselves. Now, the range of
\
NQMI is [0, 1]. However, in practice, \
NQMI may be higher than 1. 2.1.
LSQMI estimation (1) In this paper, we use the Gaussian kernel for both K(x, xℓ) and L(y, yℓ) as both x and y are continuous: In this paper, we use the Gaussian kernel for both K(x, xℓ) and L(y, yℓ) as both x and y are continuous: K(x, xℓ) := exp
−∥x −xℓ∥2
2σ2
,
L(y, yℓ) := exp
−∥y −yℓ∥2
2σ2
, so that the integral in H (1) can be computed analytically as so that the integral in H (1) can be computed analytically as Hℓ,ℓ′ = (πσ2)dx/2 exp
−∥xℓ−xℓ∥2
4σ2
× (πσ2)dy/2 exp
−∥yℓ−yℓ′∥2
4σ2
. Thus, the solution bθ can be obtained as Thus, the solution bθ can be obtained as bθ = (H + λI)−1bh, bθ = (H + λI)−1bh, where I denotes the identity matrix. Finally, the least-squares quadratic mutual information (LSQMI) is given
by [
QMI := max(0, 2bθ
⊤bh −bθ
⊤Hbθ). [
QMI := max(0, 2bθ
⊤bh −bθ
⊤Hbθ). [
QMI := max(0, 2bθ
⊤bh −bθ
⊤Hbθ). Unsupervised feature selection with least-squares quadratic mutual information (Janya Sainui) ISSN: 2502-4752 1622 Ì 2.2. The normalisation of LSQMI The inputs of the algorithm are a set of features, {f1, ..., fd}, the number of needed features, m, and
a threshold, τ. While the output is a subset of selected features S = {f ∗
1 , . . . , f ∗
m}. The algorithm starts by 3.
THE PROPOSED UNSUPERVISED FEATURE SELECTION METHOD In an unsupervised scenario, the objective function that is used to evaluate the important feature is
very important. However, the objective function of the existing methods tends to be less reliable because its
approximation depends on the parameters included in the objective function, and there is no automatic way to
choose those parameters. In this paper, we propose an unsupervised feature selection by using a more reliable
objective function called the least-squares quadratic mutual information (LSQMI). The benefits of LSQMI are
that the parameters needed in the objective function can be chosen by cross validation, and it is less sensitive
to noises and outliers that may cause the estimation of the selected criteria unreliable [26], [27]. The proposed
method is described as follows. Given a set of n samples denoted as {xi}n
i=1, where each sample xi consists of d features (i.e.,
xi = {f1, . . . , fd}), our goal here is to select the most m informative features from d features, while the
redundant features should be discarded. To address this problem, we apply the LSQMI [27] as our criteria for
both selecting the most m important features as well as ignoring the redundant ones. Let fj ∈Rn be the jth
feature, and f\j ∈Rn×(d−1) be all features excluding fj. To select the important but not redundant features,
our proposed algorithm is shown in Algorithm 1. Algorithm 1 The unsupervised feature selection with least-squares quadratic mutual information (UFSLSQMI)
Input: A set of features, {f1, ..., fd}, fj ∈Rn, the number of needed features, m, and a threshold, τ. Output: A subset of selected features, S = {f ∗
1 , . . . , f ∗
m}. 1: S ←∅
2: for j = 1, . . . , d do
3:
compute \
NQMI (Equation (2)) between fj and f\j
4: end for
5: sort features {f1, ..., fd} according to their \
NQMI in descending order resulting in {f ∗
1 , ..., f ∗
d }
6: S ←{S ∪f ∗
1 }
7: k ←1
8: for j = 2, . . . 3.
THE PROPOSED UNSUPERVISED FEATURE SELECTION METHOD In other words, if the ratio of the current score and the previous
one is higher than the given threshold τ, (line 10), the feature according to the smaller score is ignored. The
algorithm is repeated until the m features (lines 8 - 17) are obtained. The performance of the proposed method depends on the parameters used for approximating the
LSQMI. However, thanks to the least-squares method, we can choose the appropriate parameters by cross-
validation method. Although the selected features are also depended on the threshold τ, we show you through
the experiments that our objective function well defines the redundant features and the noise features. In other
words, if two features are redundancy, their LSQMI scores tend to be closed to each other, and if a feature is
the noise feature, the LSQMI score tends to be small. 4.
RESULTS AND DISCUSSION In this section, we demonstrate the effectiveness of the proposed unsupervised feature selection. We
compared the proposed method with three competitive methods, including UFSMI [9], LSFS [10], and MCFS
[11]. All methods are based on the k-nearest neighbor, and here we set k = 5 for all methods as their default
setting. For MCFS, there is another parameter that is the number of eigenvectors, and we set this parameter at
5 as the same reason. For the proposed method, the threshold τ is set at 0.95. The competitive methods including the proposed method rank the features according to the criteria to
select the top m features. This means that the objective function mainly affect a subset of selected features. Thanks to the reliability of LSQMI, our method considers not only the importance of features but the redun-
dancy also. Therefore, in the experiment setups, we firstly show you that our objective function is effective for
selecting the important features as well as discarding the redundant features on the synthetic data. Secondly,
we illustrate the clustering performance after feature selection using six UCI benchmark datasets to confirm
the usefulness of our proposed method. 3.
THE PROPOSED UNSUPERVISED FEATURE SELECTION METHOD , d do
9:
if (k < m) then
10:
if (
\
NQMI(f ∗
j ,f ∗
\j)
\
NQMI(f ∗
j−1,f ∗
\j−1) < τ) then
11:
S ←{S ∪f ∗
j }
12:
k ←k + 1
13:
end if
14:
else
15:
break
16:
end if
17: end for 5: sort features {f1, ..., fd} according to their \
NQMI in descending order resulting in {f ∗
1 , ..., f ∗
d }
S
{S ∪f ∗} The inputs of the algorithm are a set of features, {f1, ..., fd}, the number of needed features, m, and
a threshold, τ. While the output is a subset of selected features S = {f ∗
1 , . . . , f ∗
m}. The algorithm starts by Indonesian J Elec Eng & Comp Sci, Vol. 22, No. 3, June 2021 : 1619 – 1628 1623 Indonesian J Elec Eng & Comp Sci ISSN: 2502-4752 Ì computing the normalisation of LSQMI as (2) for all features (lines 2 - 4). Then, the features are sorted in
descending order according to their score (line 5). Lastly, the features are selected according to the ranking;
a feature with the highest score is firstly selected and the feature with the second high score is then selected
and so on. However, the redundant features are ignored by discarding the feature that has the normalisation
of LSQMI score closed to the previous one. In other words, if the ratio of the current score and the previous
one is higher than the given threshold τ, (line 10), the feature according to the smaller score is ignored. The
algorithm is repeated until the m features (lines 8 - 17) are obtained. computing the normalisation of LSQMI as (2) for all features (lines 2 - 4). Then, the features are sorted in
descending order according to their score (line 5). Lastly, the features are selected according to the ranking;
a feature with the highest score is firstly selected and the feature with the second high score is then selected
and so on. However, the redundant features are ignored by discarding the feature that has the normalisation
of LSQMI score closed to the previous one. 4.1.
Synthetic data In the first experiment, we would like to demonstrate the reliability of the LSQMI comparing to the
competitive methods. Specifically, we would like to show you that the LSQMI is more reliable for evaluating
both the informative feature and the redundant features. To do so, we conducted a synthetic data of 3 clusters
with 3-dimensionality illustrated in Figure 1. We generated 100 instances with standard deviation = 1 for each
class. Figure 1 (a)-(c) shows that the 3rd feature is the most important for underlying clusters, while the 1st
feature and the 2nd feature are redundancy, because they have the same mean. Therefore, if we would like to choose two features (i.e., m = 2), the first selected one should be the
3rd feature, and the second one may be the 1st feature or the 2nd feature. In other words, the set of selected
features should not be the 1st feature and the 2nd feature, because they can capture only 2 clusters of the
original data as shown in Figure 1 (a). (a)
(b)
(c)
Figure 1. Relationship between features in the synthetic data: (a) Feature 1 vs. Feature 2, (b) Feature 1 vs. Feature 3, (c) Feature 2 vs. Feature 3 (b) (c) (a) (b) (a) (c) (b) Figure 1. Relationship between features in the synthetic data: (a) Feature 1 vs. Feature 2, (b) Feature 1 vs. Feature 3, (c) Feature 2 vs. Feature 3 Unsupervised feature selection with least-squares quadratic mutual information (Janya Sainui) 1624 Ì ISSN: 2502-4752 We ran all methods using this toy data, where each method was repeated 10 times with different
random data. We found that UFSMI performs the worst, because the 3rd feature was not selected 4 times. LS
missed selecting the 3rd feature 1 time, while the proposed method and MCFS always selected the 3rd feature. The average scores between each feature and the rest features obtained from each objective function are shown
in Table 1, showing that our objective function, \
NQMI, tends to be more sensible than other ones. As can be
seen, the \
NQMI score between the 3rd feature and the rest features is significantly highest, while the \
NQMI
scores according to the 2nd feature and the 1st feature are close to each other. In contrast to other objective
functions, the scores according to each feature are almost the same for all features. Indonesian J Elec Eng & Comp Sci, Vol. 22, No. 3, June 2021 : 1619 – 1628 4.1.
Synthetic data Notice that the scores of
LS showing in Tables 1 and 2 are the scores of (1 −Laplacian score) for each feature. Thus, as same as other
objective functions, the feature with larger score is more important. Table 1. The average scores over 10 runs between the feature j and the rest of the features obtained from
different objective functions using the synthetic data
Objective functions
feature 1
feature 2
feature 3
LS
0.9935
0.9934
0.9936
MI
-9.3682
-9.3771
-9.3730
MCFS
0.7330
0.8010
0.9848
\
NQMI
0.7424
0.7404
1.9034 Table 1. The average scores over 10 runs between the feature j and the rest of the features obtained from
different objective functions using the synthetic data Table 2. The average scores over 10 runs between the feature j and the rest of the features obtained from
different objective functions after adding the noise feature (feature 4)
Objective functions
feature 1
feature 2
feature 3
feature 4
LS
0.9821
0.9818
0.9801
0.9704
MI
-8.1727
-8.1980
-8.2047
-8.8783
MCFS
0.6872
0.7244
0.8226
0.9997
\
NQMI
0.3331
0.3415
0.8952
0.0167 he average scores over 10 runs between the feature j and the rest of the features obtained from
different objective functions after adding the noise feature (feature 4) Table 2. The average scores over 10 runs between the feature j and the rest of the features obtained from
different objective functions after adding the noise feature (feature 4) Next, we added a noise feature (the 4th feature) with mean = 0 for all clusters and standard deviation
= 1 into the above toy data. We ran the experiment 10 times for each method again, and the results show
that our method outperforms other methods including MCFS. Moreover, MCFS performs the worst in this
experiment as it always selected the 4th feature, while the other three methods never selected the 4th feature. The average scores of each method are shown in Table 2, confirming that \
NQMI is more reliable than other
objective functions. Notice that MCFS works well if the number of used eigenvectors is equal to the number
of clusters; however, in an unsupervised scenario, we do not know the actual number of clusters. 4.2.
UCI datasets Here, we evaluated the clustering performance after feature selection using six UCI benchmark datasets,
namely Abalone, Sonar, Glass, Pima, Heart, and Cancer datasets. For each dataset, the number of clusters (c),
the number of features (d), and the number of samples (n) are shown in Figure 2. For each run, we randomly
chosen 90% samples from each dataset, then we performed feature selection. Finally, we performed clustering
by the k-means algorithm; we ran the k-means algorithm 10 times with random initialization and chose the
best solution with the minimum error rate. The clustering accuracy is evaluated. The results of each dataset are
shown in Figure 2, showing the average accuracies over 10 runs at different number of seleced features, m. The results indicate that the proposed method (i.e., UFSLSQMI) almost performs better than the
counterpart approaches. However, we cannot say that the proposed method is the best. Because, in practice,
there is no method that works well for all data. Moreover, it is hard to explain the reasons why it works or it
does not work. What we can say is that the proposed method can be an alternative for solving unsupervised
feature selection problem that may work well for your data. Indonesian J Elec Eng & Comp Sci
ISSN: 2502-4752 ISSN: 2502-4752 Indonesian J Elec Eng & Comp Sci Ì 1625 Ì (a)
(b)
(c)
(d)
(e)
(f)
Figure 2. The average of clustering accuracies over 10 runs on UCI datasets: (a) Abalone (c=10 ,d=8 ,
n=2493), (b) Sonar (c=2 ,d= 60, n=188), (c) Glass (c=6 ,d=9 , n= 193), (d) Pima (c= 2,d= 8, n= 692), (e) Heart
(c=2 ,d=13 , n=244), (f) Cancer (c=2 ,d=9 , n=615) (b) (a) (a) (b) (c) (d) (d) (c) (e) (f) (f) (e) Figure 2. The average of clustering accuracies over 10 runs on UCI datasets: (a) Abalone (c=10 ,d=8 ,
n=2493), (b) Sonar (c=2 ,d= 60, n=188), (c) Glass (c=6 ,d=9 , n= 193), (d) Pima (c= 2,d= 8, n= 692), (e) Heart
(c=2 ,d=13 , n=244), (f) Cancer (c=2 ,d=9 , n=615) 4.3.
Discussion In the experiments, we compared the proposed unsupervised feature selection method with the other
filter methods. All methods including the proposed method select the informative features from the rank of
features according to the scores of the objective function. This means that the objective function plays an
important role for this task. We discuss here each objective function as follows. Firstly, Laplacian score (LS) is
computed by constructing of a nearest neighbor graph with the specific-defined number of nearest neighbors,
k. This is the reason why the Laplacian score feature selection does not always perform well. In addition, as
can be seen in the experiments on toy data (Table 1 and Table 2), the Laplacian scores of the most important
feature and the noise feature are not significantly different from each other, this may cause the rank of features
relating to the important features is unreliable. Unsupervised feature selection with least-squares quadratic mutual information (Janya Sainui) 1626 Ì ISSN: 2502-4752 Secondly, the mutual information (MI) is also computed based on the k-nearest neighbors graph. The
results in Tables 1 and 2 demonstrate that this MI score does not describe the difference between the informative
feature and the noise feature well. In other words, as same as LS, the MI scores of the important feature and
the noise feature are not significantly different from each other. Thirdly, the MCFS score is also computed based on the k-nearest neighbor graph such that the suitable
k effects the good score. Not only the parameter k, the MCFS score also needs the number of used eigenvectors
that makes the MCFS score depend on these two parameters. As can be seen in the experiment on toy data
with noise feature, the MCFS score of noise feature is unreliable; however, if we change the number of used
eigenvectors to be 2 instead of 5, the MCFS score of noise feature becomes more reliable. This confirms that
the MCFS score heavily depends on the number of used eigenvectors. Lastly, the proposed method utilizes the LSQMI that approximates by least-square method so that the
cross-validation can be included, resulting in that the estimator of QMI is sensible. 4.3.
Discussion As can be seen in Tables
1 and 2, the normalized LSQMI describe the structure of the original data as well; the LSQMI is highest if
the feature is the most important as the 3rd feature of the toy data; in contrast, the LSQMI is smallest if the
feature is the noise feature that provides no information about data like the 4th feature. The main drawback of
the proposed method is the time complexity that is higher than other methods because of the cross-validation
process. To sum up, the main problem of the filter based unsupervised feature selection methods is lacking of
the ability to automatically choose the parameters included in their objective function so that the approximation
of the objective function may be unreliable. Thus, we here propose to use the LSQMI for unsupervised feature
selection, that is not only reliable for evaluating the important features but also for ignoring the redundant
features. 5.
CONCLUSION The goal of feature selection is to select a subset of features in order to reduce the dimensionality of
the original data, while the structure of data should be preserved. There are two main points that are important
in order to achieve this goal. Firstly, we need to select the most informative features underlying preserving the
structure of the original data. Secondly, we have to remove the redundant features because they do not provide
more information about data. However, the existing approaches may not select the important features because
the use of unreliable objective functions. Moreover, they do not ignore the redundant features. In this paper, we
focus on these two points by exploring the reliable objective function that is the most important for evaluating
the informative features as well as the redundant features in an unsupervised manner. Specifically, we propose
to use the least-squares quadratic mutual information (LSQMI) as the objective function to select the valuable
features excluding the redundant features. The benefit of LSQMI is that the needed parameters can be chosen
by cross validation resulting that the estimation of the objective function will be more reliable, while most of
the other objective functions lack of the systematic way to choose their parameters. Through the experiments,
we show that the LSQMI can be used to determine the important features including the redundant feature as
well. Indonesian J Elec Eng & Comp Sci, Vol. 22, No. 3, June 2021 : 1619 – 1628 [5]
Ma. Dash, K. Choi, P. Scheuermann, and H. Liu, ”Feature selection for clustering—a filter solution,”
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national Conference on Data Mining, 2014, pp. 977-982, doi: 10.1109/ICDM.2014.58. [14] F. Nie, W. REFERENCES Zhu, and X. Li, ”Unsupervised feature selection with structured graph optimiza-
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10.5555/3015812.3016004 . 5] S. S.-Fern´andez, J. A. C.-Ochoa, and J. Fco. M.-Trinidad, ”A review of unsupervised feature selectio
methods,” Artificial Intelligence Review, vol. 53, pp. 907–948, 2020, doi: 10.1007/s10462-019-09682-y methods,” Artificial Intelligence Review, vol. 53, pp. 907–948, 2020, doi: 10.1007/s10462-019-09682-y. [16] S. Alelyani, J. Tang, and H. Liu, ”Feature selection for clustering: a review,” Data Cluster Algorithms
Applications, pp.29-60, 2018. [16] S. Alelyani, J. Tang, and H. Liu, ”Feature selection for clustering: a review,” Data Cluster Algorithms
Applications, pp.29-60, 2018. [17] M. Dash and Y. Ong, ”RELIEF-C: efficient feature selection for clustering over noisy data,” 2011 IEEE
23rd International Conference on Tools with Artificial Intelligence, 2011, pp. 869-872, doi: 10.1109/IC-
TAI.2011.135. [18] V. M. Rao and V. N. Sastry, ”Unsupervised feature ranking based on representation entropy,” 2012 1st
International Conference on Recent Advances in Information Technology (RAIT), 2012, pp. 421-425, doi:
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Research, vol. 5, pp. 845–889, 2004. 0] P. Y. Lee, W. P. Loh, and J. F. Chin, ”Feature selection in multimedia: the state-of-the-art review,” Imag
Vision Computing, vol. 67, pp. 29–42, 2017, doi: 10.1016/j.imavis.2017.09.004. 1] R. Varshavsky, A. Gottlieb, M. Linial, and D. Horn, ”Novel unsupervised feature filtering of biologic
data,” Bioinformatics, vol.22, no. 14, pp. e507–e513, 2006, doi: 10.1093/bioinformatics/btl214. f
pp
[22] M.i Banerjee and N. R. Pal, ”Feature selection with SVD entropy: some modification and extension,”
Information Sciences, vol. 264, pp. 118–134, 2014, doi: 10.1016/j.ins.2013.12.029. [23] S. S.-Fern´andez, J. F. Mart´ınez-Trinidad, and J. A. Carrasco-Ochoa, ”A new unsupervised spectral feature
selection method for mixed data: a filter approach,” Pattern Recognition, vol. 72, pp. 314–326, 2017, doi:
10.1016/j.patcog.2017.07.020. [24] X. Zhu, Y. Wang, Y. Li, Y. Tan, G. Wang, and Q. Song, ”A new unsupervised feature selection algorithm
using similarity-based feature clustering,” Computational Intelligence, vol. 35, no. 1, pp. 2-22, 2018, doi:
10.1111/coin.12192. [25] H. Wang, Y. Zhang, J. Zhang, T. Li, and L. Peng, ”A factor graph model for unsupervised feature selec-
tion,” Information Sciences, vol. 480, pp. 144-159, 2019, doi: 10.1016/j.ins.2018.12.034. 6] K. Torkkola, ”Feature extraction by non-parametric mutual information maximization,” The Journal
Machine Learning Research, vol. 3, pp. 1415-1438, 2003. g
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[27] J. Sainui and M. Indonesian J Elec Eng & Comp Sci, Vol. 22, No. 3, June 2021 : 1619 – 1628 REFERENCES Sugiyama, ”Direct approximation of quadratic mutual information and its application to
dependence maximization clustering,” IEICE Transactions on Information and Systems, vol. E96-D, no. 10, pp. 2282-2285, 2013, doi: 10.1587/transinf.E96.D.2282. Unsupervised feature selection with least-squares quadratic mutual information (Janya Sainui) 1628 Ì ISSN: 2502-4752 [28] J. Sainui and M. Sugiyama, ”Unsupervised key frame selection using information theory and colour
histogram difference,” International Journal of Business Intelligence and Data Mining, vol. 16, no. 3, pp. 324-344, 2020, doi: 10.1504/ijbidm.2020.106137. [28] J. Sainui and M. Sugiyama, ”Unsupervised key frame selection using information theory and colour
histogram difference,” International Journal of Business Intelligence and Data Mining, vol. 16, no. 3, pp. 324-344, 2020, doi: 10.1504/ijbidm.2020.106137. [28] J. Sainui and M. Sugiyama, ”Unsupervised key frame selection using information theory and colour
histogram difference,” International Journal of Business Intelligence and Data Mining, vol. 16, no. 3, pp.
324-344, 2020, doi: 10.1504/ijbidm.2020.106137. BIOGRAPHIES OF AUTHORS Janya Sainui received her BS and MS in Computer Science from Prince of Songkla University, Hat
Yai, Songkhla, Thailand, in 2005 and 2009, respectively. She is currently a Lecturer in Computer
Science at Prince of Songkla University, Thailand. She has conducted research on topics such as
bitmap indexing, video indexing and detection, clustering and unsupervised dimension reduction. She is interested in algorithm and application of machine learning, especially, applying machine
learning techniques to real world applications such as image/video processing, medical imaging,
bioinformatics, as well as information retrieval. Chouvanee Srivisal is currently a Lecturer in Computer Science at Faculty of Science Prince of
Songkla University, Thailand. She received her BS in Computer Science from Prince of Songkla
University and MS in Information Technology from King Mongkut’s Institute of Technology Lad-
krabang, Thailand in 1997 and 2002, respectively. She has interested in algorithm and application of
machine learning that for conducting research on topics such as image processing and information
retrieval.
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Review of: "Ancient Houses in Ben Tre City: A Multifaceted Approach to Preserve Artistic Architectural Heritage and Boost Local Tourism"
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Qeios, CC-BY 4.0 · Review, March 17, 2024 Review of: "Ancient Houses in Ben Tre City: A Multifaceted
Approach to Preserve Artistic Architectural Heritage and
Boost Local Tourism" Rute Matos1 Rute Matos1 Rute Matos1 1 Universidade de Evora 1 Universidade de Evora 1 Universidade de Evora Potential competing interests: No potential competing interests to declare. The topic is of great interest in terms of preserving a region's heritage and identity. The topic is of great interest in terms of preserving a region's heritage and identity. However, the structure of the article is not clear. It needs an introduction that includes: It needs an introduction that includes: - Contextualisation of the article. Why and how does it come about? - Its objectives. What is the aim of this article? - The methodology used - The methodology used - The results obtained or expected The methodology is missing. The bibliographical research could be more consistent and broader. Although there is no
specific information on the Ben Tre City case, information could be used by analogy; for example, what has been done in
the French neighbourhood of Ha Noi or in Hoi An city. Both are examples of heritage and identity preservation with
touristic and locals utilisation, which are widely recognised and publicised. There is a lack of overall images. There is a lack of urban cartography that allows us to read the relationship between the
houses and the existing urban fabric, as well as the relationship/articulation between the houses and the surrounding
space and landscape. Results are lacking. If there are no results at the end, those expected at the conclusion of this article should be presented. For example, the completion of the aforementioned inventory could be a result, as well as a proposal for the
use/dissemination of these houses and their ensemble. Of this landscape space. Qeios ID: O0X1UB · https://doi.org/10.32388/O0X1UB 1/1
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Adoption and continued use of mobile contact tracing technology: multilevel explanations from a three-wave panel survey and linked data
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Strengths and limitations of this study Objective To identify the key individual-level
(demographics, attitudes, mobility) and contextual
(COVID-19 case numbers, tiers of mobility restrictions,
urban districts) determinants of adopting the NHS
COVID-19 contact tracing app and continued use overtime. Design and setting A three-wave panel survey
conducted in England in July 2020 (background survey),
November 2020 (first measure of app adoption) and March
2021 (continued use of app and new adopters) linked with
official data. ►Our data captures reported behaviour at two points
to assess within-subject changes over time. ►Our results are based on a large, nationally repre-
sentative sample, as opposed to the convenience
and limited-N samples of previous contact tracing
studies. on October 23, 2024 by guest. P
http://bmjopen.bmj.com/
17 January 2022. Downloaded from ►Integrating demographic/structural and attitudinal
explanations relating to technology acceptance with
questions adopted from the results of a deliberative
poll. ►Prepublication history and
additional supplemental material
for this paper are available
online. To view these files,
please visit the journal online
(http://dx.doi.org/10.1136/
bmjopen-2021-053327). Participants N=2500 adults living in England,
representative of England’s population in terms of regional
distribution, age and gender (2011 census). Primary outcome Repeated measures of self-reported
app usage. Participants N=2500 adults living in England,
representative of England’s population in terms of regional
distribution, age and gender (2011 census). Primary outcome Repeated measures of self-reported
app usage. ►Studied population is England (see section 2.3)
where overall mobility is restricted in wave 3 during
national lockdown, allowing for limited opportunities
for app usage for example, venue check-ins.i Received 11 May 2021
Accepted 23 December 2021 Analytical approach Multilevel logistic regression linking
a range of individual level (from survey) and contextual
(from linked data) determinants to app usage. Results We observe initial app uptake at 41%, 95% CI
(0.39% to 0.43%), and a 12% drop-out rate by March
2021, 95% CI (0.10% to 0.14%). We also found that 7% of
nonusers as of wave 2 became new adopters by wave 3,
95% CI (0.05% to 0.08%). Initial uptake (or failure to use)
of the app associated with social norms, privacy concerns
and misinformation about third-party data access, with
those living in postal districts with restrictions on mobility
less likely to use the app. Perceived lack of transparent
evidence of effectiveness was associated with drop-out of
use. In addition, those who trusted the government were
more likely to adopt in wave 3 as new adopters. 1Politics, Birkbeck College,
University of London, London,
UK
2Politics, University of Exeter,
Exeter, UK
3Institute for Community
Research and Development,
University of Wolverhampton,
Wolverhampton, UK
4School of Geography, Politics
and Sociology, Newcastle
University, Newcastle upon
Tyne, UK
5Faculty of Laws, UCL, London,
UK
6Sociology, Philosophy and
Anthropology, University of
Exeter, Exeter, UK
Correspondence to
Dr Laszlo Horvath;
L.Horvath@bbk.ac.uk
© Author(s) (or their
employer(s)) 2022. Re-use
permitted under CC BY. Published by BMJ. sending users a notification of possible expo-
sure to the virus, along with health advice. Public acceptance is key to efficiency: Several
studies have shown that the app’s ability to
suppress the epidemic depends on the level
of overall uptake. An early estimate indi-
cated app usage of 56%1 could have helped
to avoid a second nationwide lockdown in
the UK. Another study indicated 15% uptake
would decrease the death toll if combined
with effective human contact tracing.2 On the
other hand, the rejection of contact tracing
apps by some may suggest that the govern-
ment failed to secure the public’s trust that
is crucial for compliance with restrictions on
mobility and social contact.3 4 ober 23, 2024 by guest. Protected by copyright. Current evidence about who uses contact
tracing apps and why is limited in several
ways. First, prior to their introduction,
studies were only able to measure intention
to use apps because they had not been devel-
oped and rolled out.5 The studies relied on To cite: Horvath L, Banducci S,
Blamire J, et al. Adoption
and continued use of mobile
contact tracing technology:
multilevel explanations from
a three-wave panel survey
and linked data. BMJ Open
2022;12:e053327. doi:10.1136/
bmjopen-2021-053327 Original research Original research Original research Open access on October 23, 2024 by guest. Protected by
http://bmjopen.bmj.com/
BMJ Open: first published as 10.1136/bmjopen-2021-053327 on 17 January 2022. Downloaded from BMJ Open: first published as 10.1136/bmjopen-2021-053327 on 17 Janu Adoption and continued use of mobile
contact tracing technology: multilevel
explanations from a three-wave panel
survey and linked data Adoption and continued use of mobile
contact tracing technology: multilevel
explanations from a three-wave panel
survey and linked data http://bmjo
blished as 10.1136/bmjopen-2021-053327 on 17 January 2022. Downloaded from Laszlo Horvath ,1,2 Susan Banducci,2 Joshua Blamire,3 Cathrine Degnen,4
Oliver James,2 Andrew Jones,5 Daniel Stevens,2 Katharine Tyler6 on October 23, 2024 by guest. Protected by copyright. http://bmjopen.bmj.com/
36/bmjopen-2021-053327 on 17 January 2022. Downloaded from 1Politics, Birkbeck College,
University of London, London,
UK
2Politics, University of Exeter,
Exeter, UK
3Institute for Community
Research and Development,
University of Wolverhampton,
Wolverhampton, UK
4School of Geography, Politics
and Sociology, Newcastle
University, Newcastle upon
Tyne, UK
5Faculty of Laws, UCL, London,
UK
6Sociology, Philosophy and
Anthropology, University of
Exeter, Exeter, UK
Correspondence to
Dr Laszlo Horvath;
L.Horvath@bbk.ac.uk
© Author(s) (or their
employer(s)) 2022. Re-use
permitted under CC BY.
Published by BMJ. Strengths and limitations of this study Conclusions Successful uptake of the contact tracing
app should be evaluated within the wider context of the UK
Government’s response to the crisis. Trust in government is
key to adoption of the app in wave 3 while continued use
is linked to perceptions of transparent evidence. Providing
clear information to address privacy concerns could
increase uptake, however, the disparities in continued
use among ethnic minority participants needs further
investigation. ►Drawing on our findings, an ethnic minority booster
sample will in the future allow us to better under-
stand inequalities across and within diverse ethnic
populations. Subjects, setting and data linkage Our panel vendor is ORB International. We use a sample
of 2500 respondents across three waves of data collection. We consulted the vendor and planned attrition so that the
first wave of surveys were completed by 5000 respondents
in July 2020, the second wave by 3700 in November 2020,
and the final wave by 2500 in March 2021—consisting of
those who completed all three surveys. Our study of adop-
tion is embedded in a larger population survey about
people’s lived experience in ‘Brexit-Covid-19 Britain (For
more information, see https://brexit-studies.org/covid-
19)’ thus our sample size is not determined by power
calculations for this specific study. Other than compli-
ance with quota sampling demographics (managed by
panel vendor) and participation in all three waves, there
were no exclusion criteria for this study. While the NHS
COVID-19 app is used by citizens living in England and
Wales, we needed to restrict our study to England’s popu-
lation on the funder’s request (We used the Revised Stan-
dards for Quality Improvement Reporting Excellence
(SQUIRE) reporting guidelines12). g
p
y
We report uptake at 41% in November 2020 with a
12% drop-out rate by March 2021, and that 7% of non-
adopters in November 2020 had installed the app by
March 2021. Of the predictors of the uptake, we find
that individual-level attitudinal measures best capture the
reasons why some adopted the technology while others
have not (privacy and norms). We also report concern
and misinformation about third party data access among
nonusers, and that trust in government was a significant
predictor of new adoption in March 2021 which, we spec-
ulate, could be related to the severity of the January–
March 2021 wave and/or perceptions about the UK
Government’s early success in its vaccination programme. Explaining continued use specifically, we highlight the
role of perceived usefulness and concern about trans-
parent evidence. on October 23, 2024 by guest. Protected by copyright. mjopen.bmj.com/ We provide an overview of the study design in figure 1
below. Matching the survey dates and respondents’
self-reported postal districts (first part of postcodes),
we merged COVID-19 cases data, regional closure and
restrictions data (three-tier system overlapping with wave
2), and urban/rural neighbourhood data from external
sources, as detailed below. Subjects, setting and data linkage Our data linkages are proba-
bilistic as neither units of analyses across the official data
constitute an exact match to postal districts, however,
asking for more granular location data (eg, postal area)
from our respondents would have potentially compro-
mised privacy. ber 23, 2024 by guest. Protected by copyright. METHODS
S
j In this study, our objective is to address these limita-
tions with a large-scale multiwave study in England, drawn
from a probability-based research panel, with represen-
tative sample demographics. We measured adoption of
contact tracing apps first in November 2020 and again in
March 2021. To explain adoption and continued use, we
link data from this survey (demographics, attitudes and
reported behaviour) with their postal districts’ COVID-19
case numbers, urban versus rural majority population,
as well as policy restrictions on social gatherings and
mobility. Our model specification is informed by the liter-
ature on Technology Acceptance particularly of health
technology, trust and findings from a deliberative public
forum with UK residents.10 Our predictor on views of the
app in respondents’ social networks echoes additional
fieldwork insights reported recently.11 INTRODUCTION As a tool in national COVID-19 Track and
Trace systems, mobile contact tracing apps
automate the process of contact tracing by Horvath L, et al. BMJ Open 2022;12:e053327. doi:10.1136/bmjopen-2021-053327 1 Open access on October 23, 2024 by guest. Protected by
http://bmjopen.bmj.com/
BMJ Open: first published as 10.1136/bmjopen-2021-053327 on 17 January 2022. Downloaded from experimental scenarios looking at the potential proper-
ties of the apps that could influence adoption, such as
data storage and sponsors.6 7 Second, of the limited obser-
vational evidence available, studies have been restricted to
convenience sampling which tends to overestimate adop-
tion rates.8 A recent study looking at user feedback on
Google Play fails to capture nonusers entirely.9 Important
qualitative work has identified key areas of citizen concern
(eg, transparency and the needs of vulnerable groups10 or
social norms or pressure11) but the distribution of these
concerns remains to be investigated at the national level. Third, studies have been limited to exploring adoption at
a single time point given the relatively short time since the
roll-out of the technology in many countries. Continued
use and drop-out rates, thus, remain to be investigated. We include additional notes about our theoretical expec-
tations of the relevant predictors in section 2.3. to the virus being determined locally on the users’ phones,
minimising data sharing (see also ‘Third party data
access’ under section 2.3 for additional details). In prac-
tice, this also means that, as opposed to direct interven-
tion by NHS Test and Trace (human contact tracing or
receipt of a positive test result), there is ‘no legal duty’
to self-isolate if instructed by the app (see https://faq.
covid19.nhs.uk/article/KA-01398/ and https://www.nhs.
uk/conditions/coronavirus-covid-19/self-isolation-and-
treatment/if-youre-told-to-self-isolate-by-nhs-test-and-
trace-or-the-covid-19-app/). The advice to self-isolate and
count-down lasts for ten days after predicted exposure. Additional features of the app include routine venue
check-ins (pubs, restaurants), local public health advice,
a symptom checker—encouraging continued use. http://bmjop
ublished as 10.1136/bmjopen-2021-053327 on 17 January 2022. Downloaded from Contact tracing in the UK The roll-out of the government-backed NHS COVID-19
app on 24 September 2020, by National Health Services
(NHS) England and Wales, makes the UK a relatively late
adopter of digital contact tracing, 6 months behind their
first recorded use globally (Singapore) and 4 months
behind the first adopters in Europe (Italy and France). It
is built on a decentralised system, with potential exposure Coronavirus cases are published on the UK Govern-
ment’s official website, and updated on a weekly basis
(week’s end) on the Middle Layer Super Output Area
(MSOA) level. While sometimes MSOAs are entirely
contained within a district constituting an exact match,
often a number of these overlap with a district. For
simplicity, we link data from the largest overlapping 2 Horvath L, et al. BMJ Open 2022;12:e053327. doi:10.1136/bmjopen-2021-053327 p
Figure 1 Research design. on October 23, 2024 by guest. Protected by copyright. http://bmjopen.bmj.com/
irst published as 10.1136/bmjopen-2021-053327 on 17 January 2022. Downloaded from http://bmjopen
shed as 10.1136/bmjopen-2021-053327 on 17 January 2022. Downloaded from Figure 1 Research design. launched an NHS contact tracing app for England
and Wales that will notify you if you have been in
close contact with someone who has tested positive
for Covid-19. Are you using this app? MSOA in terms of population wherever there is ambiguity
(For example, people living in an EX4 postcode may
be counted across thirteen MSOAs but the percentage
overlap ranges between one per cent (Mid Devon 10) to
100% of MSOA located within EX4 (Exeter 002) thus we
merged it with the latter. We tried an alternative method
of estimating case numbers by weighting the MSOA totals
according to the proportion of overlap with postcode
districts and this produced similar results.). Based on the responses submitted in wave 2, we split the
sample for analysis in wave 3 to examine continued use
separately from new adoption, see section 2.6. Predictors Tiers data were published on the UK Government’s
official website when changes occurred, with restrictions
applied on the local authority district (LAD) level. Postal
districts were linked to LADs and a tier assigned where a
postcode was situated wholly within a single tier. Where
a postcode overlapped with LADs assigned to different
tiers, we used the respondent’s self-reporting of their tier. For exact question wording and additional information
about these variables, see data availability statement. Demographics: wave 1 Among standard sociodemographic questions were age,
gender, education level, and identification with a list of 14
ethnic minority groups (including ‘mixed’) in addition
to ‘Whites’, following the recommendation by the Office
of National Statistics England-specific list (https://www.
ons.gov.uk/methodology/classificationsandstandards/
measuringequality/ethnicgroupnationalidentityandre
ligion). Location is provided as the first section of the
postcode (first three to four digits), which we refer to as
the postal district. Official urban–rural classification data (Census 2011) is
available on the more granular Output Area level that are
linked to postcodes by the ONS. We aggregated these into
districts comprising a number of urban and rural loca-
tions, of which we took the modal category for merging
with our dataset. on October 23, 2024 by guest. Protected by copyright. mjopen.bmj.com/ The linked, final dataset is publicly available, see also
data availability statement.1 App attitudes: wave 2 Patient and public involvement The first set of attitudinal questions, measured on five-
point agreement scales about the app itself tap into four
aspects of an extended version of the Technology Accep-
tance Model to health tech including apps and wearable
devices14 (These variables are subject to missingness,
see 2.4 Analytical framework and online supplemental
appendix figure A1): Patients or the public were not involved in the design,
conduct, or reporting plans of this research. However, a
range of public impact activities connected to the broader
project about inequalities linked to COVID-19 in Britain
will include a summary of this research. Third-party data access: wave 2 Third-party data access: wave 2 We asked both users and nonusers ‘Who do you think
will have access to the data collected by the NHS
COVID-19 app?’ In response, they could use a check-
list of up to eight items or ‘none of these.’ The parties
were listed as follows: the NHS, UK Government,
Local health authority, UK Police, Apple, Google,
Your telecommunications provider, Your internet
network provider. For simplicity, we use concern about
privacy (see above) in our complex multilevel models
predicting app usage and scrutinise privacy further in
section 3.3 (see the Results section) using this measure
separately. The app’s primary purpose is the automa-
tion of contact tracing locally on phone, third-party
access is kept to the minimum by sharing anonymised
data only. Apps match a list of ‘broadcast codes’ and
venues encountered by the app with a list curated by
public health officials showing evidence of infection.15 y
p
p
►The number of new cases recorded by the end of the
week while the survey was in the field, available for all
waves. on October 23, 2024 by guest. Prot
http://bmjopen.bmj.com/
nuary 2022. Downloaded from ►The temporary local restriction tiers at the time of
wave 2 coded tier 1 (medium alert), tier 2 (high alert),
tier 3 (very high alert). ►The dichotomous urban location measure derived
from ONS Rural-Urban Classifications data (in which
a location is classified urban if 74% or more of the
resident population living in urban areas). Trust: all waves In each survey wave, we asked about general trust in
government on a 0–10 scale; we predict wave 2 adoption
with wave 2 trust and wave 3 continued use with wave 3
trust. We expect that the government’s ability to gain and
maintain its citizens’ trust will motivate uptake of contact
tracing apps.3 4 Analytical approach We combine the measures listed above in two sets of
multilevel logistic regression models, first estimating
adoption at wave 2, then depending on wave 2 response
either continued use in wave 3 or new adoption in wave
3. In all cases, we first fit null models estimating variance
of uptake across second-level units (postal districts) as
random intercepts, and continue to add individual and
district-level predictors as appropriate. We scale and
mean-centre all continuous predictors. As we observed
a pattern of non-random missingness on the attitudinal
predictors, we carried out multiple imputation and
pooled the estimates across five imputed datasets, see
online supplemental appendix for further details (As
we show in online supplemental appendix figure A1),
missing data particularly on ease of use is related to non-
adoption thus exclusion of these cases would be inappro-
priate. In practice, omitting these observations had little
impact on our initial uptake and new uptake models, but
had an effect on the significance of three technology
acceptance variables in the continued use model.). For
parsimony, we analyse respondents’ perceptions of third-
party data access by adoption in section 3.3 separately as
their inclusion in the regression models would add eight
additional categorical predictors relating to a similar
underlying concept (privacy). Outcomes pp
g
►The app’s perceived ease of use (or judgement
whether it would be easy to use if respondent has not
adopted the app yet). The dependent variable is adoption measured first in
wave 2, shortly after the app’s roll-out on 24 September
2020 by the Department of Health and Social Care; and
again, in wave 3. We provided the following description
along with a close-ended question: ►Its perceived usefulness to slow the spread of the virus. ►Whether and how concerned respondent is about
privacy when using the app. Contact tracing is a tried and tested method used to
slow down the spread of infectious diseases. Contact
tracing can be done by public health officials or dig-
itally with mobile phone applications or wearable
devices. On the 24th of September, the government ►Social norms in terms of whether people in respond-
ents’ social networks think it is a good idea to use the
app. pp
The second set of questions expand on the above with
questions adopted from the results of the qualitative work Horvath L, et al. BMJ Open 2022;12:e053327. doi:10.1136/bmjopen-2021-053327 3 Second-level (postal district) variables Second-level (postal district) variables The procedure of data linkage is described in section 2.3. District-level characteristics such as case numbers, stricter
local lockdowns or higher population density in urban
and metropolitan locations may affect overall anxiety
and uncertainty that can generate more compliance with
health interventions. We include the following measures
varying across respondents’ postal districts: Compliance: all waves Compliance: all waves Across a set of 20 questions, we asked about the ways in
which respondents have been affected by the corona-
virus. One of these options was ‘Have worn a face mask
when out in public,’ which we use as a proxy for compli-
ance with other non-pharmaceutical public health
interventions to control the spread of COVID-19. on October 23, 2024 by guest. Protected by copyright. http://bmjopen.bmj.com/
s 10.1136/bmjopen-2021-053327 on 17 January 2022. Downloaded from ►Whether respondent needs transparent evidence that
the app is indeed effective. ►Whether respondent needs further information about
how the app treats and uses data. ►Whether respondent needs further information about
how the needs of vulnerable groups (eg, older age)
are addressed. Open access on October 23, 2024 by guest. Pro
http://bmjopen.bmj.com/
BMJ Open: first published as 10.1136/bmjopen-2021-053327 on 17 January 2022. Downloaded from of app users8 on consultation with the study’s authors. The study mode was a ‘rapid online discussion’ event
with a deliberative format (deliberative poll) where
28 members of the public were selected to discuss and
consider a variety of viewpoints about the app while crys-
tallising their own opinions. The additional questions in
our survey reflect the concerns that emerged from this
event (p. 4)8 and are similarly measured on five-point
scales: mobile (followed 100% or mostly complied with stay at
home) and those who likely remained mobile (those who
did not/could not comply in addition to those who did
not need to comply). Uptake and geographical variation We observe uptake in 2020 November at 41%, 95% CI
(0.39% to 0.43%). Of the initial adopters, 124 or 12%
of respondents no longer said they used the app by wave
3, 95% CI (0.10% to 0.14%); while of those initially not
adopting, 98 respondents or 7% reported usage by wave 3,
95% CI (0.05% to 0.08%) (including five who responded
with ‘Don’t know’ in wave 2). Of those not using the app
in wave 2, 36%, 95% CI (0.34% to 0.38%) reported that
they did not own a suitable device (This might seem high,
but given (1) that iPhone 6 and earlier as well as Android
6 (Marshmallow) and earlier phones cannot run the app
(https://faq.covid19.nhs.uk/article/KA-01116/en-us);
(2) the high level of misinformation we report about third
party data access among nonusers and (3) that notable
segments of the UK population perceive the Govern-
ment’s COVID-19 communication as ‘low’ in clarity
according to recent reports,17 we think it 36% of nonusers
may very well have concerns about device compatibility),
1% (16 people) that they were discouraged to use it by
their employer (eg, reports in September 2020 confirmed
that police officers were asked not to instal app on work
phones or ignore advice on personal phones, see https://
www.bbc.co.uk/news/technology-54328644), while the
rest may be linked with other reasons including what we
report in Section 3.2. Although not part of our theoret-
ical framework, we note that respondents who had the
coronavirus were just as likely to be using the app as not
using it: 2.39% (95% CI 1.61% to 3.53%) and 2.71%
(95% CI 1.99% to 3.69%), respectively. By wave 3, people
who had the coronavirus were users of the app in only
slightly higher proportion than non-users, 3.41% (95%
CI 2.44% to 4.75%) and 2.82% (95% CI 2.10% to 3.80%),
respectively, with 95% CIs. on October 23, 2024 by guest. Protected by copyright. http://bmjopen.bmj.com/
bmjopen-2021-053327 on 17 January 2022. Downloaded from Open access on October 23, 2024 by guest. Protected by
http://bmjopen.bmj.com/
BMJ Open: first published as 10.1136/bmjopen-2021-053327 on 17 January 2022. Downloaded from across postcode districts, SD=0.34, shown in figure 2. This
is similar to the magnitude of mobility and compliance
effects and about half of the magnitude of the most influ-
ential attitudinal effects. By contrast, we find no variation
across districts in wave 3 either for continued use or for
new adoption. We explain this by comparing the two time
points in terms of mobility and social contact: while in
November 2020, these districts belonged to different tiers
of restrictions (less open districts in tiers 2 and 3 with
lower mobility thus lower adoption), in March 2021 all
districts faced similar restrictions under a national lock-
down. Beyond local tiers of restrictions, we find little
evidence that COVID-19 case numbers influenced adop-
tion but we found that initial enthusiasm to adopt the app
was higher in urban locations. Mobility: all waves ber 23, 2024 by guest. Protected by copyright. As for behavioural predictors, we asked a set of ques-
tions about stay at home orders including ‘working
from home.’ This predictor draws on an influential
contact study showing that high infection rates particu-
larly in disadvantaged neighbourhoods were explained
by mobility patterns due to these residents’ inability to
work from home.16 The response options were ‘followed
100%’, ‘mostly complied’, ‘mostly not complied’, ‘was
not possible to comply’ and ‘does not apply to me’. We
used working from home as a proxy of more substantial
and regular mobility (for work rather than recreational
purposes). We dichotomised this measure so that we
obtained a group of respondents who were likely not Horvath L, et al. BMJ Open 2022;12:e053327. doi:10.1136/bmjopen-2021-053327 4 Individual-level predictors
W
id
i We provide an overview of the results visually in figure 3
below, and summarise all fitted models and list ORs in
online supplemental appendix table A1. The individual-
level predictors of initial adoption in wave 2 are drawn
from a multilevel model accounting for the postal district-
level variation as shown above. The individual-level predic-
tors of wave 3 continued use (subset of respondents who
were adopters in wave 2) and of new adoption (subset of
respondents who were non-adopters in wave 2) are drawn
from simpler linear models as we found no comparable
variation on the postal district-level (In both cases, the
null model consisting of an intercept and random effects
only, the variance component is either zero (singular)
or would not reach convergence.) and thus multilevel
modelling was not appropriate. on October 23, 2024 by guest. Protecte
http://bmjopen.bmj.com/
nuary 2022. Downloaded from Attitudinal predictors, particularly technology accep-
tance model variables, appear to be the most powerful
predictors of adoption. When it comes to demographics,
we find older respondents less likely to be adopters in
both waves but not more likely to drop out of usage. on October 23, 2024 by guest. Protected by copyright. p://bmjopen.bmj.com/ Looking at the initial measure of adoption in wave
2, our random intercept model detects some variation Figure 2 Geographical variation of uptake percentage (left, plotted here across postal area eg, BH) and log odds and 95% CI
of group-level predictors. Figure 2 Geographical variation of uptake percentage (left, plotted here across postal area eg, BH) and log odds and 95% CI
of group-level predictors. Horvath L, et al. BMJ Open 2022;12:e053327. doi:10.1136/bmjopen-2021-053327 5 on October 23, 2024 by guest. Protected by
http://bmjopen.bmj.com/
BMJ Open: first published as 10.1136/bmjopen-2021-053327 on 17 January 2022. Downloaded from http://bmjopen.bmj.com
BMJ Open: first published as 10.1136/bmjopen-2021-053327 on 17 January 2022. Downloaded from Open access BMJ Open: first published as 10.1136/bmjopen-2021-053327 on 17 January 20 Figure 3 Individual-level predictors: log odds and 95% CI. TAM, technology acceptance model. on October 23, 2024 by guest. Protected by copyright. http://bmjopen.bmj.com/
6/bmjopen-2021-053327 on 17 January 2022. Downloaded from Figure 3 Individual-level predictors: log odds and 95% CI. TAM, technology acceptance model. Education has an impact on new adoption in wave 3 only
with respondents higher than the median education level
more likely to opt into usage. Individual-level predictors
W
id
i We find that ethnic minority
respondents were somewhat less likely to be adopters and
more likely to drop out of initial usage, as opposed to
those identifying with the group ‘Whites’ only. The small
group size (8.20% of the sample) is, however, reflected
in the large uncertainty around the estimate not meeting
conventional thresholds of statistical significance. The items adopted from the deliberative poll have
only small impacts on initial adoption. We expected that
concern about the lack of transparent evidence would
predict non-usage from the outset, but our results suggest
that initial adopters of the app are more likely to think in
these terms. When it comes to continued use, however,
we find that those who had concern about the lack of
transparent evidence were indeed more likely to drop out
of usage. We find small effects regarding information on
data usage with initial adopters who were more likely to
miss this kind of information. We find negligible effects
relating to the vulnerable groups steer. However, we find
that concern about the needs of vulnerable groups is
related to age and work status with retired respondents
a little more likely to express concern, t(1316.4)=−3.18,
p<0.01, mean difference of 0.20; and also people in the
highest age group, t(1191.9)=−2.68, p<0.01, mean differ-
ence of 0.13 on a five point scale. on October 23, 2024 by guest. Protected by copyright. mjopen.bmj.com/ g
In terms of technology acceptance, we find that the
perceived usefulness of digital contact tracing to slow the
spread of the virus is not influential on initial adoption
or wave 3 new adoption. When it comes to continued
use, however, respondents who thought the app was
useful were somewhat more likely to continue using it by
wave 3. Related variables also have large effects: people
concerned about privacy were less likely to adopt the app,
while those who agreed that people in their social circles
(family, friends, work) thought it was a good idea to use
the app were also likely to adopt. See also section 3.2 for
additional insights on privacy. Perceived or expected ease
of use also appears important although the direction of
causality is less clear; users exposed to the app may have
become more confident in its usability. ober 23, 2024 by guest. Protected by copyright. Individual-level predictors
W
id
i Of the rest of the individual-level characteristics, we
note that trust in the UK government, although not
influential in the first decision to adopt was predictive of
new adoption in wave 3. In terms of mobility, people not
working from home were less likely to adopt in wave 2
which may be cause for concern, potentially making the 6 Horvath L, et al. BMJ Open 2022;12:e053327. doi:10.1136/bmjopen-2021-053327 6 Figure 4 Respondents’ perceptions of third-party data
access, wave 2. NHS, National Health Services. Open access on October 23, 2024 by guest. Protected by
http://bmjopen.bmj.com/
BMJ Open: first published as 10.1136/bmjopen-2021-053327 on 17 January 2022. Downloaded from data collection (see https://www.adalovelaceinstitute.
org/project/covid-19-digital-contact-tracing-tracker),
strengthening our survey’s external validity. The observed
drop-out rate between waves 2 and 3 was 12%. We note
that at the timing of our follow-up survey, England was
under national lockdown, thus mobility and social
contact decreased overall, making the app less needed for
regular use (eg, venue check-ins). Similarly, while there
was a lot more overall mobility in wave 2 during initial
uptake, the linked contextual variables showed that the
app was less used where there were some regional restric-
tions. Notably, we found that new adoption (people not
using the app in wave 2 but opting into usage in wave 3
of the survey, 7%) was facilitated by high trust in the UK
government, in line with emerging literature linking trust
to compliance during times of crisis.18 We speculate this
is linked to the increased severity of crisis including new
variants by wave 3, and perceptions about the UK govern-
ment’s early success in its vaccination programme. y
p
g
We found that attitudinal characteristics, notably social
norms and privacy concerns, were powerful predictors of
(non-)adoption. With additional analysis, we also found
that non-users particularly overestimated the potential
for third-party data access including non-health actors
such as broadband and telecom providers. This contrasts
with earlier experimental findings on intention to use
in the UK7 which polled respondents in June 2020 thus
well before the app’s roll-out, documenting little concern
about varying privacy features and or potential data
breaches. We speculate these differences are, on the one
hand, due to study design: In the experimental scenario,
respondents were given complete and transparent infor-
mation about the app’s data usage and storage settings,
whereas in the field this information is likely to be more
opaque, with citizens more risk-averse. Third-party data access Further scrutinising privacy, we ask if users and non-
users have different perceptions about who has access
to the data collected by the app (We also asked about
the kind of data collected by the app, but only from
app adopters. The top answers were: over 60% of users
correctly identified venue check-ins; approximately 40%
incorrectly identified exact location; approximately 20%
correctly identified user-provided health data and the
same percentage incorrectly identified ‘contacts from
phone.’). While above we found that overall ‘concern
about privacy’ is a powerful predictor of non-adoption,
figure 4 below looking at third-party data demonstrates
how this concern may translate into perceptions about
data access. on October 23, 2024 by guest. Protected by copyright. mjopen.bmj.com/ Among other variables, we found work-related mobility
is also associated with adoption16 which partially explains
why indicators of structural inequality (eg, ethnicity) are
less relevant. Yet, after controlling for mobility, ethnic
minority participants are still slightly less likely to adopt,
a phenomenon that might be better explained with
targeted data collection with a larger pool of ethnic
minority respondents. ber 23, 2024 by guest. Protected by copyright. While a plurality of both users and non-users think the
NHS has access to data collected by the app, stark differ-
ences emerge when it comes to other parties such as the
police or broadband providers producing a ¼–¾ split
between users and non-users. While in itself the relation-
ship may not be causal, it is consistent with the model-
based results above regarding privacy concern. Our findings contribute to the understanding of
inequalities around the adoption and public acceptance
of digital technologies supporting the public health
response to the pandemic globally, extending beyond
contract tracing to telemedicine, digital health passports
or targeted public health messaging. To tap inequalities, Individual-level predictors
W
id
i Indeed, in this
study we found transparency of evidence about effective-
ness was a concern that predicted drop-out. On the other
hand, the early versions of the app would have used an
NHS-centralised system as opposed to the final decen-
tralised Exposure Notifications System created by Google
and Apple jointly. While the latter has better overall
privacy preserving characteristics, high public trust in
the NHS may have mitigated concern about privacy more
effectively. on October 23, 2024 by guest. Protecte
http://bmjopen.bmj.com/
nuary 2022. Downloaded from Figure 4 Respondents’ perceptions of third-party data
access, wave 2. NHS, National Health Services. app less effective, but in wave 3, this effect is no longer
significant. Mask wearing predicts adoption in wave 2
only, suggestive of masks becoming increasingly norma-
tive and less contentious in the population as a whole. REFERENCES 1 Hinch R, Probert W, Nurtay A, et al. Effective configurations of a
digital contact tracing APP: a report to NHSX, 2020. 2 Abueg M, Hinch R, Wu N. Modeling the combined effect of digital
exposure notification and non-pharmaceutical interventions on the
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complacency: low concern and low political trust combine to
undermine compliance with governmental restrictions in the
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Relations 2020;81:136843022096798. Twitter Laszlo Horvath @_lhorvath and Andrew Jones @andrewtjones79 Acknowledgements We thank Cary Kind and The Ada Lovelace Institute for
recommending questions to include from their deliberative poll. Acknowledgements We thank Cary Kind and The Ada Lovelace Institute for
recommending questions to include from their deliberative poll. 4 Mohammed A, Johnston RM, van der Linden C. Public responses
to policy reversals: the case of mask usage in Canada during
COVID-19. Canadian Public Policy 2020;46:S119–26. Contributors LH is the author responsible for the overall content as the guarantor. All authors contributed to the study concept and design, and the development of the
questionnaire. LH, SB, AJ and DS have full access to the data and have performed
analysis leading to the results reported in this paper. LH, SB, JB, CD, AJ, OJ, DS
and KT contributed to the interpretation of results, writing and critical revisions. All authors approved the final version to be published and are accountable for all
aspects of the work. y
5 Milsom L, Abeler J, Altmann SM. Survey of acceptability of app-
based contact tracing in the UK, US, France, Germany and Italy
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6 Zhang B, Kreps S, McMurry N, et al. Americans' perceptions of
privacy and surveillance in the COVID-19 pandemic. PLoS One
2020;15:e0242652. 7 Horvath L, Banducci S, James O. Citizens’ Attitudes to Contact
Tracing Apps. J Exp Polit Sci 2020;12:1–13. Funding This research is funded by the Economic and Social Research Council
as part of UK Research and Innovation’s rapid response initiative to COVID-19. The
project is entitled: ‘Identity, Inequality and the Media in Brexit-Covid-19-Britain’
(Grant Ref: ES/V006320/1). Project website: https://www.brexit-studies.org/
covid-19. 8 Panchal M, Singh S, Rodriguez-Villegas E. Analysis of the factors
affecting the adoption and compliance of the NHS COVID-19 mobile
application. medRxiv 2021. pp
9 Garousi V, Cutting D, Felderer M. CONCLUSION AND DISCUSSION Our result regarding general uptake, 41% in November
2020, is consistent with other reports that followed our Horvath L, et al. BMJ Open 2022;12:e053327. doi:10.1136/bmjopen-2021-053327 7 Open access BMJ Open: first published as 10.1136/bmjopen-2021-053327 on 17 January 2 we aimed at an explanatory model with an exhaustive
set of demographic, attitudinal, behavioural and postal
district-level characteristics. of the translations (including but not limited to local regulations, clinical guidelines,
terminology, drug names and drug dosages), and is not responsible for any error
and/or omissions arising from translation and adaptation or otherwise. Open access This is an open access article distributed in accordance with the
Creative Commons Attribution 4.0 Unported (CC BY 4.0) license, which permits
others to copy, redistribute, remix, transform and build upon this work for any
purpose, provided the original work is properly cited, a link to the licence is given,
and indication of whether changes were made. See: https://creativecommons.org/
licenses/by/4.0/. We acknowledge that our study is limited to a sample
of English residents and thus not fully representative of
app usage in both England and Wales, where the NHS
COVID-19 App was released; nor of health behaviour
more generally across the UK. In England, overall
mobility was restricted during wave 3 of our survey which
allowed limited opportunities for app usage such as venue
check-ins (venues were closed). In addition, data collec-
tion in the future would benefit from a booster sample
of ethnic minority participants to better understand
inequalities across diverse ethnic populations: Our results
suggest there may be some ethnicity effects on adoption
even after controlling for a range of other predictors,
however, we lack sufficient numbers to investigate these
mechanisms. http://bmjop
blished as 10.1136/bmjopen-2021-053327 on 17 January 2022. Downloaded from ORCID iD
Laszlo Horvath http://orcid.org/0000-0003-0606-1050 ORCID iD
Laszlo Horvath http://orcid.org/0000-0003-0606-1050 ORCID iD Patient consent for publication Not applicable. 12 Ogrinc G, Davies L, Goodman D, et al. Squire 2.0 (standards for
quality improvement reporting excellence): revised publication
guidelines from a detailed consensus process. BMJ Qual Saf
2016;25:986–92. Ethics approval This study was approved by Prior to data collection, our research
design received ethical approval from the University of Exeter College of Social
Sciences and International Studies Ethics Committee on 16 July 2020, Certificate
No. 201920-131. Our panel vendor ORB International manages informed consent
from their panel respondents. 13 Stevens D, Banducci S, Horvath L, et al. Identity, inequality, and the
media in Brexit-COVID-19-Britain surveys, waves 1-3. figshare 2021. 14 Wang X, White L, Chen X. An empirical study of wearable technology
acceptance in healthcare. Industrial Management & Data Systems
2015:1704–23. REFERENCES Mining user reviews of COVID
contact-tracing apps: an exploratory analysis of nine European apps. arXiv 2012:13589. Map disclaimer The inclusion of any map (including the depiction of any
boundaries therein), or of any geographic or locational reference, does not imply
the expression of any opinion whatsoever on the part of BMJ concerning the legal
status of any country, territory, jurisdiction or area or of its authorities. Any such
expression remains solely that of the relevant source and is not endorsed by BMJ. Maps are provided without any warranty of any kind, either express or implied. 10 Ada Lovelace Institute. Confidence in a crisis? building
trust in a contact tracing APP, 2020. Available: https://www.
adalovelaceinstitute.org/wp-content/uploads/2020/08/Ada-Lovelace-
Institute_COVID-19_Contact_Tracing_Confidence-in-a-crisis-report-
3.pdf on October 23, 2024 by guest. Protected by copyright. mjopen.bmj.com/ 11 Williams SN, Armitage CJ, Tampe T, et al. Public attitudes towards
COVID‐19 contact tracing apps: a UK‐based focus group study. Health Expect 2021;24:377–85. Competing interests None declared. Patient consent for publication Not applicable. Provenance and peer review Not commissioned; externally peer reviewed. Provenance and peer review Not commissioned; externally peer reviewed. 15 Department of Health and Social Care. Nhs COVID-19 APP: privacy
notice. version 28 April 2021. Available: https://www.gov.uk/
government/publications/nhs-covid-19-app-privacy-information/nhs-
covid-19-app-privacy-notice Data availability statement Data are available in a public, open access repository. The questionnaires, case summaries, as well as replication data are available on
figshare: Stevens D, Banducci S, Horvath L, Jones A. Identity, Inequality, and the
Media in Brexit-COVID-19-Britain Surveys, Waves 1–3. figshare; 2021 (cited 9 May
2021). https://doi.org/10.6084/m9.figshare.14527188.V.1 y
16 Chang S, Pierson E, Koh PW, et al. Mobility network models
of COVID-19 explain inequities and inform reopening. Nature
2021;589:82–7. Supplemental material This content has been supplied by the author(s). It has
not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been
peer-reviewed. Any opinions or recommendations discussed are solely those
of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and
responsibility arising from any reliance placed on the content. Where the content
includes any translated material, BMJ does not warrant the accuracy and reliability 17 Abrams D, Lalot F, Broadwood J, et al. Public perceptions of UK and
local government communication about COVID-19. Available: https://
www.belongnetwork.co.uk/wp-content/uploads/2021/08/Belong_
PublicPerceptions_paper_V5.pdf p
p p
p
18 Bargain O, Aminjonov U. Trust and compliance to public health
policies in times of COVID-19. J Public Econ 2020;192:104316. 8 Horvath L, et al. BMJ Open 2022;12:e053327. doi:10.1136/bmjopen-2021-053327
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https://openalex.org/W2093810977
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https://figshare.com/articles/journal_contribution/PDC_Mediated_Tandem_Oxidative_8211_Wittig_Olefination/1009039/1/files/1476662.pdf
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English
| null |
PDC-Mediated Tandem Oxidative–Wittig Olefination
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Synthetic communications
| 2,014
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cc-by
| 2,583
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2. General Procedure To a magnetically stired suspension of pyridinium dichromate (1.85 mmol), Wittig reagent
(1.85 mmol) in anhyd CH2Cl2 (10 mL), alcohol (1.85 mmol) in anhyd CH2Cl2 (10 mL) was added in
one portion. The reaction mixture was stirred at r.t. for 24h. Et2O (30 mL) was added to
the reaction mixture and filtered. The residue was washed with ether (2 X 5 mL) and dried
over anhydrous Na2SO4. The combined filtrate was then evaporated to give the crude product. Further purification by column chromatography using petroleum ether as an eluent gave the
product. PDC mediated tandem oxidative-Wittig Olefination Kashinath L. Dhumaskar, Chinmay Bhat and Santosh G. Tilve* Department of Chemistry, Goa University, Taleigao Plateau, Goa 403 206, India
*stilve@unigoa.ac.in
Supporting Information 1. General remarks Flash chromatography was performed on silica gel (230-400 mesh). IR
spectra were recorded on Shimadzu FT-IR spectrophotometer. 1H NMR (400 MHz) and
13C NMR
(100 MHz) were recorded on a Bruker instrument. Chemical shift (ppm) are relative to the
internal standard Me4Si (0 ppm). 3. Spectral data brown oil, yield 0.33g (99%);
1H NMR (400 MHz,
CDCl3) δ = 1.252 (t, 3H, J = 7.2 Hz, CH3), 4.17 (q, 2H, J = 7.2 Hz, OCH2), 6.24 (d, 1H, J
= 16 Hz, CH), 6.39 (m, 1H, Ar-H), 6.54 (d, 1H, Ar-H), 7.35 (d, 1H, J = 16 Hz, CH), 7.41
(d, 1H, Ar-H);
13C NMR (100 MHz, CDCl3); δ = 14.3 (CH3), 60.4 (OCH2), 112.2 (CH), 114.7
(CH), 115.9 (CH), 130.9 (CH), 144.7 (CH), 150.9 (Cq), 167.0 (Cq); max (thin flim); 1712,
1639, 1209 cm
-1. (3E)-4-(furan-2-yl)but-3-en-2-one2 (3e). Yield, 0.27g (97%), Colourless oil;
1H NMR (400 MHz,
CDCl3); δ = 2.265 (s, 3H, CH3), 6.42 (m, 1H, Ar-H), 6.55 (d, 1H, J = 16 Hz, CH), 6.59 (m,
1H, Ar-H), 7.19 (d, 1H, J = 16 Hz, CH), 7.43 (m, 1H, Ar-H);
13C NMR (100 MHz, CDCl3) δ =
27.6 (CH3), 112.5 (CH), 115.7 (CH), 124.1 (CH), 129.4 (CH), 145.0 (CH), 150.8 (Cq), 197.8
(Cq); max (thin flim); 1685, 1610, 1170 cm
-1. (3E)-4-(furan-2-yl)but-3-en-2-one2 (3e). Yield, 0.27g (97%), Colourless oil;
1H NMR (400 MHz,
CDCl3); δ = 2.265 (s, 3H, CH3), 6.42 (m, 1H, Ar-H), 6.55 (d, 1H, J = 16 Hz, CH), 6.59 (m,
1H, Ar-H), 7.19 (d, 1H, J = 16 Hz, CH), 7.43 (m, 1H, Ar-H);
13C NMR (100 MHz, CDCl3) δ =
27.6 (CH3), 112.5 (CH), 115.7 (CH), 124.1 (CH), 129.4 (CH), 145.0 (CH), 150.8 (Cq), 197.8
(Cq); max (thin flim); 1685, 1610, 1170 cm
-1. Ethyl (2E)-4-(4-methoxyphenyl)but-2-enoate4 (3f). Yield 0.17g, (60%), Colourless oil;
1H NMR
(400 MHz, CDCl3); δ = 1.20 (t, 3H, J = 7.2 Hz, CH3), 3.34 (d, 2H, CH2), 3.74 (s, 3H,OCH3),
4.15 (q, 2H, J = 7.2 Hz, OCH2), 5.76 (td, 1H, J = 16 Hz, J = 1.6 Hz, CH), 6.79 (m, 2H,
Ar-H), 7.01(m, 3H, Ar-H);
13C NMR (100 MHz, CDCl3); δ = 14.3 (CH3), 37.6 (CH2), 55.2
(OCH3), 60.3 (OCH2), 114.1 (2 x CH), 122.1 (CH), 129.7 (Cq), 129.8 (2 x CH), 147.8 (CH),
158.4 (Cq), 166.6 (Cq); max (thin flim); 1716, 1604, 1174 cm
-1 Ethyl (2E)-non-2-enoate1 (3g). 3. Spectral data Ethyl (2E)-3-phenylprop-2-enoate1 (3a). Yield 0.15g (90%), Colourless oil;
1H NMR (400 MHz,
CDCl3); δ = 1.36 (t, 3H, J = 7.2 Hz, CH3), 4.29 (q, 2H, J = 7.2 Hz, CH2), 6.46 (d, 1H, J =
16 Hz, CH), 7.42-7.71 (m, 2H, Ar-H), 7.53-7.58 (m, 2H, Ar-H), 6.46 (d, 1H, J = 16 Hz, CH);
13C NMR (100 MHz, CDCl3); δ = 14.3 (CH3), 60.5(CH2), 118.3 (CH), 128.1 (2 x CH), 128.9 (2 x
CH), 130.23 (CH), 134.5 (Cq), 144.6 (Cq), 167.0 (Cq). max (thin flim); 1705, 1637, 1203cm
-1. Ethyl (2E)-3-(4-nitrophenyl)prop-2-enoate2 (3b). Yield 0.23g (82%), yellow solid;
1H NMR (400
MHz, CDCl3); δ = 1.28 (t, 3H, J = 7.2 Hz, CH3), 4.22 (q, 2H, J = 7.2 Hz, OCH2), 6.49 (d,
1H, J = 16 Hz, CH), 7.59-7.66 (m, 3H, Ar-H and CH), 8.18 (m, 2H, Ar-H),
13C NMR (100 MHz,
CDCl3); δ = 14.3 (CH3), 60.0 (CH2), 122.6 (CH), 124.2 (2 x CH), 128.6 (2 x CH), 140.6
(Cq), 141.6 (CH), 148.4 (Cq), 166.2 (Cq). max (KBr); 1710, 1643, 1253 cm
-1. Ethyl (2E)-3-(4-methoxyphenyl)prop-2-enoate3 (3c). Yield 0.24g (81%), Colourless oil;
1H NMR
(400 MHz, CDCl3); δ = 1.25 (t, 3H, J = 7.2 Hz, CH3), 3.77 (s, 3H,OCH3), 4.17 (q, 2H, J =
7.2 Hz, OCH2), 6.24 (d, 1H, J = 16 Hz, CH), 6.83 (d, 2H, J = 8.8 Hz, Ar-H), 7.41 (d, 2H, J
= 8.8 Hz, Ar-H), 7.575 (d, 1H, J = 16 Hz, CH),
13C NMR (100 MHz, CDCl3) δ = 14.3 (CH3),
55.2 (OCH3), 60.2 (CH2), 114.2 (2 x CH), 115.6 (CH), 121.3 (Cq),129.6 (2 x CH), 144.2 (CH),
161.3 (Cq), 167.4 (Cq); max (thin flim); 1707, 1602, 1170 cm
-1. Ethyl (2E)-3-(4-methoxyphenyl)prop-2-enoate3 (3c). Yield 0.24g (81%), Colourless oil;
1H NMR
(400 MHz, CDCl3); δ = 1.25 (t, 3H, J = 7.2 Hz, CH3), 3.77 (s, 3H,OCH3), 4.17 (q, 2H, J =
7.2 Hz, OCH2), 6.24 (d, 1H, J = 16 Hz, CH), 6.83 (d, 2H, J = 8.8 Hz, Ar-H), 7.41 (d, 2H, J
= 8.8 Hz, Ar-H), 7.575 (d, 1H, J = 16 Hz, CH),
13C NMR (100 MHz, CDCl3) δ = 14.3 (CH3),
55.2 (OCH3), 60.2 (CH2), 114.2 (2 x CH), 115.6 (CH), 121.3 (Cq),129.6 (2 x CH), 144.2 (CH),
161.3 (Cq), 167.4 (Cq); max (thin flim); 1707, 1602, 1170 cm
-1. Ethyl (2E)-3-(furan-2-yl)prop-2-enoate2 (3d). 3. Spectral data Ethyl (2E)-4-methylpent-2-enoate2 (3j). Yield 0.25g (66%); Colourless oil;
1H NMR (400 MHz,
CDCl3); δ = 1.00 (d, 6H, J = 6.8 Hz, CH3), 1.22 (t, 3H, J = 7.2 Hz, CH3), 2.39 (m, 1H,
CH), 4.12 (q, 2H, J = 7.2 Hz, CH2), 5.71 (dd, 1H, J = 16 Hz, J = 1.6 Hz, CH), 6.89 (dd,
1H, J = 16 Hz, J = 1.6 Hz, CH);
13C NMR (100 MHz, CDCl3) δ = 14.2 (CH3), 23.9 (CH), 29.8 (2
x CH3), 60.5 (OCH2), 117.9 (CH), 154.6 (CH), 167.0 (Cq); max (thin flim); 1705, 1654, 1157
cm
-1. Ethyl (2E)-4-methylpent-2-enoate2 (3j). Yield 0.25g (66%); Colourless oil;
1H NMR (400 MHz,
CDCl3); δ = 1.00 (d, 6H, J = 6.8 Hz, CH3), 1.22 (t, 3H, J = 7.2 Hz, CH3), 2.39 (m, 1H,
CH), 4.12 (q, 2H, J = 7.2 Hz, CH2), 5.71 (dd, 1H, J = 16 Hz, J = 1.6 Hz, CH), 6.89 (dd,
1H, J = 16 Hz, J = 1.6 Hz, CH);
13C NMR (100 MHz, CDCl3) δ = 14.2 (CH3), 23.9 (CH), 29.8 (2
x CH3), 60.5 (OCH2), 117.9 (CH), 154.6 (CH), 167.0 (Cq); max (thin flim); 1705, 1654, 1157
cm
-1. Ethyl (2E,4E)-5-phenylpenta-2,4-dienoate2 (3k). Yield 0.22g (73%); Colourless oil;
1H NMR (400
MHz, CDCl3); δ = 1.34 (t, 3H, J = 7.2 Hz, CH3), 4.25 (q, 2H, J = 7.2 Hz, OCH2), 6.01 (d,
1H, J = 16 Hz, CH), 6.90 (d, 1H, J = 8.4 Hz, CH), 7.3-7.50 (m, 7H, Ar-H and vinylic);
13C
NMR (100 MHz, CDCl3) δ = 14.3 (CH3), 60.3 (OCH2), 121.3 (CH), 126.2 (CH), 127.4 (2 x CH),
128.8 (2 x CH), 129.0 (CH), 136.0 (Cq), 140.4 (CH), 144.6 (CH), 167.1 (Cq); max (thin
flim); 1718, 1510, 1176 cm
-1. Ethyl (2E)-penta-2,4-dienoate6 (3l). Yield 0.30g (70%); Colourless oil;
1H NMR (400 MHz,
CDCl3); δ = 1.22 (t, 3H, J = 7.2 Hz, CH3), 4.16 (q, 2H, J = 7.2 Hz, CH2), 5.42 (d, 1H, J =
10 Hz, CH), 5.52 (dd, 1H, J = 17 Hz, 0.4 Hz, CH), 5.82 (dd, 1H, J = 15.2 Hz, 0.4 Hz, CH),
6.38 (m, 1H, CH), 7.19 (m, 1H, J = 11 Hz, CH);
13C NMR (100 MHz, CDCl3); δ = 14.2 (CH3),
60.6 (OCH2), 122.9 (CH), 125.5 (CH), 134.7 (CH), 144.7 (CH), 166.9 (Cq); max (thin flim);
1720, 1651, 1178 cm
-1. Ethyl (2E)-pent-2-en-4-ynoate6 (3m). 3. Spectral data Yield 0.28g (87%), Colourless oil;
1H NMR (400 MHz, CDCl3); δ
= 1.19-1.26 (m, 11H, C5H11), 1.38 (t, 3H, J = 7.2 Hz, CH3), 2.09 (m, 2H, CH2), 4.12 (q, 2H,
J = 7.2 Hz, CH2), 5.73 (td, 1H, J = 16 Hz, 1.6 Hz, CH), 6.89 (td, 1H, J = 16 Hz, 6.8 Hz,
CH);
13C NMR (100 MHz, CDCl3); δ = 14.04 (CH3), 14.25 (CH3), 22.54 (CH2), 27.96 (CH2), 28.99
(CH2), 31.43 (CH2), 32.19 (CH2), 60.11(CH2), 121.16 (CH), 149.5 (CH), 166.8 (Cq); max (thin
flim); 1720, 1654, 1174 cm
-1. Diethyl (2E,6E)-octa-2,6-dienedioate5 (3h). Yield 0.31g (62%); Colourless oil;
1H NMR (400
MHz, CDCl3); δ = 1.30 (t, 6H, J = 7.2 Hz, CH3), 2.39 (d, 4H,CH2), 4.24 (q, 4H, J = 7.2 Hz,
OCH2), 5.87 (d, 1H, J = 16 Hz, CH), 6.95 (d, 1H, J = 16 Hz, CH);
13C NMR (100 MHz, CDCl3) δ
= 14.2 (2 x CH3), 30.4 (2 x CH2), 60.3 (2 x OCH2), 122.3 (2 x CH), 133.6 (2 x Cq), 146.9 (2
x Cq); max (thin flim); 1720, 1654, 1182 cm
-1 Diethyl (2E,4E)-hexa-2,4-dienedioate1 (3i). Yield 0.25 ( 40%); low melting solid;
1H NMR (400
MHz, CDCl3); δ = 1.32 (d, 6H, J = 6 Hz, CH3), 4.24 (t, 4H, J = 6 Hz, OCH2), 6.20 (m, 2H,
CH), 7.32 (m, 2H, CH);
13C NMR (100 MHz, CDCl3) δ = 14.2 (2xCH3), 60.8 (2 x OCH2), 128.3 (2
x CH), 140.8 (2 x CH), 165.9 (2 x Cq); max (KBr); 1730, 1714, 1176 cm
-1. Diethyl (2E,4E)-hexa-2,4-dienedioate1 (3i). Yield 0.25 ( 40%); low melting solid;
1H NMR (400
MHz, CDCl3); δ = 1.32 (d, 6H, J = 6 Hz, CH3), 4.24 (t, 4H, J = 6 Hz, OCH2), 6.20 (m, 2H,
CH), 7.32 (m, 2H, CH);
13C NMR (100 MHz, CDCl3) δ = 14.2 (2xCH3), 60.8 (2 x OCH2), 128.3 (2
x CH), 140.8 (2 x CH), 165.9 (2 x Cq); max (KBr); 1730, 1714, 1176 cm
-1. Diethyl (2E,4E)-hexa-2,4-dienedioate1 (3i). Yield 0.25 ( 40%); low melting solid;
1H NMR (400
MHz, CDCl3); δ = 1.32 (d, 6H, J = 6 Hz, CH3), 4.24 (t, 4H, J = 6 Hz, OCH2), 6.20 (m, 2H,
CH), 7.32 (m, 2H, CH);
13C NMR (100 MHz, CDCl3) δ = 14.2 (2xCH3), 60.8 (2 x OCH2), 128.3 (2
x CH), 140.8 (2 x CH), 165.9 (2 x Cq); max (KBr); 1730, 1714, 1176 cm
-1. 3. Spectral data Yield 0.25g (56%); Colourless oil;
1H NMR (400 MHz,
CDCl3); δ = 1.35 (t, 3H, J = 7.2 Hz, CH3), 3.26 (s, 1H, CH) 4.14 (q, 2H, J = 7.2 Hz,
OCH2), 6.25 (d, 1H, J = 16 Hz, CH), 6.64 (d, 1H, J = 16 Hz, CH);
13C NMR (100 MHz, CDCl3);
δ = 14.2 (CH3), 60.95 (OCH2), 80.2 (Cq), 85.81 (CH), 123.9 (CH), 132.5 (CH), 164.4 (Cq);
max (thin flim); 1720, 1651, 1178 cm
-1. tert-Butyl 2-[(1E)-3-ethoxy-3-oxoprop-1-en-1-yl]pyrrolidine-1-carboxylate7 (3n). Yield, 0.21g (
84%); [α]D
21=79 (c = 1 MeOH); Colourless oil;
1H NMR (400 MHz, CDCl3); δ = 1.21 (t, 3H, J
= 7.2 Hz, CH3), 1.36 (s, 9H, CH3), 1.80 (m, 2H, CH2), 2.02 (m, 2H, CH2), 3.35 (t, 2H, J =
5.2 Hz, CH2), 4.12 (q, 2H, J = 7.2 Hz, OCH2), 4.352 (br, 1H, CH), 5.75 (d, 1H, J = 15.6 Hz,
CH), 6.75 (dd, 1H, J = 15.6 Hz, J = 5.6 Hz, CH);
13C NMR (100 MHz, CDCl3) δ = 14.2 (CH3),22.9 (CH2), 28.2 (3 x CH3), 31.7 (CH2), 46.2 (CH2), 57.8 (CH), 60.4 (OCH2), 79.7 (Cq),
120.4 (CH), 148.5 (CH), 154.3 (Cq), 166.5 (Cq); max (thin flim); 1720, 1693, 1168 cm
-1. (CH3),22.9 (CH2), 28.2 (3 x CH3), 31.7 (CH2), 46.2 (CH2), 57.8 (CH), 60.4 (OCH2), 79.7 (Cq),
120.4 (CH), 148.5 (CH), 154.3 (Cq), 166.5 (Cq); max (thin flim); 1720, 1693, 1168 cm
-1. References 1. Shet, J.; Desai, V.; Tilve, S. Domino Primary Alcohol Oxidation-Wittig Reaction: Total Synthesis
of ABT-418 and (E)-4-Oxonon-2-enoic Acid. Synthesis 2004, 11, 1859-1863. 2. Chintareddy, V. R.; Arkady, E.; Verkade, J. G. P[N(i-Bu)CH2CH2]3N: Nonionic Lewis Base for
Promoting the 1. Shet, J.; Desai, V.; Tilve, S. Domino Primary Alcohol Oxidation-Wittig Reaction: Total Synthesis
of ABT-418 and (E)-4-Oxonon-2-enoic Acid. Synthesis 2004, 11, 1859-1863. 2. Chintareddy, V. R.; Arkady, E.; Verkade, J. G. P[N(i-Bu)CH2CH2]3N: Nonionic Lewis Base for
Promoting the Room-Temperature Synthesis of -Unsaturated Esters, Fluorides, Ketones, and Nitriles Using
Wadsworth-Emmons Phosphonates. J. Org. Chem. 2010, 75, 7166–7174. 3. Cao, P.; Li, C-Y.; Kang, Y-B.; Xie, Z.; Sun, X-L.; Tang, Y. Ph3As-Catalyzed Wittig-Type
Olefination of Aldehydes with Diazoacetate in the Presence of Na2S2O4. J. Org. Chem. 2007, 72, 6628-
6630 4. Narahashi, H.; Shimizu, I.; Yamamoto, A. Synthesis of benzylpalladium complexes through C–O bond
cleavage of benzylic carboxylates: Development of a novel palladium-catalyzed benzylation of
olefins. J. Organomet. Chem. 2008, 693, 283–296. 4. Narahashi, H.; Shimizu, I.; Yamamoto, A. Synthesis of benzylpalladium complexes through C–O bond
cleavage of benzylic carboxylates: Development of a novel palladium-catalyzed benzylation of
olefins. J. Organomet. Chem. 2008, 693, 283–296. 5. Phillips, D. J.; Pillinger, K. S.; Li, W.; Taylor, A. E.; Graham, A. E. Diol desymmetrization as
an approach to the 5. Phillips, D. J.; Pillinger, K. S.; Li, W.; Taylor, A. E.; Graham, A. E. Diol desymmetrization as
an approach to the synthesis of unsymmetrical dienyl diesters. Tetrahedron 2007, 63, 10528–10533. y
y
y
,
,
6. Barrett, A. G. M.; Hamprecht, D.; Ohkubo, M. Dess−Martin Periodinane Oxidation of Alcohols in
the Presence of Stabilized Phosphorus Ylides: A Convenient Method for the Homologation of Alcohols
via Unstable Aldehydes. J. Org. Chem. 1997, 62, 9376–9378. 7. Dieter, R. K.; Oba, G.; Chandupatla, K. R.; Topping, C. M.; Lu, K.; Watson, R. T. Reactivity and
Enantioselectivity in the Reactions of Scalemic Stereogenic -(N-Carbamoyl)alkylcuprates. J. Org. Chem. 2004, 69, 3076-3086. 6. Barrett, A. G. M.; Hamprecht, D.; Ohkubo, M. Dess−Martin Periodinane Oxidation of Alcohols in
the Presence of Stabilized Phosphorus Ylides: A Convenient Method for the Homologation of Alcohols
via Unstable Aldehydes. J. Org. Chem. 1997, 62, 9376–9378. 6. Barrett, A. G. M.; Hamprecht, D.; Ohkubo, M. Dess−Martin Periodinane Oxidation of Alcohols in
the Presence of Stabilized Phosphorus Ylides: A Convenient Method for the Homologation of Alcohols
via Unstable Aldehydes. J. Org. Chem. 1997, 62, 9376–9378. 6. Barrett, A. References G. M.; Hamprecht, D.; Ohkubo, M. Dess−Martin Periodinane Oxidation of Alcohols in
the Presence of Stabilized Phosphorus Ylides: A Convenient Method for the Homologation of Alcohols
via Unstable Aldehydes. J. Org. Chem. 1997, 62, 9376–9378. 7. Dieter, R. K.; Oba, G.; Chandupatla, K. R.; Topping, C. M.; Lu, K.; Watson, R. T. Reactivity and
Enantioselectivity in the Reactions of Scalemic Stereogenic -(N-Carbamoyl)alkylcuprates. J. Org. Chem. 2004, 69, 3076-3086. 4. Spectra 4. Spectra 4. Spectra Ethyl (2E)-3-phenylprop-2-enoate (3a). Ethyl (2E)-3-phenylprop-2-enoate (3a). COOEt COOEt
O2N
Ethyl (2E)-3-(4-nitrophenyl)prop-2-enoate (3b). O2N 2
Ethyl (2E)-3-(4-nitrophenyl)prop-2-enoate (3b). MeO
O
O
Ethyl (2E)-3-(4-methoxyphenyl)prop-2-enoate (3c). MeO Ethyl (2E)-3-(4-methoxyphenyl)prop-2-enoate (3c). O
COOEt
Ethyl (2E)-3-(furan-2-yl)prop-2-enoate (3d). O
Ethyl (2E)-3-(furan-2-yl)prop-2-enoate (3d). Ethyl (2E)-3-(furan-2-yl)prop-2-enoate (3d). Ethyl (2E)-3-(furan-2-yl)prop-2-enoate (3d). O
COMe
(3E)-4-(furan-2-yl)but-3-en-2-one (3e). O
COMe
(3E)-4-(furan-2-yl)but-3-en-2-one (3e). (3E)-4-(furan-2-yl)but-3-en-2-one (3e). (3E)-4-(furan-2-yl)but-3-en-2-one (3e). MeO
COOEt MeO
Ethyl (2E)-4-(4-methoxyphenyl)but-2-enoate (3f). MeO
Ethyl (2E)-4-(4-methoxyphenyl)but-2-enoate (3f). H13C6
COOEt
Ethyl (2E)-non-2-enoate (3g). 13
6
Ethyl (2E)-non-2-enoate (3g). Ethyl (2E)-non-2-enoate (3g). COOEt
EtOOC
Diethyl (2E,6E)-octa-2,6-dienedioate (3h). COOEt
EtOOC
Diethyl (2E,6E)-octa-2,6-dienedioate (3h). COOEt
EtOOC
Diethyl (2E,6E)-octa-2,6-dienedioate (3h). COOEt
EtOOC
diethyl (2E,4E)-hexa-2,4-dienedioate (3i). COOEt
EtOOC
diethyl (2E,4E)-hexa-2,4-dienedioate (3i). yl (2E,4E Ethyl (2E)-4-methylpent-2-enoate (3j). Ethyl (2E)-4-methylpent-2-enoate (3j). Ethyl (2E)-4-methylpent-2-enoate (3j). Ethyl (2E)-4-methylpent-2-enoate (3j). COOEt
Ethyl (2E,4E)-5-phenylpenta-2,4-dienoate (k). COOEt Ethyl (2E,4E)-5-phenylpenta-2,4-dienoate (k). Ethyl (2E,4E)-5-phenylpenta-2,4-dienoate (k). Ethyl (2E)-penta-2,4-dienoate (3l). Ethyl (2E)-penta-2,4-dienoate (3l). Ethyl (2E)-penta-2,4-dienoate (3l). Ethyl (2E)-pent-2-en-4-ynoate (m). Ethyl (2E)-pent-2-en-4-ynoate (m). Ethyl (2E)-pent-2-en-4-ynoate (m). Ethyl (2E)-pent-2-en-4-ynoate (m). Ethyl (2E)-pent-2-en-4-ynoate (m). N
COOEt
O
O N
COOEt
O
O tert-Butyl 2-[(1E)-3-ethoxy-3-oxoprop-1-en-1-yl]pyrrolidine-1-carboxylate (n). tert-Butyl 2-[(1E)-3-ethoxy-3-oxoprop-1-en-1-yl]pyrrolidine-1-carboxylate (n). tert-Butyl 2-[(1E)-3-ethoxy-3-oxoprop-1-en-1-yl]pyrrolidine-1-carboxylate (n).
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English
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Classifying simulated wheat yield responses to changes in temperature and precipitation across a european transect
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International Crop Modelling Symposium
15-17 March 2016, Berlin 15-17 March 2016, Berlin Introduction A wide variety of dynamic crop growth simulation models have been developed over
the past few decades that can differ greatly in their treatment of key processes and
hence in their response to environmental conditions. Here, multi-model ensemble
approaches have been adopted to quantify aspects of uncertainty in simulating yield
responses to climate change (e.g. Asseng et al., 2013). We use a large ensemble of
wheat models applied at sites across a European transect to compare their sensitivity
to changes in climate by plotting them as impact response surfaces (IRSs; Fronzek et
al., 2010). A previous paper using the same simulated yield dataset (Pirttioja et al.,
2015) presented ensemble medians and inter-quartile ranges, focusing on long-term
averages. This paper extends that work by classifying the responses of individual mod-
els and attempting to interpret differences in response between groups of models by
examining results from selected extreme years in addition to the long-term average. Classifying simulated wheat yield responses to changes in temperature
and precipitation across a european transect S. Fronzek
1*
– N. Pirttioja
1
– T. R. Carter
1
– M. Bindi2 – H. Hoffmann2 – T. Palosuo2 –
M. Ruiz-Ramos2 – F. Tao2 – M. Trnka2 – M. Acutis2 – S. Asseng2 – P. Baranowski2 –
B. Basso2 – P. Bodin2 – S. Buis2 – D. Cammarano2 – P. Deligios2 – M.-F. Destain2 –
B. Dumont2 – F. Ewert2 – R. Ferrise2 – L. François2 – T. Gaiser2 – P. Hlavinka2 –
I. Jacquemin2 – K. C. Kersebaum2 – C. Kollas2 – J. Krzyszczak2 – I. J. Lorite2 – J. Minet2 –
M. I. Minguez2 – M. Montesino2 – M. Moriondo2 – C. Müller2 – C. Nendel2 – I. Öztürk2 –
A. Perego2 – A. Rodríguez2 – A. C. Ruane2 – F. Ruget2 – M. Sanna2 – M. A. Semenov2 –
C. Slawinski2 – P. Stratonovitch2 – I. Supit2 – K. Waha2 – E. Wang2 – L. Wu2 – Z. Zhao2 –
R. P. Rötter2 S. Fronzek
1*
– N. Pirttioja
1
– T. R. Carter
1
– M. Bindi2 – H. Hoffmann2 – T. Palosuo2 –
M. Ruiz-Ramos2 – F. Tao2 – M. Trnka2 – M. Acutis2 – S. Asseng2 – P. Baranowski2 –
B. Basso2 – P. Bodin2 – S. Buis2 – D. Cammarano2 – P. Deligios2 – M.-F. Destain2 –
B. Dumont2 – F. Ewert2 – R. Ferrise2 – L. François2 – T. Gaiser2 – P. Hlavinka2 –
I. Jacquemin2 – K. C. Kersebaum2 – C. Kollas2 – J. Krzyszczak2 – I. J. Lorite2 – J. Minet2 –
M. I. Minguez2 – M. Montesino2 – M. Moriondo2 – C. Müller2 – C. Nendel2 – I. Öztürk2 –
A. Perego2 – A. Rodríguez2 – A. C. Ruane2 – F. Ruget2 – M. Sanna2 – M. A. Semenov2 –
C. Slawinski2 – P. Stratonovitch2 – I. Supit2 – K. Waha2 – E. Wang2 – L. Wu2 – Z. Zhao2 –
R. P. Rötter2 1 Finnish Environment Institute (SYKE), 00251 Helsinki, Finland; * Corresponding author
2 Affiliations of co-authors as in Pirttioja et al., (2015) 1 Finnish Environment Institute (SYKE), 00251 Helsinki, Finland; * Corresponding author
2 Affiliations of co-authors as in Pirttioja et al., (2015) International Crop Modelling Symposium
15-17 March 2016, Berlin International Crop Modelling Symposium
15-17 March 2016, Berlin 15-17 March 2016, Berlin Results and Discussion Ensemble median responses showed declining yields with higher temperatures and
decreased precipitation and yield increases with higher precipitation. However, indi-
vidual models departed considerably from the average. An illustration of how respons-
es are classified is given in Fig. 1, which distinguishes three patterns of winter wheat
response across all three sites: (1) maximum yield at temperatures lower than the
baseline, (2) stronger sensitivity to precipitation than temperature changes, and (3)
large yield decreases with cooling and for strong warming. While some models were
grouped into the same classes of response patterns for the different locations and crop
varieties, a single factor could not be identified to explain common model responses. IRSs for anomalous weather-years showed larger model differences than for 30-year
averages (e.g. in a cool year some models simulated crop failure over large parts of the
IRS and others only small reductions relative to the baseline). Figure 1. Ensemble mean changes in winter wheat grain yield ( %) relative to the 1981-2010 baseline for the
three dominant patterns of response identified using a hierarchical clustering approach across all study sites
-2
0
2
4
6
8
-40
-20
0
20
40
Cluster 1 (n=39)
Temperature change ( C)
Precipitation change (%)
-60
-50
-40
-30
-20
-10
0
-2
0
2
4
6
8
-40
-20
0
20
40
Cluster 2 (n=24)
Temperature change ( C)
Precipitation change (%)
-50
-50
-40
-30
-20
-10
0
10
20
-2
0
2
4
6
8
-40
-20
0
20
40
Cluster 6 (n=4)
Temperature change ( C)
Precipitation change (%)
-40
-40
-30
-30
-20
-20
-10
-10
0
0
10
-50
-40
-30
-20
-10
0
10
20 Figure 1. Ensemble mean changes in winter wheat grain yield ( %) relative to the 1981-2010 baseline for the
three dominant patterns of response identified using a hierarchical clustering approach across all study sites
Temperature change ( C)
Temperature change ( C)
Temperature change ( C) Conclusions At the time of writing, analysis of the modelled patterns of response were still ongoing. Preliminary results indicate that the study site is an important determinant of the posi-
tioning of the response pattern for a given crop with respect to baseline climate. Dif-
ferences in the shape and strength of the response pattern, especially under high-end
changes and in anomalous weather-years, appear to be related to the model represen-
tation of processes such as heat stress, moisture stress and vernalisation. Differences
in calibration methods may also contribute to inter-model discrepancies. Acknowledgements This work is part of the FACCE-JPI Knowledge Hub MACSUR. For funding sources see Pirttioja et al., (2015). References
Asseng, S., F. Ewert, C. Rosenzweig et al., (2013). Nature Climate Change 3 (9): 827–32. Fronzek, S., T.R. Carter, J. Räisänen et al., (2010). Climatic Change 99 (3): 515–534. Pirttioja, N., T.R. Carter, S. Fronzek et al., (2015). Climate Research, 65: 87–105. This work is part of the FACCE-JPI Knowledge Hub MACSUR. For funding sources see Pirttioja et al., (2015 This work is part of the FACCE-JPI Knowledge Hub MACSUR. For funding sources see Pirttioja et al., (2015). References Materials and Methods An ensemble of 26 process-based crop models was used to simulate yields of winter
and spring wheat at three sites: in Finland (mainly temperature-limited), Germany
(close to optimal conditions) and Spain (precipitation limited). The sensitivity of simu-
lated yield to systematic increments of changes in temperature (−2 to +9°C) and pre-
cipitation (−50 to +50 %) was tested by modifying values of baseline (1981 to 2010)
daily weather. The results were plotted as IRSs that show the changes in yields relative
to the baseline. IRSs of 30-year averages and selected extreme years were classified
using a hierarchical clustering method and a second approach based on the location of
the maximum yield and strength of the model response. IRSs were classified and com-
pared to aspects of model performance, structure and genealogy (indicating the de-
velopment history and relationships among some of the models). 64 nternational Crop Modelling Symposium
15-17 March 2016, Berlin References 65
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Interactions Increase Forager Availability and Activity in Harvester Ants
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RESEARCH ARTICLE Data Availability Statement: All relevant data are
within the paper and its Supporting Information files. Data Availability Statement: All relevant data are
within the paper and its Supporting Information files. Funding: Field work for EP in 2012 was funded by a
Stanford UAR Student Small Grant, and field work for
JQ in 2013 was funded by a Stanford UAR Student
Major Grant (https://undergrad.stanford.edu/
opportunities/research/get-funded/apply-student-
grants/grant-types). Other expenses were funded by
the grant DMG received from the National Institutes
of Health (grant number R01GM105024, http://www. nih.gov/). The funders had no role in study design,
data collection and analysis, decision to publish, or
preparation of the manuscript. OPEN ACCESS Citation: Pless E, Queirolo J, Pinter-Wollman N,
Crow S, Allen K, Mathur MB, et al. (2015) Interactions
Increase Forager Availability and Activity in Harvester
Ants. PLoS ONE 10(11): e0141971. doi:10.1371/
journal.pone.0141971 Evlyn Pless1☯¤a*, Jovel Queirolo1☯¤b, Noa Pinter-Wollman2, Sam Crow3, Kelsey Allen4,
Maya B. Mathur5, Deborah M. Gordon1 1 Department of Biology, Stanford University, Stanford, California, United States of America, 2 BioCircuits
Institute, University of California, San Diego, La Jolla, California, United States of America, 3 Department of
Computer Science and Engineering, University of Washington, Seattle, Washington, United States of
America, 4 Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology,
Cambridge, Massachusetts, United States of America, 5 Quantitative Sciences Unit, Stanford University,
Stanford, California, United States of America a1111 ☯These authors contributed equally to this work. ¤a Current address: Department of Ecology and Evolution, Yale University, New Haven, Connecticut, United
States of America
¤b Current address: Stanford Teacher Education Program, Stanford University, Stanford, California, United
States of America
* evlyn.pless@yale.edu ☯These authors contributed equally to this work. ¤a Current address: Department of Ecology and Evolution, Yale University, New Haven, Connecticut, United
States of America
¤b Current address: Stanford Teacher Education Program, Stanford University, Stanford, California, United
States of America
* evlyn.pless@yale.edu ☯These authors contributed equally to this work. ¤a Current address: Department of Ecology and Evolution, Yale University, New Haven, Connecticut, United
States of America
¤b Current address: Stanford Teacher Education Program, Stanford University, Stanford, California, United
States of America
* evlyn.pless@yale.edu ¤a Current address: Department of Ecology and Evolution, Yale University, New Haven, Connecticut, United
States of America ¤b Current address: Stanford Teacher Education Program, Stanford University, Stanford, California, United
States of America
*
l
l
@
l
d Abstract Social insect colonies use interactions among workers to regulate collective behavior. Har-
vester ant foragers interact in a chamber just inside the nest entrance, here called the
'entrance chamber'. Previous studies of the activation of foragers in red harvester ants
show that an outgoing forager inside the nest experiences an increase in brief antennal con-
tacts before it leaves the nest to forage. Here we compare the interaction rate experienced
by foragers that left the nest and ants that did not. We found that ants in the entrance cham-
ber that leave the nest to forage experienced more interactions than ants that descend to
the deeper nest without foraging. Additionally, we found that the availability of foragers in
the entrance chamber is associated with the rate of forager return. An increase in the rate of
forager return leads to an increase in the rate at which ants descend to the deeper nest,
which then stimulates more ants to ascend into the entrance chamber. Thus a higher rate of
forager return leads to more available foragers in the entrance chamber. The highest den-
sity of interactions occurs near the nest entrance and the entrances of the tunnels from the
entrance chamber to the deeper nest. Local interactions with returning foragers regulate
both the activation of waiting foragers and the number of foragers available to be activated. Editor: Nicolas Chaline, Universidade de São paulo,
BRAZIL Received: June 4, 2015
Accepted: October 15, 2015
Published: November 5, 2015
Copyright: © 2015 Pless 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. Interactions Regulate Foraging in Harvester Ants [2,3]. Social insect colonies offer a compelling example of collective behavior, as they operate
without any central control [4]. Competing Interests: The authors have declared
that no competing interests exist. In social insect colonies, individual workers use local interactions to perform and regulate
collective behavior [4,5], such as nest construction [6,7], nest relocation [8,9,10], and foraging
[11,12,13,14,15]. Many studies suggest that wasps, honeybees, and ants all use interaction rate
to activate foraging [14,15, 16, 17]. In wasps, foragers may be activated by the biting of incom-
ing foragers and other nestmates, and foragers are bitten more frequently than other wasps
[14]. In honeybees, forager arrival and interactions inside the hive [15], such as the waggle
dance [18] and vibration signals [19], are associated with an increase in outgoing foraging
[15,19], while another 'stop signal’ may counter the waggle dance [18]. Local encounters also play an important role for the organization of foraging in ants, for exam-
ple to respond to the intensity of crowding [20,21,22]. Ants appear to use the rate of antennal con-
tact as an indication of local density [23,24,25] and adjust recruitment and trail networks
accordingly [20,21,22]. For example, Lasius niger ants downregulate the production of recruitment
signals [22] and bifurcate their trail during crowding to maintain a high rate of food return [20]. It appears that interactions also regulate the availability of foragers. For example, interac-
tions among honeybees affect hormonal factors that determine the onset of foraging and there-
fore the number of bees available to forage [26]. Depleting a colony of the older foragers
induces an early onset of foraging in younger bees [26]. Removal experiments in ants also sug-
gest that the availability of foragers depends on interactions, because the removal of foragers
leads other ants to switch tasks and become foragers [27, 28]. Red harvester ants (Pogonomyrmex barbatus) use interaction rates to decide which task to
perform, including whether and when to forage [12,13,29]. Interactions between harvester ants
are tactile and chemical and take the form of brief antennal contacts [30,31]. During an anten-
nal contact, one ant assesses the cuticular hydrocarbon profile of the other, which reveals an
ant’s task and whether it is carrying food [30,31,32]. Foraging poses trade-offs for seed-eating
ants in the desert. Ants must spend water to gain water because foraging in the desert heat can
lead to desiccation, and they obtain some water by metabolizing the fats in the seeds they col-
lect [33,34]. In addition, colonies compete with their neighbors for foraging area [35,36]. How
well a colony manages these trade-offs influences its reproductive success [37]. In harvester ants, returning foragers come into the nest entrance through a tunnel and into
a chamber just inside the entrance that we call the 'entrance chamber' (Fig 1). In previous work
we referred to the 'vestibule' as the area just inside the nest entrance [13], but in this study we
excavated more deeply to observe ants in the full chamber at the end of the entrance tunnel. Foragers deposit the food they collected in this chamber or carry it down tunnels leading from
the entrance chamber to the deeper nest. The sizes and shapes of tunnels and chambers vary
among colonies, and these differences probably influence the rate and location of interactions. Nest structure affects ant movement [38], which affects the rate of interaction [39,40]. In turn,
rate of interaction can influence traffic flow [41,42]. Here we consider three questions about the regulation of foraging through interaction rate
in harvester ants: Introduction A fundamental question in the study of animal behavior and other networks is how simple
individual behaviors add up to complex collective behaviors [1]. Distributed networks, includ-
ing those found in natural populations, are regulated using feedback based on local interactions 1 / 18 PLOS ONE | DOI:10.1371/journal.pone.0141971
November 5, 2015 Question 1: Do outgoing foragers experience a different interaction rate
from other ants? Studies on harvester ants have shown that interactions with successful returning foragers stim-
ulate foraging activity [12,43]. The chemical odors of both a returning forager and the seed it is
carrying are required in combination to stimulate foraging [32]. A previous study showed that
outgoing foragers experience a substantial increase in interactions 3–8 seconds before they 2 / 18 PLOS ONE | DOI:10.1371/journal.pone.0141971
November 5, 2015 Interactions Regulate Foraging in Harvester Ants Fig 1. Diagram of the entrance chamber. A schematic vertical cross section of a typical P. barbatus ant
nest. The solid line indicates the ground surface, and the dashed line indicates where a horizontal section
was made to observe activity inside the nest. Interactions occur in the entrance chamber, connected to the
outside by the entrance tunnel, with tunnels leading to the deeper nest. Fig 1. Diagram of the entrance chamber. A schematic vertical cross section of a typical P. barbatus ant
nest. The solid line indicates the ground surface, and the dashed line indicates where a horizontal section
was made to observe activity inside the nest. Interactions occur in the entrance chamber, connected to the
outside by the entrance tunnel, with tunnels leading to the deeper nest. doi:10.1371/journal.pone.0141971.g001 doi:10.1371/journal.pone.0141971.g001 choose to leave the nest [13]. However, this study examined the interaction rates only of the
foragers that left the nest. Here we test whether ants that left the nest to forage experienced a
higher interaction rate than ants that did not leave the nest to forage and instead descended
from the entrance chamber to the deeper nest without foraging. Question 2: What regulates the number of outgoing foragers available in
the entrance chamber? Previous work on harvester ants shows that while foragers inside the nest respond to interac-
tions within seconds, the pool of ants available to become foragers is regulated on the order of
minutes [13]. Inhibiting forager return for more than four minutes leads to a decrease in the
number of foragers inside the nest entrance that are available to forage (S1 Movie) [13]. Here we examine how the rate of returning foragers regulates the number of ants in the
entrance chamber available to forage. We hypothesized that the descent into the deeper nest of
returning foragers, or other ants transporting food within the nest, influences the rate at which
ants ascend from the deeper nest to the entrance chamber to become available to forage. We
manipulated the rate of forager return to determine the effect on the rate at which ants ascend
from the deeper nest, and return to the deeper nest, thus regulating the numbers in the
entrance chamber available to forage. Question 3: What determines the spatial distribution of interactions? Previous work shows that an ant’s interaction frequency depends on its path shape and loca-
tion [35,38,39,40,41,42,44,45]. By influencing interactions, the shape and size of the entrance
chamber, which varies among colonies, probably affects the regulation of foraging in harvester
ants. Previous studies show that most interactions occur at the entrances of the tunnels from
the entrance chamber to the deeper nest [13]. We asked how manipulating the rate of incoming
foragers would change the spatial distribution and frequency of interactions in the nest. Materials and Methods Data were collected over the course of one week in August 2012 and two weeks in August 2013
at the site of a long-term study in Rodeo, New Mexico [37]. Prof. Gordon’s long-term study 3 / 18 PLOS ONE | DOI:10.1371/journal.pone.0141971
November 5, 2015 Interactions Regulate Foraging in Harvester Ants site is owned by Stanford University, and no permission was required to work on the site. This
study did not involve any endangered or protected species. Nests of P. barbatus have an entrance approximately 2cm in diameter, which leads to one or
more entrance tunnels approximately 5cm long. The entrance tunnels lead into an entrance
chamber approximately 8cm wide, and from this chamber, tunnels descend into the deeper
nest (Fig 1). To observe interactions inside the entrance chamber, we excavated the soil above the
entrance chamber, as in Pinter-Wollman et al. [13]. We positioned a rectangular piece of ply-
wood (23cm x 28cm) such that one of the short edges of the wood was directly over the nest
entrance. The rest of the wood was positioned over the entrance tunnel and entrance chamber. We drew an outline around the wood, removed the wood, and dug out approximately 4cm of
soil from the rectangular area for all films made in 2012 and approximately 9cm of soil for
films made in 2013. The area exposed in 2012 corresponds to the area inside the nest entrance
described as the 'vestibule' in Pinter-Wollman et al. [13]. The area we exposed in the 2013
observations described here included more of the entrance chamber than in 2012. Here we
refer to both as the 'entrance chamber'. We covered the area with the wood overnight so that
the ants would acclimate to their new nest ceiling. Each morning between 6am and 11am, we
removed the wood and covered the excavated area with a rectangular glass sheet (23cm x
28cm) for filming (Fig 2). Previous studies show that the incoming light does not disturb the
ants [13]. In 2012 we filmed three colonies for three consecutive days, and in 2013 we filmed six colo-
nies over a period of 10 days (S1 Table). We converted the films into JPEGs (30 frames/second)
using Adobe Premiere Elements 7.0 in 2012 and Java software that we developed in 2013. PLOS ONE | DOI:10.1371/journal.pone.0141971
November 5, 2015 Materials and Methods We
observed the films of ants in the entrance chamber, which included all area covered by the glass
sheet and all area immediately around the glass window which was depressed at the same level
as the rest of the ant chamber. When observing the films for each of our analyses, we used the following definitions for cat-
egorizing ant activities: 1. Outgoing foragers: ants that leave the entrance chamber and go outside not carrying
anything 2. Returning foragers: ants that come from outside of the nest and enter the nest (Observations
were made at a time when most ants outside the nest were foragers, but it is possible that
some of the ants considered to be returning foragers were ants of another task that did not
carry a food item.) 3. Descending ants: ants that were first observed in the entrance chamber and go down a tun-
nel into the deeper nest 4. Ascending ants: ants that were in the deeper nest, come up a tunnel, and emerge into the
entrance chamber 5. Nest maintenance workers: ants carrying dirt or debris inside the nest To test whether outgoing foragers experience a different interaction rate from other ants
(Question 1), we made 3 sets of measurements from the films made in 2012. First, we deter-
mined what percentage of the ants that ascended from the deeper nest into the entrance cham-
ber (‘ascending ants’) later performed each of the following: outgoing foraging, descending into
the deeper nest, or nest maintenance. The focal ants for this analysis were chosen by selecting
the first ant to emerge from the deeper nest at five-second intervals. Second, we observed out-
going foragers and recorded the time and location of all their interactions, in the form of brief PLOS ONE | DOI:10.1371/journal.pone.0141971
November 5, 2015 4 / 18 Interactions Regulate Foraging in Harvester Ants Fig 2. Labeled entrance chamber. A photograph of the entrance chamber of colony N_4 covered with the window used during filming. The tunnels to the
deeper nest are labeled, as is the trail of returning and outgoing foragers. doi:10 1371/journal pone 0141971 g002 Fig 2. Labeled entrance chamber. A photograph of the entrance chamber of colony N_4 covered with the window
deeper nest are labeled, as is the trail of returning and outgoing foragers. Fig 2. Labeled entrance chamber. PLOS ONE | DOI:10.1371/journal.pone.0141971
November 5, 2015 Interactions Regulate Foraging in Harvester Ants Fig 3. Comparison of activity distribution by colony. Each bar shows the mean proportion of ascending ants that foraged (black), did nest maintenance
(white), or ascended into the nest entrance but then descended back into the deeper nest without leaving the nest to forage (grey), over the course of three
days. Error bars show standard errors of the mean. Fig 3. Comparison of activity distribution by colony. Each bar shows the mean proportion of ascending ants that foraged (black), did nest maintenance
(white), or ascended into the nest entrance but then descended back into the deeper nest without leaving the nest to forage (grey), over the course of three
days. Error bars show standard errors of the mean. doi:10.1371/journal.pone.0141971.g003 obstruction, and ants that did not perform any of the indicated behavior until the end of the
film were excluded from analysis. Out of 147 possible focal ants per colony (pooled over the
three days), 16 were excluded from the analysis of N_2, 21 from the analysis of N_4, and 9
from the analysis of N_13 (S1 Dataset). To record the interactions of outgoing foraging and descending ants, we analyzed the JPEGs
from the films using a custom written MATLAB script as in Pinter-Wollman et al. [13] (code
provided in S1 Appendix). We observed ten outgoing foragers and four or five descending ants
per film, the first foraging and descending ants in the film segment that were clearly visible,
from when the ant entered the entrance chamber from the deeper nest to when she either left
the entrance chamber or descended to the deeper nest. (Five descending ants were selected for
each film except for the film of N_2 on August 15th, which showed only four descending ants.)
We recorded the time each ant entered and left the entrance chamber, and the time and loca-
tion of each interaction of the focal ant. Interactions were recorded when the focal ant made
antennal contact with the head or body of another ant, and the location of the interaction was
defined as the point in between the head of the first ant and the place of contact on the second
ant. Our analysis did not examine interactions with other outgoing foragers, because previous
work showed that only successful returning foragers stimulated foraging [43]. PLOS ONE | DOI:10.1371/journal.pone.0141971
November 5, 2015 Materials and Methods A photograph of the entrance chamber of colony N_4 covered with the window used during filming. The tunnels to the
deeper nest are labeled, as is the trail of returning and outgoing foragers. doi:10.1371/journal.pone.0141971.g002 doi:10.1371/journal.pone.0141971.g002 doi:10.1371/journal.pone.0141971.g002 antennal contacts, before leaving the entrance chamber. Third, we recorded the time and loca-
tions of the interactions of descending ants. The first clearly visible foraging and descending
ants from each video were chosen as the focal ants for the second and third analyses. To determine the sample size of outgoing foragers, we considered the possibility that if the
proportion of foragers were extremely different in the three colonies observed, a given sample
size would not be equally representative in all three colonies. We found that the proportions
foraging in the three colonies were not significantly different (ANOVA, F2,6 = 3.854; p = 0.08)
(Fig 3) (S1 Dataset). Therefore, we decided to measure interaction rates in the same number of
foragers and descending ants in all three colonies, rather than choose different sample sizes for
each colony. To determine what percentage of the ants that ascended from the deeper nest into the
entrance chamber were outgoing foragers, descending ants, or nest maintenance workers
within our 5–7 minutes of film, we observed randomly selected ants in each of the nine films
(three per colony for three colonies taken over the course of three days, S1 Table). Every five
seconds for the first four minutes of film, the first ant to ascend from the entrance tunnel was
tracked until it left the nest, performed nest maintenance by moving dirt or debris around the
nest entrance, or descended to the deeper nest. Ants that were lost from view due to glare or an 5 / 18 PLOS ONE | DOI:10.1371/journal.pone.0141971
November 5, 2015 3. rates of ascending ants and descending ants 3. rates of ascending ants and descending ants We tested for cross-correlations in each of the three combinations of variables listed above,
to determine whether the two variables tended to be correlated in time, and if so, with what lag. For example, if an ant in the entrance chamber often descended to the deeper nest 10 seconds
after a returning forager came in, then there would be a positive correlation at a lag of 10 sec-
onds in the rates of returning foragers and descending ants. To normalize each time series for
differences in numbers of ants, we subtracted from each point the moving average of each time
series using a time window that was half the duration of each film (S2 Table). The timescale of
the smoothing window was minutes (median 7 minutes), whereas observed lags were on the
order of seconds (median ~15 seconds), and thus the results were probably not affected by this
normalization procedure. To identify significant cross-correlations, indicating a time dependence for two rates, we
calculated the likelihood of a correlation for each possible lag time, relative to the null hypothe-
sis of no time dependence. We report lag times that were statistically significant at p < 0.01. In
addition, we ran 1000 simulations in which the time of each event (ant returning, ascending, or
descending) was randomly assigned from a uniform distribution covering the average length of
the videos. We then compared the proportion of significant cross correlations in the simulated
data with that proportion in the observed data. A smaller proportion of significant events in
the simulated data compared with the observed indicates that the observed correlations were
not found by chance. We next investigated the spatial distribution of interactions (Question 3). Using Java software we developed, we split the films into JPEG frames (at 30 frames per sec-
ond) and marked the location in the entrance chamber of each interaction of the ants we
tracked. The locations of interactions were recorded in two instantaneous images, one at the
start of the removal of returning foragers, and the other immediately after forager removals
ended 3–5 minutes later. To illustrate the spatial pattern of interactions, we used a 2D Gaussian kernel density esti-
mator in R package MASS to produce 13 pairs of utilization distribution maps for the 13 trials
with forager removals [46]. An outgoing for-
ager was identified as an ant that moved directly toward the outside of the nest and immedi-
ately left the nest as soon as it reached the edge of the entrance chamber. We used a linear multilevel model with normal errors, fit via REML using R’s lme4 package,
to determine if the rate of interactions and time spent in the entrance chamber differed
between outgoing foragers and descending ants [46]. To account for colony and day effects, ant 6 / 18 PLOS ONE | DOI:10.1371/journal.pone.0141971
November 5, 2015 Interactions Regulate Foraging in Harvester Ants activity was a fixed effect with colony and day as random effects with normally distributed
error terms. To investigate what regulates the number of outgoing foragers available in the entrance
chamber (Question 2), we filmed the entrance chamber in the morning when the ants were for-
aging and relatively undisturbed for about 6 minutes in 2013 (S1 Table). To test how changes
in the rate of forager return influence the rate at which ants ascend into and descend from the
entrance chamber, we manipulated forager return rate. Returning foragers were removed for
3–5 minutes (depending on the amount of time needed to collect most of the returning forag-
ers) and kept in a plastic box (and returned to the nest after observations were completed), as
in Gordon et al. [12]. We observed and filmed behavior inside the nest during and after forager
removals. We performed removal experiments in 13 out of the 16 trials (S1 Table). We counted from the films the numbers of returning and outgoing foragers at the nest
entrance and the number of ascending and descending ants at all tunnel entrances. We used
MATLAB to test for cross-correlations (p < 0.01) among: 1. rates of returning foragers and ascending ants 2. rates of returning foragers and descending ants PLOS ONE | DOI:10.1371/journal.pone.0141971
November 5, 2015 1. Interaction rates of outgoing foragers The mean (±SD) time outgoing foragers were tracked was 7.51 (±7.43) seconds (range 0.401 to
36.6) and the mean time descending ants were tracked was 16.0 (±10.8) seconds (range 2.91 to
47.6). Although sampling times varied widely, the distribution was similar across each colony
(S1 Fig). The mean (±SD) interaction rate for outgoing foragers was 1.47 (±1.09) interactions per sec-
ond, while the mean interaction rate for descending ants was 0.83 (±0.587) interactions per sec-
ond (S2 Dataset). Taking into account differences in colony- and day-specific interaction rates,
the interaction rates of foragers were significantly higher than those of descending ants (linear
multilevel model, b = 0.64; 95% CI: 0.33, 0.95; p < 0.001). (The coefficient (b) represents the
model-based estimate for the increase in interaction rate between foragers and descending
ants.) Foragers also spent a shorter time in the entrance chamber than ants that eventually
descended to the deeper nest (linear multilevel model with normal errors, b = -8.55; 95% CI:
11.65, -5.44; p<0.001). For illustrative purposes, interaction rates and time in the entrance
chamber by colony (pooling across all three days) are shown in Fig 4. The three colonies differed in the interaction rates of outgoing foragers (ANOVA, F2,27 =
8.42; p = 0.0004) and descending ants (ANOVA, F2,11 = 4.83; p = 0.0002), and all differed in
pairwise tests except colonies N_2 and N_13 (N_2 vs N_13 Tukey multiple comparison of
means, p = 0.622 for outgoing foragers, p = 0.92 for descending). The three days also differed
in the interaction rates of outgoing foragers (ANOVA, F2,27 = 5.64; p = 0.005) and descending
ants (ANOVA, F2,11 = 12.21; p <0.0001), due to a difference between August 13 and August 14
(Tukey multiple comparisons of means, p = 0.0033 for outgoing foragers, p < 0.0001 for
descending ants). Within the 5–7 minutes of each of our films, the proportion of ascending ants that left the
nest ranged from 0.31–0.89, (mean (±SD) 0.59 (±0.18)) (Fig 3) (S1 Dataset). The proportion of
ascending ants that performed nest maintenance, carrying soil or debris out of or around the
nest, ranged from 0.00–0.083 (mean (±SD) 0.067 (±0.048)), and the proportion that returned
to the deeper nest ranged from 0.056–0.65 (mean (±SD) 0.34 (±0.19)) (Fig 3). 1. Interaction rates of outgoing foragers In all colonies
and days, significantly more ascending ants left the nest (Student’s t-test, p<0.0001) or
returned to the deeper nest (Student’s t-test, p = 0.0036) than did nest maintenance. In seven of
the nine colonies and days, more ants left the nest than returned to the deeper nest without
foraging. 3. rates of ascending ants and descending ants These maps show the spatial density of interactions in the entrance
chamber at the start and at the end of the forager removals. 7 / 18 PLOS ONE | DOI:10.1371/journal.pone.0141971
November 5, 2015 Interactions Regulate Foraging in Harvester Ants PLOS ONE | DOI:10.1371/journal.pone.0141971
November 5, 2015 2. Availability of foragers in entrance chamber There was evidence of time-dependence among the rates at which ants returned to the nest,
ascended from the deeper nest to the entrance chamber, and descended from the entrance
chamber to the deeper nest (S3 Dataset). We found significant cross-correlations between the
rates of returning foragers and ascending ants, returning foragers and descending ants, and
ascending ants and descending ants in some of the trials, when removals were performed, for
each of the 3 colonies. (Table 1; Cross-correlation, p < 0.01). The cross-correlation between
rates of returning foragers and ascending ants was significant in one out of three trials for col-
ony 367 and one out of two trials for colony 229 (2/5 trials) (Table 1). The cross-correlation
between rates of returning foragers and descending ants was significant in one out of three tri-
als for colony N_5, two out of three trials for colony 367, one out of one trial for colony 25, and
one out of one trials for colony 242 (5/7 trials) (Table 1). The cross-correlation between rates
of ascending ants and descending ants was significant in two out of three trials for colony N_5, 8 / 18 PLOS ONE | DOI:10.1371/journal.pone.0141971
November 5, 2015 Interactions Regulate Foraging in Harvester Ants Table 1. Lag times found after removals were performed. The lag values in seconds between returning foragers and ascending ants, returning foragers
and descending ants, and ascending ants and descending ants. Mean and standard deviation across all colonies and days also shown. For cross-correla-
tions in which more than one lag time was detected, all significant lags are reported. A “—” indicates that no significant lags were found. Colony name
Date
filmed
Lag times between returning
foragers and ascending (seconds)
Lag times between returning foragers
and descending ants (seconds)
Lag times between ascending and
descending ants (seconds)
N_5
8-17-
2013
-
13
-
N_5
8-18-
2013
-
-
5
N_5
8-20-
2013
-
-
30, 68
367
8-18-
2013
-
6, 26
-
367
8-20-
2013
11, 61
2, 17, 45
13, 29, 69
367
8-21-
2013
-
-
-
868
8-19-
2013
-
-
43
868
8-20-
2013
-
-
4
868
8-21-
2013
-
-
-
25
8-24-
2013
-
22
-
229
8-24-
2013
-
-
-
229
8-26-
2013
58
-
62, 68
242
8-25-
2013
-
13
-
Mean
43.33
18
39.1
Standard
Deviation
28.04
13.42
26.59
doi:10.1371/journal.pone.0141971.t001 cross-correlations: colony 367 on August 20, 2013. There were two statistically significant lag
times found between the returning foragers and ascending ants, three for returning foragers
and descending ants, and three for ascending ants and descending ants. Fig 5 shows all the sta-
tistically significant cross correlations from this trial. The rate at which ants descended into the deeper nest depended significantly on the rate of
returning foragers. Of the three relationships considered, the shortest lag time was between for-
agers returning and ants descending into the deeper nest. The mean (±SD) lag, 18.00 (±13.42)
seconds, between returning foragers and descending ants was significantly shorter than the lag
between ascending ants and descending ants, 39.10 (±26.59) seconds (Student’s t-test,
p = 0.048) (Fig 6). It was also significantly shorter than the lag between returning foragers and
ascending ants, 43.33 (±28.04) seconds (Student’s t-test, p = 0.032) (Fig 6). Some colonies
showed significant lag times between more than one of the tested relationships, while other col-
onies had only one (Table 1). Interactions Regulate Foraging in Harvester Ants Fig 4. Comparison of interaction rate and time in entrance chamber in outgoing foragers and descending ants. A) Each bar shows the mean
brief antennal contacts, in interactions per second, of ants in the entrance chamber. B) Each bar shows the mean time in seconds that ants spent in
entrance chamber. Black: ants that subsequently left the nest to forage (outgoing foragers); White: ants that descended from the entrance chamber
deeper nest (descending ants). Error bars show standard errors of the mean. doi:10.1371/journal.pone.0141971.g004 Fig 4. Comparison of interaction rate and time in entrance chamber in outgoing foragers and descending ants. A) Each bar shows the mean rate of
brief antennal contacts, in interactions per second, of ants in the entrance chamber. B) Each bar shows the mean time in seconds that ants spent in the
entrance chamber. Black: ants that subsequently left the nest to forage (outgoing foragers); White: ants that descended from the entrance chamber to the
deeper nest (descending ants). Error bars show standard errors of the mean. doi:10.1371/journal.pone.0141971.g004 doi:10.1371/journal.pone.0141971.g004 one out of three trials for colony 367, two out of three trials for colony 868, and one out of two
trials for colony 229 (9/11 trials) (Table 1). The simulations in which ascending, descending,
and returning ant times were chosen from a uniform distribution produced only 76/1000 sig-
nificant cross correlations, a smaller proportion than any of the observed cross correlations
(0.076 for the randomization simulations compared with 0.4, 0.71, and 0.82 for the observed
data). There was one removal trial that produced statistically significant lag times for all three PLOS ONE | DOI:10.1371/journal.pone.0141971
November 5, 2015 PLOS ONE | DOI:10.1371/journal.pone.0141971
November 5, 2015 9 / 18 doi:10.1371/journal.pone.0141971.t001 3. Spatial distribution of interactions Considering each of the thirteen films individually, eight films
showed significantly fewer interactions after removals than before removals: colony 367 on 8-
20-13 and 8-21-13, colony 868 on 8-19-13 and 8-21-13, colony 25 on 8-24-13, colony 229 on
8-24-13 and 8-26-13, and colony 242 on 8-25-13 (Binomial test, p < 0.05) (Fig 7). 8) (S4 Dataset, S1 Movie). Considering each of the thirteen films individually, eight films
showed significantly fewer interactions after removals than before removals: colony 367 on 8-
20-13 and 8-21-13, colony 868 on 8-19-13 and 8-21-13, colony 25 on 8-24-13, colony 229 on
8-24-13 and 8-26-13, and colony 242 on 8-25-13 (Binomial test, p < 0.05) (Fig 7). The interaction utilization distribution maps show that the highest number of interactions
occurs at the entrances to the tunnels to the deeper nest, both before and after removals (Fig 8)
(S4 Dataset). 3. Spatial distribution of interactions Several minutes after the rate of returning foragers was experimentally reduced, there were sig-
nificantly fewer ant interactions in the entrance chamber (Paired t-test, p = 0.003) (Figs 7 and 10 / 18 PLOS ONE | DOI:10.1371/journal.pone.0141971
November 5, 2015 Interactions Regulate Foraging in Harvester Ants Fig 5. Comparison of significant cross-correlations in lag times for a single colony. All lag times are
from colony 367 on August 20, 2013, the only trial that showed significant lag times for all three cross-
correlations. Each bar shows the lag of a significant cross-correlation between 1) rate of forager return and
rate at which ants ascended to the entrance chamber from the deeper nest, 2) rate of forager return and rate
at which ants descended to the deeper nest, and 3) rate at which ants ascended to the entrance chamber and
rate at which ants descended to the deeper nest. Fig 5. Comparison of significant cross-correlations in lag times for a single colony. All lag times are
from colony 367 on August 20, 2013, the only trial that showed significant lag times for all three cross-
correlations. Each bar shows the lag of a significant cross-correlation between 1) rate of forager return and
rate at which ants ascended to the entrance chamber from the deeper nest, 2) rate of forager return and rate
at which ants descended to the deeper nest, and 3) rate at which ants ascended to the entrance chamber and
rate at which ants descended to the deeper nest. Fig 5. Comparison of significant cross-correlations in lag times for a single colony. All lag times are
from colony 367 on August 20, 2013, the only trial that showed significant lag times for all three cross-
correlations. Each bar shows the lag of a significant cross-correlation between 1) rate of forager return and
rate at which ants ascended to the entrance chamber from the deeper nest, 2) rate of forager return and rate
at which ants descended to the deeper nest, and 3) rate at which ants ascended to the entrance chamber and
rate at which ants descended to the deeper nest. doi:10.1371/journal.pone.0141971.g005 8) (S4 Dataset, S1 Movie). PLOS ONE | DOI:10.1371/journal.pone.0141971
November 5, 2015 Interactions Regulate Foraging in Harvester Ants Fig 6. Comparison of time lags between ant activities. Each bar shows the duration in seconds of the lag between two rates, with data pooled across six
colonies and eight days: 1) rate of forager return and rate at which ants ascended to the entrance chamber from the deeper nest, 2) rate of forager return and
rate at which ants descended to the deeper nest from the entrance chamber, and 3) rate at which ants ascended to the entrance chamber from the deeper
nest and rate at which ants descended to the deeper nest from the entrance chamber. The figure shows lag values only for the cross-correlations that were
statistically significant. Error bars show standard errors of the mean. Numbers above bars signify sample sizes. Fig 6. Comparison of time lags between ant activities. Each bar shows the duration in seconds of the lag between two rates, with data pooled across six
colonies and eight days: 1) rate of forager return and rate at which ants ascended to the entrance chamber from the deeper nest, 2) rate of forager return and
rate at which ants descended to the deeper nest from the entrance chamber, and 3) rate at which ants ascended to the entrance chamber from the deeper
nest and rate at which ants descended to the deeper nest from the entrance chamber. The figure shows lag values only for the cross-correlations that were
statistically significant. Error bars show standard errors of the mean. Numbers above bars signify sample sizes. doi:10.1371/journal.pone.0141971.g006 interactions per sec). Ants that left the nest to forage also spent significantly less time in the
entrance chamber (mean 7.51 sec) than those that descended to the deeper nest (mean 16.03
sec). Our results show that during the period of foraging activity, the rate of forager return helps
to regulate the rate at which foragers become available to forage. Most of the ants in the
entrance chamber are foragers: of the ants that came from the deeper nest into the entrance
chamber, most left the nest to forage. Almost half the ants that did not forage went back down
into the deeper nest (Fig 2). The regulation of the number of ants in the entrance chamber allows the colony to adjust
forager availability quickly in response to changing conditions. PLOS ONE | DOI:10.1371/journal.pone.0141971
November 5, 2015 Discussion Ants in the entrance chamber that leave the nest to forage have experienced more interactions,
in the form of brief antennal contacts, than ants that descend to the deeper nest without forag-
ing. The availability of foragers in the entrance chamber is regulated by another process that
determines the flow of ants into and out of the entrance chamber from the deeper nest. This
movement of ants in and out of the entrance chamber from the deeper nest, which determines
the availability of foragers, is associated with the rate of forager return. The more frequently
that returning foragers come in, the more frequently ants descend from the entrance chamber
to the deeper nest, and in turn, the more frequently foragers ascend to the entrance chamber
from the deeper nest. In this way, higher rates of forager return lead to the presence of more
available foragers, as previously observed [13]. Interactions occur at highest density in the nest
entrance and the entrances of the tunnels leading to the deeper nest. Our result supports previous work showing that ants in the entrance chamber determine
whether to forage based on the rate of interactions with returning, successful foragers
[11,12,32] and showed that the rate of forager return is correlated with the rate of interactions
inside the nest [13]. Here we show directly that the ants that left the entrance chamber to forage
had experienced a significantly higher rate of interactions (mean 1.47 interactions per sec) than
ants that descended from the entrance chamber to the deeper nest without foraging (mean 0.83 PLOS ONE | DOI:10.1371/journal.pone.0141971
November 5, 2015 11 / 18 When food availability is high,
foragers find food quickly, leading to a high rate of forager return [47]. The number of ants in
the entrance chamber depends on the rate of forager return [13]. The rate of forager return is
linked with a short lag (mean 18 seconds) to the rate at which ants descend to the deeper nest
(Table 1). The returning foragers sometimes deposit their food in the entrance chamber for
other ants to carry to the lower nest, but in the exposed entrance chambers that we filmed,
more often returning foragers descended still carrying the food items they brought back (more 12 / 18 PLOS ONE | DOI:10.1371/journal.pone.0141971
November 5, 2015 Interactions Regulate Foraging in Harvester Ants Fig 7. Comparison of number of interactions in the entrance chamber before and after removals. Black: the number of interactions observed from one film frame at the start of forager removals; White: the
number of interactions observed from one film frame 3–5 minutes after removal of returning foragers was
completed. doi 10 1371/jo rnal pone 0141971 g007 Fig 7. Comparison of number of interactions in the entrance chamber before and after removals. Black: the number of interactions observed from one film frame at the start of forager removals; White: the
number of interactions observed from one film frame 3–5 minutes after removal of returning foragers was
completed. doi:10.1371/journal.pone.0141971.g007 doi:10.1371/journal.pone.0141971.g007 discussion below). The rate at which foragers ascend is linked with a significantly longer lag
(mean 38.25 seconds) to the rate at which ants descend (Table 1). When the rate of forager
return slows, indicating low food availability, the number of available foragers in the entrance
chamber also slows [13]. The number of interactions in the entrance chamber nest decreases
with low forager return (Figs 7 and 8), further decreasing the probability ants will leave the nest
to forage (Fig 4A). The process that regulates the numbers in the entrance chamber is very rapid; most ants in
the entrance chamber either forage or descend in less than 20 seconds. The rapid turnover of
ants entering and leaving the entrance chamber may help to limit the number of waiting ants
in the entrance chamber [13], who could slow the rate of interactions with incoming, successful
foragers. The different lags between rates of forager return, ascending ants, and descending ants may
be due to different processes. PLOS ONE | DOI:10.1371/journal.pone.0141971
November 5, 2015 Interactions Regulate Foraging in Harvester Ants Fig 8. Spatial distribution of interactions. Each interaction utilization distribution map shows the location of interactions, brief antennal cont
ants, that were observed from instantaneous images. The left figure of each pair shows interactions at the very beginning of an experiment tha
the rate of incoming foragers, and the right figure shows interactions 3–5 minutes after the experiment was complete. White shows the areas o
interaction density for each pair of heat maps, yellow shows areas with some interactions, and red shows areas with no interactions. The tunn
is indicated with a T and the nest entrance/exit is indicated with an N. The scales for size and color are relative to each pair of maps. doi:10.1371/journal.pone.0141971.g008 Fig 8. Spatial distribution of interactions. Each interaction utilization distribution map shows the location of interactions, brief antennal contacts between
ants, that were observed from instantaneous images. The left figure of each pair shows interactions at the very beginning of an experiment that decreased
the rate of incoming foragers, and the right figure shows interactions 3–5 minutes after the experiment was complete. White shows the areas of highest
interaction density for each pair of heat maps, yellow shows areas with some interactions, and red shows areas with no interactions. The tunnel entrance/ex
is indicated with a T and the nest entrance/exit is indicated with an N. The scales for size and color are relative to each pair of maps. Fig 8. Spatial distribution of interactions. Each interaction utilization distribution map shows the location of interactions, brief antennal contacts between
ants, that were observed from instantaneous images. The left figure of each pair shows interactions at the very beginning of an experiment that decreased
the rate of incoming foragers, and the right figure shows interactions 3–5 minutes after the experiment was complete. White shows the areas of highest
interaction density for each pair of heat maps, yellow shows areas with some interactions, and red shows areas with no interactions. The tunnel entrance/exit
is indicated with a T and the nest entrance/exit is indicated with an N. The scales for size and color are relative to each pair of maps. Fig 8. Spatial distribution of interactions. Each interaction utilization distribution map shows the location of interactions, brief antennal contacts between
ants, that were observed from instantaneous images. For example, the lag times between ascending ants and descend-
ing ants were approximately factors of one another in two cases (30 and 68 seconds for colony
N_5 on 8-20-13; and 13, 29, and 69 seconds for colony 367 on 8-20-13). This suggests a period-
icity in the behavior of ascending into and descending from the entrance chamber. It is inter-
esting to note that the lag between returning foragers and ascending ants tends to be larger
than both the lags between returning foragers and descending ants and between ascending and
descending ants. This is what we would expect if the same ants return from foraging, descend
to the deeper nest, drop off their food, and then ascend back to the entrance chamber to be
available to leave on the next foraging trip. More work is needed to determine how often
returning foragers carry their food into the deeper nest, or instead deposit it in the entrance
chamber for other ants to transport down to the seed chambers in the deeper nest. A number of factors influence the spatial distribution and frequency of interactions. Har-
vester ant colonies vary consistently from year to year in foraging activity [12,37]. These PLOS ONE | DOI:10.1371/journal.pone.0141971
November 5, 2015 13 / 18 The left figure of each pair shows interactions at the very beginning of an experiment that decreased
the rate of incoming foragers, and the right figure shows interactions 3–5 minutes after the experiment was complete. White shows the areas of highest
interaction density for each pair of heat maps, yellow shows areas with some interactions, and red shows areas with no interactions. The tunnel entrance/exit
is indicated with a T and the nest entrance/exit is indicated with an N. The scales for size and color are relative to each pair of maps. doi:10.1371/journal.pone.0141971.g008 doi:10.1371/journal.pone.0141971.g008 variations may arise from colony-specific differences in how interaction rates affect the forag-
ing decisions of ants. The three colonies filmed in 2013 differed in interaction rates, and inter-
action rates differed between two of the days (August 14th and August 15th). Colony
differences in interaction rate could be related to variation in nest structure [38,41,42]. Nests
vary in the size and shape of the entrance chamber and in the number of tunnels leading from
the chamber to the deeper nest (Fig 8). This variation may lead to differences among colonies PLOS ONE | DOI:10.1371/journal.pone.0141971
November 5, 2015 PLOS ONE | DOI:10.1371/journal.pone.0141971
November 5, 2015 14 / 18 Interactions Regulate Foraging in Harvester Ants in the timing of interactions and in the relation between the rate of forager return and of the
flow of ants into the entrance chamber. In another species of harvester ant, the rate of recruit-
ment to food increased with the number of tunnels leading from the entrance chamber to the
deeper nest [48]. Our heat maps of interaction rate (Fig 8) show that interactions in the
entrance chamber tend to occur at the exit to the surface and at the entrances to tunnels leading
down to the deeper nest. Conclusions Individual workers in social insect colonies respond to rates of interaction with other workers. Collectively, this allows a colony to regulate its behavior and respond to changing conditions. Here we show that interactions regulate both the activation of foragers and the availability of
foragers to be activated. Variation among harvester ant colonies in the regulation of foraging is
associated with variation in reproductive success [37]. Investigating the allocation of effort in
ant colonies contributes to a fundamental question in biology: how local interactions produce
the collective behavior of the whole system. Supporting Information S1 Appendix. Matlab script for recording information about ant interactions. This is a
Matlab function that opens up a stack of JPEG images and allows a user to move through the
images and ‘click’ anywhere on any image to record x-y coordinates of events in the frame. The
output is a csv file with 6 columns: xy coordinates of the click, time = frame/image number, ant
ID, ant type, and activity—both of which are pre-defined by the user, see below. Copyright (C)
2015 Noa Pinter-Wollman. (PDF) S1 Dataset. 2012 Ant Activity Data. We observed ants in two minutes of each of the nine
films to determine what percentage of the ants that ascended from the deeper nest into the
entrance chamber were later outgoing foragers, descending ants, or nest maintenance workers. Every five seconds, the first ant to ascend from the entrance tunnel was tracked until it left the
nest, performed nest maintenance by moving dirt or debris around the nest entrance, or
descended to the deeper nest. This dataset shows the activity type of each of the tracked ants. (PDF) S2 Dataset. 2012 Ant Interactions Data. We analyzed the JPEGs from the films using a cus-
tom written MATLAB script as in Pinter-Wollman et al. [13] (code provided in S1 Appendix). We observed ten outgoing foragers and four or five descending ants per film, the first foraging
and descending ants in the video segment that were clearly visible. This dataset shows the time
each ant entered and left the entrance chamber, and the time and location of each interaction
of the focal ants. (PDF) S2 Dataset. 2012 Ant Interactions Data. We analyzed the JPEGs from the films using a cus-
tom written MATLAB script as in Pinter-Wollman et al. [13] (code provided in S1 Appendix). We observed ten outgoing foragers and four or five descending ants per film, the first foraging
and descending ants in the video segment that were clearly visible. This dataset shows the time
each ant entered and left the entrance chamber, and the time and location of each interaction
of the focal ants. (PDF) S3 Dataset. 2013 Correlation Data. We observed and filmed behavior inside the nest during
and after forager removals. This dataset shows our counts made from the films of the numbers
of returning and outgoing foragers at the nest entrance and the number of ascending and
descending ants at all tunnel entrances. Acknowledgments We thank LeAnn Howard, Andrew Merrell, Stefan Popp, Jack Rasiel, Wallis Robinson, Brett
Salazar, Daniel Washington, and Charlotte Wayne for assistance in the field. We are grateful to
Luke Frishkoff, Aaron Goodman, and Lane McIntosh for help with coding and plotting data. Mark Goldman provided very helpful discussion and advice. We thank Tomer Czackes and an
anonymous reviewer whose comments greatly improved the manuscript. Author Contributions Conceived and designed the experiments: EP JQ NP-W DMG. Performed the experiments: EP
JQ. Analyzed the data: EP JQ NP-W SC KA MBM DMG. Contributed reagents/materials/anal-
ysis tools: NP-W SC KA. Wrote the paper: EP JQ NP-W DMG. Supporting Information (ZIP) S3 Dataset. 2013 Correlation Data. We observed and filmed behavior inside the nest during
and after forager removals. This dataset shows our counts made from the films of the numbers
of returning and outgoing foragers at the nest entrance and the number of ascending and
descending ants at all tunnel entrances. (ZIP) S4 Dataset. 2013 Heat Maps Data. After splitting the 2013 videos into JPEG frames, we
marked the location in the entrance chamber of each interaction of the ants we tracked. This
dataset shows the locations of interactions from two instantaneous images, one at the start of S4 Dataset. 2013 Heat Maps Data. After splitting the 2013 videos into JPEG frames, we
marked the location in the entrance chamber of each interaction of the ants we tracked. This
dataset shows the locations of interactions from two instantaneous images, one at the start of PLOS ONE | DOI:10.1371/journal.pone.0141971
November 5, 2015 15 / 18 Interactions Regulate Foraging in Harvester Ants the removal of returning foragers, and the other immediately after forager removals ended 3–5
minutes later. (PDF) the removal of returning foragers, and the other immediately after forager removals ended 3–5
minutes later. (PDF) S1 Fig. Sampling time distribution histograms. These histograms show the distribution of
times that foraging and descending ants filmed in 2012 remained in the entrance chamber. (TIF) S1 Fig. Sampling time distribution histograms. These histograms show the distribution of
times that foraging and descending ants filmed in 2012 remained in the entrance chamber. (TIF) S1 Movie. Interactions over time for a single colony. The attached clip shows the location of
interactions, in the form of brief antennal contacts between ants, in colony 367 on August 20,
2013. Removal of returning foragers began at 00:37 and ended at 05:41. There is an observable
difference between 00:37 seconds and 2:37. By 2:37, as the rate of forager return decreases, the
ants are no longer as spread out in the entrance chamber, and interactions occur only at the
interaction hotspots (Fig 8), the entrance to tunnels to the deeper nest and the exit from the
chamber to outside the nest. The circular metal device is a thermometer and humidity sensor. Some of the ants were painted previously for a different experiment. (MP4) S1 Table. Red harvester ant colonies filmed in August 2012 and August 2013.
(DOCX) S1 Table. Red harvester ant colonies filmed in August 2012 and August 2013. (DOCX) PLOS ONE | DOI:10.1371/journal.pone.0141971
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ences in the foraging activity of harvester ants. Behavioral Ecology. 2009; 20: 633–638. 48. Pinter-Wollman N. Nest architecture shapes the collective behavior of harvester ants. Biology Letter. 2015, in press. 18 / 18
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Rezension von: Schmidt, Ernst, Heimatgeschichtliches Lesebuch
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Städte und Orte
555
Linke, Juden, „Zigeuner“, Zwangsarbeiter, Homosexuelle und andere so genannte „Volksschädlinge“ geprägt; Folterungen und willkürliche Exekutionen in großer Zahl waren an
der Tagesordnung. Betroffene haben ihn beschrieben als „fauchenden Zwerg und zappelnden Sadisten mit kreischender Stimme“. Bei Kriegsende kam er als Kriegsverbrecher in
US-Untersuchungshaft, wo er 1946 seinem Leben selbst ein Ende setzte.
Der leidvolle Lebensweg des unbeugsamen Widerständlers Hans Gasparitsch (1918 – 2002)
wird von Peter Poguntke behandelt. Gasparitsch wuchs im proletarischen Milieu des Stuttgarter Ostens auf, einer Hochburg von Sozialisten und Kommunisten. Mit wacher Sorge
beobachteten er und sein Freundeskreis den Aufstieg des Nationalsozialismus. Die Machtergreifung Hitlers war für Gasparitsch ein Signal zum offenen Widerstand durch das Anbringen nazifeindlicher Parolen im öffentlichen Raum. Er wurde gefasst, womit ein über
zehnjähriger lebensgefährlicher Leidensweg durch Gefängnisse und Konzentrationslager
begann, welcher erst im April 1945 im KZ Buchenwald endete. Auch nach der Befreiung
setzte Gasparitsch sein reges politisches Engagement im Sinn seiner sozialistisch-kommunistischen Überzeugung in vielfältiger Weise fort. In der jungen DDR holte er das Abitur
nach und studierte Journalismus und Bauingenieurwesen. Den letztgenannten Beruf übte er,
nun wieder oft auch im Westen, bis 1980 aus. Erst 2000 wurde er mit einem Bundesverdienstkreuz gewürdigt.
Herausgeber und Autoren haben den flüssig zu lesenden Sammelband bewusst genau
siebzig Jahre nach Zusammenbruch und Befreiung vom Nazi-Regime publiziert. Ein wisHelmut Gerber
senschaftlicher Apparat rundet das Buch ab.
Ernst Schmidt, Hohenlohe Waldenburg. Heimatgeschichtliches Lesebuch, überarb. Fassung 2016, kommentiert und hg. vom Waldenburger Kreis für Kulturgeschichte, zum
Gedenken an Ernst Schmidt (Veröffentlichungen zur Ortsgeschichte und Heimatkunde
in Württembergisch Franken, Bd. 26), Waldenburg 2016. 346 S. mit 83 meist farb. Abb.
€ 16,–
Ernst Schmidt (1874 – 1952), in Waldenburg geboren, war bis 1937 Postbeamter im württembergischen Staats- und später im Reichsdienst. Auch im Ruhestand arbeitete er während
des 2. Weltkrieges eine Zeit lang als kaufmännischer Angestellter in einer Bedarfsgüter
für den Krieg herstellenden Fabrik. Gegen Kriegsende nach Hohenlohe zurückgekehrt,
widmete sich Ernst Schmidt ganz der Geschichte seiner Heimatstadt Waldenburg, die durch
den Krieg schwer zerstört worden war. Zunächst entstanden die für seine Kinder und Geschwister abgefassten persönlichen Jugenderinnerungen und seit Winter 1944/45 das heimatgeschichtliche Lesebuch, das er 1951 im Manuskript abschloss. Schmidt stellte ihm die
Widmung „Meiner leidgeprüften Heimatstadt und dem angestammten Fürstenhaus Hohenlohe-Waldenburg“ (Titelseite) voran. Die Arbeit wurde jedoch nie gedruckt, sondern stand
nur als maschinenschriftliches Manuskript mit einer „Auflage“ von ca. 300 Stück für einen
heimatgeschichtlich interessierten Leserkreis zur Verfügung.
Dank des Engagements von ehrenamtlich engagierten Mitgliedern des Waldenburger
Kreises für Kulturgeschichte konnte der Wunsch nach Veröffentlichung nach 45 Jahren realisiert werden. Die Arbeit Schmidts wurde sorgfältig überarbeitet, etwas aktualisiert und
liegt nun als gedrucktes Buch vor. Erläuterungen und Ergänzungen sowie Anmerkungen
ergänzen den ursprünglichen Text, wobei immer deutlich bleibt, was vom Autor und was
von den Bearbeitern stammt. Im Druck bleibt sogar die Seitenverteilung der Original
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© Kommission für geschichtliche Landeskunde in Baden-Württemberg und
Württembergischer Geschichts- und Altertumsverein e.V.
ISSN 0044-3786
556
Buchbesprechungen
fassung erkennbar, um das Auffinden von Zitaten zu ermöglichen. Durch Beigabe zahl
reicher Abbildungen illustrierten die Bearbeiter den Text und lockerten ihn dadurch auf.
Sie fügten im Anhang des Buches die Jugenderinnerungen Schmidts „Bilderbuch aus meiner
Jugendzeit“ (S. 296 – 311) und einen Auszug aus dem Schriftwechsel 1945 bis 1953 zur
Entstehung des Lesebuches bei (S. 312 – 331). Am Schluss befindet sich eine Stammtafel der
Familie Schmidt (vom 18. bis 20. Jh.); ein Personenindex, ein Ortsindex sowie ein „Stichwortverzeichnis“ erleichtern dem Leser die Orientierung.
Somit wurde die Arbeit eines Waldenburger Hobbyhistorikers durch ein gedrucktes
Buch einem größeren Leserkreis zugänglich gemacht. Es handelt sich um eine Ehrung für
Ernst Schmidt, der durch die zurückhaltende Redaktion seines Textes und durch die Bei
fügung der – persönlichen – Jugenderinnerungen und die Stammtafel seiner Familie sehr in
den Mittelpunkt gerät. Das „Heimatgeschichtliche Lesebuch“ richtet sich an Leser, die sich
– wie Ernst Schmidt – der Region Hohenlohe und ganz besonders der Stadt Waldenburg
eng verbunden fühlen und daher auch an ihrer Geschichte besonders interessiert sind.
Peter Schiffer
Markus Josef Maier, Würzburg zur Zeit des Fürstbischofs Julius Echter von Mespelbrunn
(1570 – 1617). Neue Beiträge zu Baugeschichte und Stadtbild (Veröffentlichungen des
Stadtarchivs Würzburg, Bd. 20), Würzburg: Verlag Ferdinand Schöningh 2016. XVII,
579 S., 80 Abb. Mit CD-ROM. ISBN 978-3-87717-857-7. € 39,90
Stadtbilder verändern sich. Trotz der intensiven Bautätigkeit in den letzten Jahrhunderten
und auch der massiven Zerstörung ihrer Bausubstanz im Bombenangriff vom 16. März 1945
zum Trotz ist das Wirken des Fürstbischofs Julius Echter als Bauherr für das Erscheinungsbild der Stadt Würzburg bis heute prägend geblieben. Der Bautätigkeit in der fürstbischöflichen Residenzstadt in den Jahrzehnten um 1600 geht Markus Josef Maier in seiner von
Stephan Kummer betreuten Dissertationsschrift nach. Dabei geht er höchst kenntnisreich
und mit sehr viel Sinn für Details vor, ohne sich darin zu verlieren.
Die vorliegende Studie kann an einige ältere Forschungs- und Überblickswerke anknüpfen. Indem Markus Josef Maier städtische Bestände sowie die Akten des Domkapitels und
einzelner Stifte zu zentralen Ausgangspunkten seiner Forschung macht, kann er nicht
nur die Begrenzungen der Überlieferung in den fürstlichen Unterlagen sprengen, sondern
auch eine weite Perspektive auf die gesamte Bautätigkeit in der Stadt während der langen
Herrschaftszeit Julius Echters einnehmen. Außerdem hat er sich eine Vielzahl von Bilddokumenten erschlossen, die er kompakt gebunden in der Mitte des Bandes zur Illustration –
allerdings mit sehr knappen Bildunterschriften – präsentiert. Maiers Fragestellung bezieht
sich auf der Basis dieser Quellenlage und -auswahl nicht nur auf die fürstbischöfliche Bautätigkeit, sondern auf eine umfängliche Erfassung des Stadtbildes von Würzburg und seinen
Wandlungen in der Echterzeit – bis hin zu Fragen der Pflasterung und Abwasserbeseitigung
(S. 45).
Die eigentliche Darstellung beginnt mit einem höchst suggestiven, aber anschaulichen
fiktiven Rundgang durch die Stadt Würzburg vor 1570, der auf guter Quellenkenntnis basiert. Dieser geht über eine detaillierte Aufnahme aller Bautätigkeiten zur Zeit Julius Echters
hinaus, weil er die Vorstellungskraft und Phantasie des Lesers anregt. Dabei gilt, dass sich
der Autor in seinen Schilderungen auf belegbare Einzelheiten konzentriert. Zugleich gewährt die Darstellung einen Einblick in die Dynamik des Untersuchungszeitraums. Diese
Zeitschrift für Württembergische Landesgeschichte 76 (2017)
© Kommission für geschichtliche Landeskunde in Baden-Württemberg und
Württembergischer Geschichts- und Altertumsverein e.V.
ISSN 0044-3786
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Behavioral aspects of Helicoverpa armigera in the cotton vegetative phase1
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RESUMO Aspectos comportamentais de Helicoverpa
armigera na fase vegetativa do algodoeiro Helicoverpa armigera (Hübner) is part of an important
complex of insects-pests that attack the cotton crop. This
study aimed to identify the preferential plant parts for the
oviposition of moths, as well as the movement and feeding
behavior of caterpillars, in the vegetative phase of the cotton
cultivar BRS 369 RF. Plants emitting floral buds were used to
study the oviposition behavior of moths. For this, a completely
randomized design, with three treatments (adaxial and abaxial
sides of the leaf and floral buds) and twenty replications (plants
in pots), was used. In order to evaluate the behavior of the
caterpillars in the cotton vegetative phase, another 3 × 5 factorial
design, with nine replications, was used. The treatments were
three ages (first, second and third instars) combined with five
behaviors (walking, resting, waving, eating and drop-off). In
this trial, the period of the day when caterpillars are more active
was also evaluated. It was observed that H. armigera moths
prefer to lay their eggs on the adaxial leaf side and on the last
two nodes of the plant, in a descending direction. The adaxial
leaf side is also the part of the plant preferred by caterpillars
for walking, eating and resting, being this last behavior the
most frequent in all instars. In general, the caterpillars walk
more in the morning. Helicoverpa armigera (Hübner) faz parte de um importante
complexo de insetos-pragas que atacam a cultura do algodoeiro. Objetivou-se identificar partes da planta preferenciais para a
oviposição das mariposas, bem como os comportamentos de
movimentação e alimentação das lagartas, na fase vegetativa da
cultivar de algodão BRS 369 RF. Plantas emitindo botões florais
foram utilizadas para estudar o comportamento de oviposição
das mariposas. Para isso, utilizou-se delineamento inteiramente
casualizado, com três tratamentos (faces adaxial e abaxial da folha
e botões florais) e vinte repetições (plantas em vasos). Para avaliar
o comportamento das lagartas na fase vegetativa do algodoeiro,
adotou-se outro delineamento fatorial 3 × 5, com nove repetições. Os
tratamentos foram três idades (1º, 2º e 3º ínstares) combinadas com
cinco comportamentos (caminhar, repousar, acenar, comer e cair). Neste ensaio, o período do dia em que as lagartas são mais ativas
também foi avaliado. Constatou-se que as mariposas de H. armigera
preferem ovipositar na face adaxial da folha e nos dois últimos nós
da planta, em direção descendente. 1. Received: Oct. 30, 2018. Accepted: Apr. 17, 2019. Published: Oct. 15, 2019. DOI: 10.1590/1983-40632019v4955628.
2. Empresa Brasileira de Pesquisa Agropecuária (Embrapa Algodão), Campina Grande, PB, Brasil.
E-mail/ORCID: sandra.rodrigues@embrapa.br/0000-0001-9043-4694.
3. Universidade Federal de Mato Grosso, Instituto de Ciências Agrárias e Ambientais, Sinop, MT, Brasil.
E-mail/ORCID: nubiadocarmo@hotmail.com/0000-0001-9923-4437, janadenadai@gmail.com/0000-0002-1013-5414.
4. Empresa Brasileira de Pesquisa Agropecuária (Embrapa Agrossilvipastoril), Sinop, MT, Brasil.
E-mail/ORCID: rafael.pitta@embrapa.br/0000-0002-8503-7106. e-ISSN 1983-4063 - www.agro.ufg.br/pat - Pesq. Agropec. Trop., Goiânia, v. 49, e55628, 2019 Research Article RESUMO A face adaxial da folha também
é a parte da planta preferida pelas lagartas para caminhar, comer e
repousar, sendo este último comportamento o mais frequente em todos
os ínstares. Em geral, as lagartas caminham mais no período matutino. PALAVRAS-CHAVE: Gossypium hirsutum, Heliothinae,
etologia. KEYWORDS: Gossypium hirsutum, Heliothinae, ethology. PALAVRAS-CHAVE: Gossypium hirsutum, Heliothinae,
etologia. MATERIAL AND METHODS The experiments were carried out at the
Embrapa Agrossilvipastoril (Sinop, Mato Grosso
state, Brazil), in 2017. The moths and caterpillars
used were obtained from those reared in laboratory,
fed with an artificial diet proposed by Parra (2001). The use of an artificial diet removed the possibility
of alterations in the adult behavior due to host plant
experience in the larval stage. Seeds of the BRS 369
RF cotton cultivar (not Bt) were sown into 5 L pots
and kept in a greenhouse, and the cotton plant received
the necessary cultural treatment for its development. l Heliothinae moths prefer to feed and oviposit on
plants that contain flowers and nectar (Cunninghan &
Zalucki 2014). The females oviposit in cotton at night
and eggs are laid single (Topper 1987, Ahmad 2004). The attack may begin in the vegetative phase, with
small caterpillars consuming the leaves (Pascua &
Pascua 2002), and then later in the reproductive phase,
with larger caterpillars feeding on flower buds, flowers
and bolls (Gomes et al. 2017). Experimental evidence
for learning of H. armigera has demonstrated that a
previous experience with a host species increases the
relative attractiveness of that host (Cunningham et
al. 1998). Therefore, preferences for different hosts
displayed by adult moths which have encountered
a host species may be vastly different from the
preferences of moths without any host experience
(Cunningham et al. 1999). When the plants began to emit flower buds,
they were then taken to the laboratory and housed in
a cage (1 m × 1 m × 1 m), in groups of five plants, and
kept in a controlled environment (25 ± 1 ºC and 60 ±
1 % RH), with a 13-h scotophase, since oviposition
occurs at night (Topper 1987). The moths used had
72 h of emergence and were previously mated and
fed with a honey solution. At 6 p.m., the moths were
released into the cages and remained in the plants
for 13 h. The ratio used was one moth to each plant
and, in the following morning, the number of eggs
deposited on the adaxial and abaxial surface of the
leaves and floral buds were counted, as well as the
vertical distribution of the eggs on the plants. The main method for controlling this pest
has been to use synthetic insecticides, what is
often performed inadequately (Costa et al. 2018). INTRODUCTION Mato Grosso state is the largest producer, with 777.8
thousand hectares of cotton, corresponding to 66.1 %
of the area planted in the country. The projected cotton
seed production for this state is 3,213.9 thousand tons,
27.1 % higher than in the 2016/2017 harvest, with a
yield of 4,132 kg ha-1 (Conab 2018). Brazil is among the largest cotton producers
and the planted area in the 2017/2018 crop season
was 1,176.1 thousand hectares, with an expected
production of 4,947,000 tons of cotton seed and
1,979.4 thousand tons of fiber. The Central-West region
is responsible for 70.4 % of this production, where the The crop is attacked by a complex of insects-
pests capable of infesting all the phenological plant S. M. M. Rodrigues et al. (2019) 2 phases (Santos 2015), and its production may be
significantly reduced if control measures are not
adopted at the right time. Within this complex is
Helicoverpa armigera (Hübner) (Lepidoptera:
Noctuidae), a pest detected in Brazil in 2013, in
soybean and cotton crops (Czepak et al. 2013). This
pest is important worldwide, because it has a wide
geographic distribution, polyphagous behavior and
high biotic potential (Zalucki et al. 1986, Behere et
al. 2013, Silva et al. 2018). the moths’ preferential oviposition plant sites in the
reproductive phase of the cotton crop, as well as the
caterpillars’ behavior of the 1st, 2nd and 3rd instars,
in the vegetative phase of the cotton crop. e-ISSN 1983-4063 - www.agro.ufg.br/pat - Pesq. Agropec. Trop., Goiânia, v. 49, e55628, 2019 MATERIAL AND METHODS The crawling, resting, waving, feeding
and drop-off behaviors were observed regarding the
frequency of use of the different plant sites. Waving,
according to Johnson & Zalucki (2005), is when the
caterpillar has its front legs raised and shakes its head
from side to side, and this indicates a search behavior. The observations were made with the aid of a pocket
magnifying glass (40x magnification) and performed
every 30 min. Furthermore, the period of the day
when the largest larval movement occurs and which
plant region is preferred by the caterpillars to carry
out the five behaviors were also evaluated. The adaxial surface (18.7 eggs) was the most
preferred, followed by the abaxial surface (13.5 eggs)
and the floral bud (3.4 eggs) (Table 1), similarly to
the results observed by Butter & Singh (1996), in
cotton. It was found that the eggs present on the
adaxial surface of the leaf were generally on the leaf
veins which have trichomes. It is known that the
plant morphological and chemical characteristics are
involved in choosing the oviposition site (Navasero &
Ramaswamy 1991). Sites with trichomes are probably
chosen by H. armigera to oviposite (Zalucki et al. 1986, Luong et al. 2016) because these structures
may hinder the movement of natural enemies
when searching for their prey or hosts, resulting in
protection for the pests’ progeny (Vendramim 2002). if A significant difference was observed between
the nodes, regarding the vertical distribution of the
eggs in the plant. All nodes presented leaves with the
presence of eggs, except the node one. The largest
ovipositions occurred at nodes 8 (6.2 eggs) and 9 (6.5
eggs), corresponding to the nodes at the top of the
plant (Figure 1). Its preference for the upper third of
the plant is closely related to its food habit, since it
mainly attacks flower buds and cotton flowers that
are concentrated in the middle and upper thirds of
the plant. Olsen & Daly (2000) also verified this
fact in cotton and Santos et al. (2017) in soybean, a
behavior also found in Chloridea virescens and H. zea
Heliothinae (Torres & Ruberson 2006). To study the caterpillar behavior in the cotton
vegetative phase, other completely randomized
design, with nine replications, was used. MATERIAL AND METHODS The continuous use of insecticides has selected
populations of pests which are resistant to various
molecules (Mota-Sanches & Wise 2018). Another
factor related to the ineffectiveness of insecticides is
the pest location in parts of the plant which hinder the
deposition of insecticide drops, thus compromising
the minimum amount of active ingredient to cause the
target death (Costa et al. 2018). In order to perform
both sampling and application of insecticides
efficiently, it is necessary to know the habits of the
target insect. Considering that caterpillars in the first
instars are more vulnerable to insecticides, knowing
the behavior of the pest in these stages is fundamental
to assist in the decision-making on managing this
pest. Such information will enable detecting how
best to sample the insect in the field, as well as in
what period of the day it will be most exposed to the
applied chemicals. To study the oviposition behavior of moths, a
completely randomized design, with three treatments
(adaxial and abaxial leaf surface and floral buds) and
twenty replications (plants in pots), was used. For the
statistical analysis, the data were transformed by √(x+1),
to correct the heterogeneity of the variances before
being submitted to the analysis of variance (Anova),
and the means were compared by the Scott-Knott test
when there were significant treatment effects (α = 0.05). The number of nodes constituted the treatments in the
vertical distribution of the eggs on the plant. The nodes
refer to the place where the branch goes out from the
stem and were counted upwards, being located just
above the cotyledon node. The data were analyzed with
the same aforementioned procedure. The separation of the caterpillars by instar was
done by observing the replacement of the cephalic
capsule. Plants ranging between V4 and V5 were Due to the importance of H. armigera in the
Brazilian cotton crop, this research aimed to study Behavioral aspects of Helicoverpa armigera in the cotton vegetative phase 3 used to evaluate the behavior in the vegetative
phase, according to the scale by Marur & Ruano
(2001). The first evaluation was carried out 20 min
after distributing the caterpillars on the leaves. The caterpillars remained on the plants for 12 h,
from 6 a.m. to 6 p.m., and the ratio used was one
caterpillar per plant, with no replacement or disposal
of caterpillars. MATERIAL AND METHODS First, the
treatments were assumed in a 3 x 5 factorial scheme,
in which three ages (1st, 2nd and 3rd instars) were
combined with five behaviors (crawling, resting,
waving, feeding and drop-off). To evaluate the period
of the day when caterpillars are more active, the
treatments assumed a 3 x 2 factorial, combining the
three instars with two periods of the day (morning
and evening). Similarly, to identify the plant region
preferred by the caterpillars, another 3 x 5 factorial
was assumed, in which the treatments were the
three ages combined with five plant parts (adaxial
surface, abaxial surface, leaflet, stem and petiole). Data analyses were the same as described before,
using transformations as √(x+0.5) (for count data)
or arcoseno√(x+0.5) (for percentage data) to correct
the heterogeneity of the variances. Regarding the behaviors of the caterpillars in
the cotton vegetative phase, no difference (p > 0.01)
among the H. armigera instars was observed (Figure 2). However, differences were observed (p < 0.01) when
analyzing the behavior within each instar. During the evaluation period of each instar,
it was observed that the caterpillars often presented
alternating behaviors. In the three studied instars,
it was noticed that the caterpillars most frequent
behavior was to rest, and this differed from the others. RESULTS AND DISCUSSION Table 1. Means and standard errors (SE) for number of eggs per
plant of Helicoverpa armigera moths in cotton plants. 1 Original data. 2 Means followed by the same letter do not differ by the Scott-Knott
test at 5 % of significance [for the analysis, data were transformed to √(x+1);
F-test. = 18.93; p < 0.001; d.f. = 37]. Treatment
Mean ± SE1,2
Adaxial leaf surface
18.7 ± 2.13 a
Abaxial leaf surface
13.5 ± 2.38 b
Floral bud
3.4 ± 0.68 c
CV (%)
33.04 Table 1. Means and standard errors (SE) for number of eggs per
plant of Helicoverpa armigera moths in cotton plants. The moths oviposited on the adaxial and
abaxial leaf surfaces and floral bud (Table 1). It was
found a significant difference between treatments
by the Scott-Knott test (p = 0.05). Even without a
prior experience and not knowing a cotton plant,
moths oviposited in all parts of the evaluated plants. Cunnighan et al. (1999) reported that the inexperience
with the host plant may influence the selection of hosts
by the moth, but this did not occur in this research. 1 Original data. 2 Means followed by the same letter do not differ by the Scott-Knott
test at 5 % of significance [for the analysis, data were transformed to √(x+1);
F-test. = 18.93; p < 0.001; d.f. = 37]. e-ISSN 1983-4063 - www.agro.ufg.br/pat - Pesq. Agropec. Trop., Goiânia, v. 49, e55628, 2019 4
S. M. M. Rodrig
0
2
4
6
8
10
1
2
3
4
5
6
7
8
9
Eggs (nº mean ± SE)
Nodes of cotton plant
b
b
b
b
b
b
a
a
Figure 1. Means and standard errors (SE) for number of eggs
of Helicoverpa armigera moths in different nodes of
cotton plants. Means followed by the same letter do
not differ by the Scott-Knott test at 1% of probability. S. M. M. Rodrigues et al. (2019) 4 0
2
4
6
8
10
1
2
3
4
5
6
7
8
9
Eggs (nº mean ± SE)
Nodes of cotton plant
b
b
b
b
b
b
a
a of finding food with higher nutritional quality, as well
as natural enemies (Johnson & Zalucki 2005). Control
tactics based on the period of day that the caterpillar
moves the most may be established, what increases
the probability of reaching the target pest, since it
will be more exposed. RESULTS AND DISCUSSION 2 Means followed by the same lowercase letter in the column,
or upper case in the row, do not differ from each other by the F-test at 5 % of
significance. Instar
Period
Mean1,2
Morning
Evening
First
7.6 ± 2.78
8.0 ± 2.21
7.8 ± 1.73a
Second
12.4 ± 3.23
5.8 ± 1.78
9.1 ± 1.96a
Third
7.6 ± 2.05
4.4 ± 2.05
6.0 ± 1.46a
Mean
9.2 ± 1.58 A
6.1 ± 1.16 B
CV (%) = 51.81
Table 2. Means and standard errors (SE) for crawling frequency
(%) of Helicoverpa armigera larvae, at two periods of
the day, on cotton plants. Table 2. Means and standard errors (SE) for crawling frequency
(%) of Helicoverpa armigera larvae, at two periods of
the day, on cotton plants. Table 2. Means and standard errors (SE) for crawling frequency
(%) of Helicoverpa armigera larvae, at two periods of
the day, on cotton plants. 1 Original values. 2 Means followed by the same lowercase letter in the column,
or upper case in the row, do not differ from each other by the F-test at 5 % of
significance. Instar
Period
Mean1,2
Morning
Evening
First
7.6 ± 2.78
8.0 ± 2.21
7.8 ± 1.73a
Second
12.4 ± 3.23
5.8 ± 1.78
9.1 ± 1.96a
Third
7.6 ± 2.05
4.4 ± 2.05
6.0 ± 1.46a
Mean
9.2 ± 1.58 A
6.1 ± 1.16 B
CV (%) = 51.81 Figure 2. Behavior of Helicoverpa armigera larvae on cotton
plants by instar. Mean frequency followed by the same
letter, within each instar, do not differ by the Scott-
Knott test at 1 % of probability. The bar above each
column is the standard error of the respective mean. 1 Original values. 2 Means followed by the same lowercase letter in the column,
or upper case in the row, do not differ from each other by the F-test at 5 % of
significance. 0
1
2
3
4
5
adaxial surface abaxial surface
leaflet
stem
petiole
Frequency (nº)
plant part
1st instar
2nd instar
3rd instar
a
b
b
b
b
a
b
b
b
a
b
c
c
0
1
2
3
4
5
adaxial
surface
abaxial
surface
leaflet
stem
petiole
Frequency (nº)
plant part
1st instar
2nd instar
3rd instar
a
b
c
a
a
b
a
a
c
bf
Figure 3. RESULTS AND DISCUSSION Preference of Helicoverpa armigera larvae to crawling
in cotton plant parts, in the vegetative phase, by instar. Mean frequency followed by the same letter, within
each instar, do not differ by the Scott-Knott test at
1 % of probability. The bar above each column is the
standard error of the respective mean. 0
1
2
3
4
5
adaxial surface abaxial surface
leaflet
stem
petiole
Frequency (nº)
plant part
1st instar
2nd instar
3rd instar
a
b
b
b
b
a
b
b
b
a
b
c
c
0
1
2
3
4
5
adaxial
surface
abaxial
surface
leaflet
stem
petiole
Frequency (nº)
plant part
1st instar
2nd instar
3rd instar
a
b
c
a
a
b
a
a
c
bf The fact that resting occurs more often is possibly
related to the digestive process of caterpillars. The
crawling and feeding behaviors were similar for
the three evaluated instars. Johnson & Zalucki
(2005, 2007) obtained results similar to those of the
present study, when they evaluated the behavior of
H. armigera in the vegetative stage of Vigna radiata L. However, the first and third instars in this research
differed for the waving and drop-off behaviors on the
plant, with waving being more frequent than drop-off. The fact that resting occurs more often is possibly
related to the digestive process of caterpillars. The
crawling and feeding behaviors were similar for
the three evaluated instars. Johnson & Zalucki
(2005, 2007) obtained results similar to those of the
present study, when they evaluated the behavior of
H. armigera in the vegetative stage of Vigna radiata L. However, the first and third instars in this research
differed for the waving and drop-off behaviors on the
plant, with waving being more frequent than drop-off. Regarding the period of the day when the
caterpillar moved the most, an absence of interaction
between period and instars in all the evaluated
behaviors was observed, except for the crawling
variable, since it was verified that all instars move
more in the morning (Table 2). The caterpillars
movement on the plant may increase the probability Figure 3. Preference of Helicoverpa armigera larvae to crawling
in cotton plant parts, in the vegetative phase, by instar. Mean frequency followed by the same letter, within
each instar, do not differ by the Scott-Knott test at
1 % of probability. The bar above each column is the
standard error of the respective mean. RESULTS AND DISCUSSION During the evaluation period in the instars, it
was observed that the caterpillars moved in different
regions of the plants, and a generally similar behavior
pattern was observed in the analysis of these regions
(Figures 3, 4 and 5). First-instar caterpillars always
preferred the adaxial surface for crawling (Figure 3),
resting (Figure 4), waving (Figure 5) and feeding
(Figure 6), differing from other sites on the plant. For the acts of crawling and waving, there was no
significant difference between the abaxial surface,
leaflet, stem and petiole, while, for resting and
feeding, the abaxial surface of the leaf differed from
the rest of the treatments. Figure 1. Means and standard errors (SE) for number of eggs
of Helicoverpa armigera moths in different nodes of
cotton plants. Means followed by the same letter do
not differ by the Scott-Knott test at 1% of probability. 0
1
2
3
4
5
crawling
resting
waving
feeding
drop off
Frequency (nº)
behavior
1st instar
2nd instar
3rd instar
a
a
a
b
b
b
b b
b
c
c
c
d
c
d
- Figure 2. Behavior of Helicoverpa armigera larvae on cotton
plants by instar. Mean frequency followed by the same
letter, within each instar, do not differ by the Scott-
Knott test at 1 % of probability. The bar above each
column is the standard error of the respective mean. 0
1
2
3
4
5
crawling
resting
waving
feeding
drop off
Frequency (nº)
behavior
1st instar
2nd instar
3rd instar
a
a
a
b
b
b
b b
b
c
c
c
d
c
d
- 0
1
2
3
4
5
adaxial surface abaxial surface
leaflet
stem
petiole
Frequency (nº)
plant part
1st instar
2nd instar
3rd instar
a
b
b
b
b
a
b
b
b
a
b
c
c
0
1
2
3
4
5
adaxial
surface
abaxial
surface
leaflet
stem
petiole
Frequency (nº)
plant part
1st instar
2nd instar
3rd instar
a
b
c
a
a
b
a
a
c
bf
the rest of the treatments. Figure 3. Preference of Helicoverpa armigera larvae to crawling
in cotton plant parts, in the vegetative phase, by instar. Mean frequency followed by the same letter, within
each instar, do not differ by the Scott-Knott test at
1 % of probability. The bar above each column is the
standard error of the respective mean. 1 Original values. Behavioral aspects of Helicoverpa armigera in the cotton vegetative phase Behavioral aspects of Helicoverpa armigera in the cotton vegetative phase 5 Figure 4. Preference of Helicoverpa armigera larvae to resting
in cotton plant parts, in the vegetative phase, by instar. Mean frequency followed by the same letter, within
each instar, do not differ by the Scott-Knott test at
1 % of probability. The bar above each column is the
standard error of the respective mean. 0
2
4
6
8
10
12
adaxial surfaceabaxial surface
leaflet
stem
petiole
Frequency (nº)
plant part
1st instar
2nd instar
3rd instar
a
b
c
c
b
a
a
b
c
c
a
b
c
0
1
2
3
4
5
adaxial
surface
abaxial
surface
leaflet
stem
petiole
Frequency (nº)
plant part
1st instar
2nd instar
3rd instar
a
b
c
a
a
b
a
a
c
b 0
2
4
6
8
10
12
adaxial surfaceabaxial surface
leaflet
stem
petiole
Frequency (nº)
plant part
1st instar
2nd instar
3rd instar
a
b
c
c
b
a
a
b
c
c
a
b
c
0
1
2
3
4
5
adaxial
surface
abaxial
surface
leaflet
stem
petiole
Frequency (nº)
plant part
1st instar
2nd instar
3rd instar
a
b
c
a
a
b
a
a
c
b When evaluating the second-instar caterpillars
for crawling (Figure 3) and feeding (Figure 6), it was
noted that the preferred region was also the adaxial
surface of the leaf. For the acts of resting (Figure 4)
and waving (Figure 5), there was no significant
difference when comparing the adaxial with the
abaxial surface, but these behaviors differed from
the others. In relation to the third-instar caterpillars, the
adaxial surface of the leaf is again observed for
crawling (Figure 3), resting (Figure 4) and feeding
(Figure 6), while the caterpillars preferred the adaxial
and abaxial surfaces, regarding the waving behavior
(Figure 5).i Figure 4. Preference of Helicoverpa armigera larvae to resting
in cotton plant parts, in the vegetative phase, by instar. Mean frequency followed by the same letter, within
each instar, do not differ by the Scott-Knott test at
1 % of probability. The bar above each column is the
standard error of the respective mean. The caterpillars of the first three instars generally
preferred the adaxial surface of the cotton leaf to move,
rest and eat. This preference may be associated with a
positive phototropism, which is a common behavior in
lepidoptera (Perkins et al. 2008). Behavioral aspects of Helicoverpa armigera in the cotton vegetative phase Johnson & Zalucki
(2007) also reported that third-instar H. armigera fed
more frequently on exposed areas of mature V. radiata
leaves. However, this result differs from that obtained
by Cribb et al. (2010), who verified first-instar
H. armigera caterpillars searching for closed spaces
and positioning themselves on the abaxial surface
of pea leaves, when there was light. The divergence
between the results obtained in this research and
those of other authors may be related to the fact that
the feeding site chosen by a caterpillar is influenced
by factors such as plant architecture, light intensity,
nutritional value, presence of trichomes and wax,
microclimate and volatiles (Navasero & Ramaswamy
1991, Cribb et al. 2010, Perkins et al. 2013). Costa
et al. (2018) reported that the highest deposition of
insecticides was observed in the upper third of cotton
plants, when they used 140 L ha-1 and 194 L ha-1 flow
rates. As the larvae of these instars are often on the
adaxial surface and prefer to move in the morning, this
indicates that they will be exposed to sprays. 0
1
2
3
4
5
adaxial
surface
abaxial
surface
leaflet
stem
petiole
Frequency (nº)
plant part
1st instar
2nd instar
3rd instar
a
b
b
b
a
a
b
a
a 0
1
2
3
4
5
adaxial
surface
abaxial
surface
leaflet
stem
petiole
Frequency (nº)
plant part
1st instar
2nd instar
3rd instar
a
b
b
b
a
a
b
a
a
Figure 5. Preference of Helicoverpa armigera larvae to waving
in cotton plant parts, in the vegetative phase, by instar. Mean frequency followed by the same letter, within
each instar, do not differ by the Scott-Knott test at
1 % of probability. The bar above each column is the
standard error of the respective mean. Figure 5. Preference of Helicoverpa armigera larvae to waving
in cotton plant parts, in the vegetative phase, by instar. Mean frequency followed by the same letter, within
each instar, do not differ by the Scott-Knott test at
1 % of probability. The bar above each column is the
standard error of the respective mean. Behavioral aspects of Helicoverpa armigera in the cotton vegetative phase 0
1
2
3
4
5
adaxial
surface
abaxial
surface
leaflet
stem
petiole
Frequency (nº)
plant part
1st instar
2nd instar
3rd instar
a
b
c
a
a
b
a
a
c
b 0
1
2
3
4
5
adaxial
surface
abaxial
surface
leaflet
stem
petiole
Frequency (nº)
plant part
1st instar
2nd instar
3rd instar
a
b
c
a
a
b
a
a
c
b
Figure 6. Preference of Helicoverpa armigera larvae to feeding
in cotton plant parts, in the vegetative phase, by instar. Mean frequency followed by the same letter, within
each instar, do not differ by the Scott-Knott test at
1 % of probability. The bar above each column is the
standard error of the respective mean. RESULTS AND DISCUSSION e-ISSN 1983-4063 - www.agro.ufg.br/pat - Pesq. Agropec. Trop., Goiânia, v. 49, e55628, 2019 REFERENCES JOHNSON, M. L.; ZALUCKI, M. P. Feeding and foraging
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each instar, do not differ by the Scott-Knott test at
1 % of probability. The bar above each column is the
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https://openalex.org/W2582221738
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https://repub.eur.nl/pub/97988/journal.pntd.0005310.pdf
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English
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Diagnosing Polyparasitism in a High-Prevalence Setting in Beira, Mozambique: Detection of Intestinal Parasites in Fecal Samples by Microscopy and Real-Time PCR
|
PLoS neglected tropical diseases
| 2,017
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cc-by
| 11,926
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RESEARCH ARTICLE Background Many different intestinal parasite species can co-occur in the same population. However,
classic diagnostic tools can only frame a particular group of intestinal parasite species. Hence, one or two tests do not suffice to provide a complete picture of infecting parasite spe-
cies in a given population. The present study investigated intestinal parasitic infections in
Beira, Mozambique, i.e. in the informal settlement of Inhamudima. Diagnostic accuracy of
five classical microscopy techniques and real-time PCR for the detection of a broad spec-
trum of parasites was compared. Editor: William Evan Secor, Centers for Disease
Control and Prevention, UNITED STATES Control and Prevention, UNITED STATES
Received: June 26, 2016
Accepted: January 9, 2017
Published: January 23, 2017 Copyright: © 2017 Meurs et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited. OPEN ACCESS Citation: Meurs L, Polderman AM, Vinkeles
Melchers NVS, Brienen EAT, Verweij JJ,
Groosjohan B, et al. (2017) Diagnosing
Polyparasitism in a High-Prevalence Setting in
Beira, Mozambique: Detection of Intestinal
Parasites in Fecal Samples by Microscopy and
Real-Time PCR. PLoS Negl Trop Dis 11(1):
e0005310. doi:10.1371/journal.pntd.0005310 Diagnosing Polyparasitism in a High-
Prevalence Setting in Beira, Mozambique:
Detection of Intestinal Parasites in Fecal
Samples by Microscopy and Real-Time PCR Lynn Meurs1,2, Anton M. Polderman1, Natalie V. S. Vinkeles Melchers1¤a, Eric A. T. Brienen1, Jaco J. Verweij1¤b, Bernhard Groosjohan3, Felisberto Mendes3,
Manito Mechendura3, Dagmar H. Hepp1¤c, Marijke C. C. Langenberg1,
Rosanne Edelenbosch1¤d, Katja Polman2, Lisette van Lieshout1* Lynn Meurs1,2, Anton M. Polderman1, Natalie V. S. Vinkeles Melchers1¤a, Eric A. T. Brienen1, Jaco J. Verweij1¤b, Bernhard Groosjohan3, Felisberto Mendes3,
Manito Mechendura3, Dagmar H. Hepp1¤c, Marijke C. C. Langenberg1,
Rosanne Edelenbosch1¤d, Katja Polman2, Lisette van Lieshout1* a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 1 Department of Parasitology, Leiden University Medical Center, Leiden, The Netherlands, 2 Department of
Biomedical Sciences, Institute of Tropical Medicine, Antwerp, Belgium, 3 Faculty of Health Science, Catholic
University of Mozambique, Beira, Mozambique ¤a Current address: Department of Public Health, University Medical Center Rotterdam (ErasmusMC),
Rotterdam, The Netherlands ¤a Current address: Department of Public Health, University Medical Center Rotterdam (ErasmusMC),
Rotterdam, The Netherlands
¤b Current address: Laboratory for Medical Microbiology and Immunology, St. Elisabeth Hospital, Tilburg,
The Netherlands
¤c Current address: Neurology Department, VU University Medical Center, Amsterdam, The Netherlands
¤d Current address: Rathenau Institute, The Hague, The Netherlands
* E.A.van_Lieshout@lumc.nl ¤a Current address: Department of Public Health, University Medical Center Rotterdam (ErasmusMC),
Rotterdam, The Netherlands
¤b Current address: Laboratory for Medical Microbiology and Immunology, St. Elisabeth Hospital, Tilburg,
The Netherlands
¤c Current address: Neurology Department, VU University Medical Center, Amsterdam, The Netherlands
¤d Current address: Rathenau Institute, The Hague, The Netherlands
* E.A.van_Lieshout@lumc.nl ¤b Current address: Laboratory for Medical Microbiology and Immunology, St. Elisabeth Hospital, Tilburg,
The Netherlands Methodology/Principal Findings A cross-sectional population-based survey was performed. One stool sample per participant
(n = 303) was examined by direct smear, formal-ether concentration (FEC), Kato smear,
Baermann method, coproculture and real-time PCR. We found that virtually all people
(96%) harbored at least one helminth, and that almost half (49%) harbored three helminths
or more. Remarkably, Strongyloides stercoralis infections were widespread with a preva-
lence of 48%, and Ancylostoma spp. prevalence was higher than that of Necator ameri-
canus (25% versus 15%), the hookworm species that is often assumed to prevail in East-
Africa. Among the microscopic techniques, FEC was able to detect the broadest spectrum
of parasite species. However, FEC also missed a considerable number of infections, notably Data Availability Statement: All relevant data are
within the paper and its Supporting Information
files. Data Availability Statement: All relevant data are
within the paper and its Supporting Information
files. Funding: Leiden University Fund (LUF; www.luf.nl/
en), the Jo Keur Fund (https://www.lumc.nl/sub/
9500/att/131107035332416.pdf) and the LUSTRA
scholarship (http://hum.leiden.edu/
internationalisation/outgoing-students/outgoing-
exchange-students/news/lustra-scholarship- internationalisation/outgoing-students/outgoing-
exchange-students/news/lustra-scholarship- 1 / 18 PLOS Neglected Tropical Diseases | DOI:10.1371/journal.pntd.0005310
January 23, 2017 Conclusions/Significance We showed intestinal parasites—especially helminths—to be omnipresent in Inhamudima,
Beira. However, it is a challenge to achieve high diagnostic sensitivity for all species. Classi-
cal techniques such as FEC are useful for the detection of some intestinal helminth species,
but they lack sensitivity for other parasite species. PCR can detect intestinal parasites more
accurately but is generally not feasible in resource-poor settings, at least not in peripheral
labs. Hence, there is a need for a more field-friendly, sensitive approach for on-the-spot
diagnosis of parasitic infections. Competing Interests: The authors have declared
that no competing interests exist. Diagnosis of Intestinal Parasites in Mozambique S. stercoralis, Schistosoma mansoni and G. intestinalis. PCR outperformed microscopy in
terms of sensitivity and range of parasite species detected. applications.html) were awarded to MCCL, DHH
and RE. The PCR analyses at Leiden University
Medical Center were partly supported by the Prof. Dr. P.C. Flu-Foundation. The funders had no role in
study design, data collection and analysis, decision
to publish, or preparation of the manuscript. Author Summary In populations living in adverse conditions due to poverty, a wide variety of intestinal par-
asite infections can be observed. These infections are usually diagnosed by stool micros-
copy, but can be easily missed if the procedures used are inaccurate or performed in a
suboptimal way. In the present study, we investigated the prevalence of intestinal parasite
infections in an informal settlement in Beira, Mozambique. We also compared the diag-
nostic performance of five different microscopic techniques and real-time PCR for the
detection of DNA of five helminth and five protozoa species. For this purpose, a single
stool sample was collected from members of 63 households, evenly distributed within the
settlement. Parasitic infections were found to be highly abundant in the 303 examined
samples. Virtually all individuals were found to be infected with at least one helminth spe-
cies and a majority of the stools showed multiple parasites. None of the classical micro-
scopic techniques was suitable to detect all different pathogenic species. Hence, the
outcome of several microscopy procedures had to be combined to get a complete picture. We found PCR to be the most accurate diagnostic approach, even in this hyper-endemic
setting. However, DNA detection is a high-tech technology, generally not applicable in
resource-poor settings. PLOS Neglected Tropical Diseases | DOI:10.1371/journal.pntd.0005310
January 23, 2017 Diagnosis of Intestinal Parasites in Mozambique In comparison to helminth infections, less is known about intestinal protozoan infections. They have been associated with persistent diarrhea in developing countries [8–10], and can
cause severe morbidity, especially in immunocompromised individuals [11]. Hundreds of mil-
lions of people may be affected by intestinal protozoa annually [12;13]. Yet, there are no reli-
able estimates of the global burden of disease [14–16]. This lack of knowledge is due to the fact
that intestinal protozoa are difficult to diagnose. Also, some diagnostic techniques cannot dis-
tinguish pathogenic from non-pathogenic species (i.e. Entamoeba histolytica versus the other
Entamoeba spp.). For some species there is no consensus on their pathogenicity (e.g. Blastocys-
tis), while for others, disease only develops in certain infected individuals but not in all (e.g. Giardia intestinalis). Loss of microscopic skills in many clinical laboratories and the general
lack of awareness on protozoon infections further add to these difficulties. The diagnosis of intestinal parasites typically relies on the microscopic detection of egg, lar-
val, trophozoite, cyst, and/or oocyst life stages in human feces samples [17;18]. The sensitivity
of stool microscopy is generally low, and for a reliable diagnosis it is important to choose the
appropriate microscopic technique [19]. For example, relatively simple techniques such as the
direct smear are known to detect high A. lumbricoides loads while underestimating the pres-
ence of other helminths such as Schistosoma mansoni [20]. Ideally, the technique with the high-
est diagnostic accuracy for the parasite of interest should be selected. In practice however, this
is difficult to achieve since many different parasite species may occur in a given population, or
even in a single individual, and resources are generally limited in countries where most of
these infections are endemic, so not all appropriate microscopic techniques can be used. In the
past decade, alternative diagnostic procedures have become available, such as the detection of
parasite DNA in stool samples using real-time PCR [21]. The disadvantage of PCR, however, is
that—in contrast to microscopy—it needs a high-tech laboratory, which is even more of a chal-
lenge for diagnostic laboratories within endemic countries. Relatively little is known about the distribution of intestinal parasites in Mozambique
[22;23]. The present study was initiated because a local hospital noticed many cases of diarrhea
in one of the informal settlements (‘bairro’) in Beira, Mozambique. Given the sanitary condi-
tions in the study area, intestinal parasites were suspected to be the cause of these complaints. However, diagnostic methods that were being used in the hospital at that time were not ade-
quate to detect these infections. Hence, the aim of this study was 1) to investigate which intesti-
nal parasite species are most common in this area, and 2) to compare diagnostic accuracy
between different microscopic techniques and real-time PCR for these intestinal parasitic
infections. Five commonly used microscopic techniques were applied and evaluated, i.e. direct
smear, formal-ether concentration (FEC), Kato smear, Baermann method, and coproculture,
for the detection of a uniquely broad spectrum of intestinal parasites: from intestinal helminths
like Strongyloides stercoralis, Ancylostoma spp., Necator americanus, A. lumbricoides, T. tri-
chiura and Schistosoma spp. blood flukes, to pathogenic intestinal protozoa such as G. intesti-
nalis, E. histolytica, the coccidium Cystoisospora belli and the microsporidia Enterocytozoon
bieneusi and Encephalitozoon spp. Microscopy and real-time PCR results were compared to
one another and to composite reference standards (CRSs). Introduction Intestinal parasitic infections are among the most prevalent infections in humans in low- and
middle-income countries. They can be largely categorized into two groups, i.e. helminthic and
protozoan infections. Intestinal parasitic infections can cause significant morbidity. Especially
children—who are generally more prone to heavy worm burdens—suffer from the sequelae of
intestinal parasitic infections, such as diarrhea, malabsorption and anemia [1;2]. The most important intestinal helminths, both in terms of abundance and disease burden,
are soil-transmitted helminths (STHs) such as hookworms, Ascaris lumbricoides, and Trichuris
trichiura [3]. It is estimated that STHs infect more than two billion people or more than a third
of the world’s population [4]. Also, the Schistosoma spp. blood flukes are of great public health
importance, with more than 250 million people infected worldwide [5;6], and an estimated
global disease burden of 4.0 million disability-adjusted life years (DALYs) [7]. 2 / 18 PLOS Neglected Tropical Diseases | DOI:10.1371/journal.pntd.0005310
January 23, 2017 were infected according to microscopy were offered treatment following standard clinical
practice at the local hospital. Samples were anonymized for further data analysis. were infected according to microscopy were offered treatment following standard clinical
practice at the local hospital. Samples were anonymized for further data analysis. Diagnosis of Intestinal Parasites in Mozambique Materials and Methods
Ethics statement Approval to perform the study was obtained from the Beira Committee of Medical Ethics,
Mozambique and the study proposal was filed by the Committee of Medical Ethics of the Lei-
den University Medical Centre (reference number CI5.151/NV/ib). Prior to the study, written
informed consent was obtained from the head of participating households. Individuals who 3 / 18 PLOS Neglected Tropical Diseases | DOI:10.1371/journal.pntd.0005310
January 23, 2017 PLOS Neglected Tropical Diseases | DOI:10.1371/journal.pntd.0005310
January 23, 2017 Diagnosis of Intestinal Parasites in Mozambique Microscopy Fecal samples were collected from all participating household members on a door-to-door
basis, 0-18h after production of the samples, and examined in Beira within 24h after collection. Three well trained microscopists performed the laboratory procedures, and on average not
more than eight stool samples were processed per day to ensure high quality microscopic
results. Multiple approaches were used for the detection of cysts and oocysts of the protozoa,
and eggs and larvae of the helminths (Table 1). Microscopic techniques included direct smear,
FEC, Kato thick smear, Baermann method, and charcoal plate coproculture [17]. Table 1. Diagnostic methods performed and definitions of composite reference standards. Parasite species
Direct smear
FEC
Kato smear
Baermann
Coproculture
PCR
STHs
Strongyloides stercoralis
+
+
-
+
+
+
Hookworm
+
+
+
-
+
+ a
Ascaris lumbricoides
+
+
+
-
-
+
Trichuris trichiura
+
+
+
-
-
-
Schistosoma mansoni b
+
+
+ c
-
-
+ d
Pathogenic protozoa
Giardia intestinalis
+
+
-
-
-
+
Entamoeba histolytica complex e
+
+
-
-
-
+
Cryptosporidium parvum/C. hominis f
-
-
-
-
-
+ g
Enterocytozoon bieneusi
-
-
-
-
-
+ g
Encephalitozoon spp. -
-
-
-
-
+ g
Cystoisospora belli f
+
+
-
-
-
-
Cyclospora cayetanensis f
-
-
-
-
-
-
Non-pathogenic protozoa
Entamoeba coli
+
+
-
-
-
-
Entamoeba hartmanni
+
+
-
-
-
-
Iodamoeba bu¨tschlii
+
+
-
-
-
-
Endolimax nana
+
+
-
-
-
-
Chilomastix mesnili
+
+
-
-
-
-
Blastocystis h
+
+
-
-
-
-
Dientamoeba fragilis h
-
-
-
-
-
-
FEC, formal-ether concentration
For each parasite species, the composite reference standard (CRS) is based on the combined findings of the test(s) indicated with a plus sign (+). The
minus sign (-) indicates the tests that were not applicable for a given parasite species. The latter tests were not included in the respective CRSs. a In contrast to the microscopic techniques, the PCR differentiates between the hookworm species Ancylostoma spp. and Necator americanus. b In addition to the microscopic methods mentioned, glycerin sedimentation was carried out [17]. However, this was stopped after the first 100 samples
because it was very labor-intensive and did not detect any additional S. mansoni cases. Microscopy d For each parasite species, the composite reference standard (CRS) is based on the combined findings of the test(s) indicated with a plus sign (+). The
minus sign (-) indicates the tests that were not applicable for a given parasite species. The latter tests were not included in the respective CRSs. a In contrast to the microscopic techniques, the PCR differentiates between the hookworm species Ancylostoma spp. and Necator americanus. b In addition to the microscopic methods mentioned, glycerin sedimentation was carried out [17]. However, this was stopped after the first 100 samples
because it was very labor-intensive and did not detect any additional S. mansoni cases. c The time interval between preparation of the Kato smear and the examination of the slides was 30–60 minutes and therefore suboptimal for the detection
of S. mansoni. d d The PCR detects DNA of both S. mansoni and S. haematobium. e Entamoeba histolytica cannot be differentiated microscopically from E. dispar and the other Entamoeba spp. of the E. histolytica complex: E. bangladeshi,
E. ecuadoriensis, E. moshkovskii, and E. nutalli [24–26]. The PCR was specific for E. histolytica. Consequently, this parasite was not included in the
analysis on diagnostic accuracy. e Entamoeba histolytica cannot be differentiated microscopically from E. dispar and the other Entamoeba spp. of the E. histolytica complex: E. bangladeshi,
E. ecuadoriensis, E. moshkovskii, and E. nutalli [24–26]. The PCR was specific for E. histolytica. Consequently, this parasite was not included in the
analysis on diagnostic accuracy. f The microscopic methods used—direct smear and FEC—are known to be inadequate for these protozoan infections. Hence, the modified Ziehl-Neelsen
staining [17] was performed but stopped after the first 201 samples because it was labor-intensive and Cryptosporidium was detected in three samples only,
while C. belli and C. cayetanensis were absent in this subsample. Because of incompleteness the modified Ziehl-Neelsen staining, data was not included in
the CRS. f The microscopic methods used—direct smear and FEC—are known to be inadequate for these protozoan infections. Hence, the modified Ziehl-Neelsen
staining [17] was performed but stopped after the first 201 samples because it was labor-intensive and Cryptosporidium was detected in three samples only,
while C. belli and C. cayetanensis were absent in this subsample. Because of incompleteness the modified Ziehl-Neelsen staining, data was not included in
the CRS. Study population The study was performed in Inhamudima (E34.86˚, S19.84˚), an informal settlement in the
city of Beira, Mozambique, and was conducted on request of the local hospital and faculty of
medicine. The area of Inhamudima is frequently flooded and is not connected to a sewage sys-
tem. The rainy season lasts from October to March. The study was performed between June
and August 2007. A geographical map of this area was prepared and households and roads were annotated. In
order to obtain a random and geographically evenly distributed sample of households and a
logistically feasible sample size, a grid with 75 x 75 meter quadrants was superimposed on this
map and the household that was closest to each of the intersections was selected. In this way,
all participants of in total 63 households were approached to participate. In the field, these
houses were located using handheld GPS devices. Infants (younger than one year) and people
who did not provide sufficient fecal material for all procedures were excluded from the study. Fig 1 shows that 303 out of the 399 individuals that had given informed consent provided suffi-
cient fecal material for inclusion into the study (i.e. participation rate of 76%). Initially, urine samples were also collected for detection of Schistosoma haematobium (by
urine filtration on one 10ml urine sample). Because of the relatively low numbers of S. haema-
tobium cases however, collection of urine samples was stopped to focus on the diagnosis of
intestinal parasites. Fig 1. Flow diagram of the selection of the study population and diagnostic procedures. doi:10 1371/journal pntd 0005310 g001 Fig 1. Flow diagram of the selection of the study population and diagnostic procedures. doi:10.1371/journal.pntd.0005310.g001 doi:10.1371/journal.pntd.0005310.g001 4 / 18 PLOS Neglected Tropical Diseases | DOI:10.1371/journal.pntd.0005310
January 23, 2017 doi:10.1371/journal.pntd.0005310.t001 Microscopy -
-
-
-
-
+ g
Cystoisospora belli f
+
+
-
-
-
-
Cyclospora cayetanensis f
-
-
-
-
-
-
Non-pathogenic protozoa
Entamoeba coli
+
+
-
-
-
-
Entamoeba hartmanni
+
+
-
-
-
-
Iodamoeba bu¨tschlii
+
+
-
-
-
-
Endolimax nana
+
+
-
-
-
-
Chilomastix mesnili
+
+
-
-
-
-
Blastocystis h
+
+
-
-
-
-
Dientamoeba fragilis h
-
-
-
-
-
-
FEC, formal-ether concentration
For each parasite species, the composite reference standard (CRS) is based on the combined findings of the test(s) indicated with a plus sign (+). The
minus sign (-) indicates the tests that were not applicable for a given parasite species. The latter tests were not included in the respective CRSs. a In contrast to the microscopic techniques, the PCR differentiates between the hookworm species Ancylostoma spp. and Necator americanus. b In addition to the microscopic methods mentioned, glycerin sedimentation was carried out [17]. However, this was stopped after the first 100 samples
because it was very labor-intensive and did not detect any additional S. mansoni cases. c The time interval between preparation of the Kato smear and the examination of the slides was 30–60 minutes and therefore suboptimal for the detection
of S. mansoni. d The PCR detects DNA of both S. mansoni and S. haematobium. Table 1. Diagnostic methods performed and definitions of composite reference standards. FEC, formal-ether concentration FEC, formal ether concentration
For each parasite species, the composite reference standard (CRS) is based on the combined findings of the test(s) indicated with a plus sign (+). The
minus sign (-) indicates the tests that were not applicable for a given parasite species. The latter tests were not included in the respective CRSs. a In contrast to the microscopic techniques, the PCR differentiates between the hookworm species Ancylostoma spp. and Necator americanus. b In addition to the microscopic methods mentioned, glycerin sedimentation was carried out [17]. However, this was stopped after the first 100 samples
because it was very labor-intensive and did not detect any additional S. mansoni cases. c The time interval between preparation of the Kato smear and the examination of the slides was 30–60 minutes and therefore suboptimal for the detection
of S. mansoni. Microscopy c The time interval between preparation of the Kato smear and the examination of the slides was 30–60 minutes and therefore suboptimal for the detection
of S. mansoni. d The PCR detects DNA of both S mansoni and S haematobium Table 1. Diagnostic methods performed and definitions of composite reference standards. Parasite species
Direct smear
FEC
Kato smear
Baermann
Coproculture
PCR
STHs
Strongyloides stercoralis
+
+
-
+
+
+
Hookworm
+
+
+
-
+
+ a
Ascaris lumbricoides
+
+
+
-
-
+
Trichuris trichiura
+
+
+
-
-
-
Schistosoma mansoni b
+
+
+ c
-
-
+ d
Pathogenic protozoa
Giardia intestinalis
+
+
-
-
-
+
Entamoeba histolytica complex e
+
+
-
-
-
+
Cryptosporidium parvum/C. hominis f
-
-
-
-
-
+ g
Enterocytozoon bieneusi
-
-
-
-
-
+ g
Encephalitozoon spp. -
-
-
-
-
+ g
Cystoisospora belli f
+
+
-
-
-
-
Cyclospora cayetanensis f
-
-
-
-
-
-
Non-pathogenic protozoa
Entamoeba coli
+
+
-
-
-
-
Entamoeba hartmanni
+
+
-
-
-
-
Iodamoeba bu¨tschlii
+
+
-
-
-
-
Endolimax nana
+
+
-
-
-
-
Chilomastix mesnili
+
+
-
-
-
-
Blastocystis h
+
+
-
-
-
-
Dientamoeba fragilis h
-
-
-
-
-
- Table 1. Diagnostic methods performed and definitions of composite reference standards. Parasite species
Direct smear
FEC
Kato smear
Baermann
Coproculture
PCR
STHs
Strongyloides stercoralis
+
+
-
+
+
+
Hookworm
+
+
+
-
+
+ a
Ascaris lumbricoides
+
+
+
-
-
+
Trichuris trichiura
+
+
+
-
-
-
Schistosoma mansoni b
+
+
+ c
-
-
+ d
Pathogenic protozoa
Giardia intestinalis
+
+
-
-
-
+
Entamoeba histolytica complex e
+
+
-
-
-
+
Cryptosporidium parvum/C. hominis f
-
-
-
-
-
+ g
Enterocytozoon bieneusi
-
-
-
-
-
+ g
Encephalitozoon spp. Diagnosis of Intestinal Parasites in Mozambique For the direct smear, ~2mg of feces was mixed with normal saline on a microscopy slide
and examined for helminth eggs. Another ~2mg of feces was mixed with a drop of iodine and
examined for protozoan cysts [17]. For FEC, the fecal parasite concentrator (FPC, Evergreen)
was used. One gram of fecal material was thoroughly mixed with 8 ml of 10% formalin. An
FPC strainer with 15 ml tube was attached to the tube containing this mixture. After having fil-
tered the suspension into the empty tube, 3 ml of ether was added to the filtrate. This mixture
was then shaken vigorously for 1 minute and centrifuged at 500 x g for 2 minutes. A thick,
unstained wet mount of the sediment was used for the detection of helminth eggs and larvae. For protozoan cysts, a thin, iodine-stained wet mount of the sediment was used. The Kato smear—also known as Kato-Katz smear—consisted of a single slide of fecal mate-
rial [18;29;30]. A 25 mg template was placed on the microscopy slide and filled with sieved
(~300 μm pore size) fecal material. Upon removal of the template, the sample was covered
with a cellophane slip soaked with glycerol and water (1:1). The sample was flattened by press-
ing it onto an even surface, and examined 30–60 minutes after preparation. For the Baermann method, fecal material (~4g) was placed on a layer of 2 hydrophilic
gauze bandages. The gauze was folded into a pouch by attaching the four perforated corners of
the gauze to a stick. Subsequently, the pouch was placed in a 50 ml tube filled with tap water
for 3h in such a way that the pouch lightly touched the water. Most of the water was decanted
and the remaining sediment was left to stand for 2 hours before being examined for nematode
larvae. For coproculture, the classical charcoal culture procedure was used [31]. Approximately 2g
of fecal material was homogenized, mixed 1:1 with vermiculite, and placed on a filter paper on
a plastic platform in a petri dish. Tap water was added to wet the filter paper and the petri dish
was covered. After incubation at room temperature for 7 days, the water was collected in a
tube and left standing for 2h. The sediment was examined for nematode larvae. For the Baermann method as well as for coproculture, two microscopy slides were pre-
pared, each with 100μl of the sediment. A drop of iodine was added if moving larvae were
detected, enabling identification and quantification of the larvae. Microscopy g These protozoa cannot be detected by any of the microscopic techniques used on all samples, and were therefore not included in the analysis on
diagnostic accuracy. h As yet, the classification of Blastocystis and D. fragilis as pathogenic or non-pathogenic species remains controversial [27;28]. PLOS Neglected Tropical Diseases | DOI:10.1371/journal.pntd.0005310
January 23, 2017 5 / 18 PLOS Neglected Tropical Diseases | DOI:10.1371/journal.pntd.0005310
January 23, 2017 Analysis IBM SPSS 22.0 (IBM Corp.) and Microsoft Excel 14.0 (Microsoft Corp.) were used for statisti-
cal analyses. GraphPad Prism 5 (GraphPad Software, Inc.) was used to prepare graphs. There is no gold standard for the detection of individual intestinal parasite species. Although microscopic techniques are known to lack sensitivity, they are, just as the PCR, sup-
posed to be 100% specific [44]. We therefore combined the results of several diagnostic meth-
ods into a composite reference standard (CRS) [45;46]. The CRS was defined in such a way
that it was negative if none of the diagnostic methods detected the parasite of interest, and pos-
itive if one or more methods detected the parasite. Table 1 shows how the CRS was defined for
the different parasite species. Infection prevalence was based on the CRS unless stated otherwise, and 95% Wald confi-
dence intervals were calculated for this parameter. For sensitivities of the different diagnostic
methods, the Wilson score method without continuity correction was used to calculate 95%
confidence intervals [47]. Differences between test sensitivities were considered statistically
significant if there was no overlap of their confidence intervals. The independent samples
Mann-Whitney U test was used to determine whether differences in Ct values between micros-
copy-positives and -negatives were statistically significant. Characteristics of the study population The study population (n = 303) consisted of 144 (48%) males and 159 (52%) females with a
median age of 17 years (range 1 to 72). These people were derived from 63 households. Per
household 1 to 11 subjects participated (median of 4 subjects). Diagnosis of Intestinal Parasites in Mozambique an additional PCR as described by Obeng et al. [38–41]. Protozoa (E. histolytica, G. intestinalis,
and C. parvum/C. hominis) DNA was detected by multiplex HGC-PCR [42]. Microsporidial
(E. bieneusi and Encephalitozoon spp.) DNA was detected in another multiplex PCR described
by Verweij et al. [43]. Negative and positive control samples were included in each PCR run. The PCR output
from this system consisted of a cycle-threshold (Ct) value, representing the amplification cycle
in which the level of fluorescent signal exceeded the background fluorescence. Hence, low Ct
values correspond to high parasite-specific DNA loads in the sample tested, and vice versa. The maximum Ct value was set at 50 indicating that DNA was not detected in the sample after
50 cycles of amplification. The Ct values of the internal Phocine Herpes Virus 1 control were
within the expected range (Ct value between 30 and 33) for all samples, indicating that there
was no evidence of inhibition of amplification in any of these samples. Real-time PCR In Beira, an aliquot (~1g) of each stool sample was sieved and mixed with 3 volumes of 96%
ethanol for preservation and shipment to Leiden, the Netherlands [32]. Here, the samples were
stored at -20˚C until detection and quantification of parasite DNA loads by real-time PCR. DNA isolation, amplification and detection were performed blinded to previous microscopic
results. For DNA isolation, 250μl of feces suspension was centrifuged and the pellet was washed
with phosphate-buffered saline, resuspended in 200μl of 2% polyvinylpolypyrolidone (Sigma)
and heated for 10 minutes at 100˚C [32;33]. After sodiumdodecyl sulfate-proteinase K treat-
ment (2h at 55˚C), DNA was isolated using QIAamp Spin Columns/Mini Kit (Qiagen, Ger-
many). In each sample, a fixed amount of Phocine Herpes Virus 1 was included within the
isolation lysis buffer as an internal control [34]. In total, 10 PCR targets were included and 5 μl DNA was used in each real-time PCR. Amplification generally comprised of 15’ at 95˚C followed by 50 cycles of 15” at 95˚C, 30” at
60˚C, and 30” at 72˚C. Parasite-specific primers and probes were used for amplification of
sequences, according to previously published protocols. Hookworm DNA (Ancylostoma spp. and N. americanus) was detected by one multiplex PCR described by Verweij et al. [35]. Ascaris
lumbricoides [36] and S. stercoralis [37] DNA was detected in separate PCRs instead of in a
multiplex format combined with other helminth targets. Schistosomal DNA was detected in 6 / 18 PLOS Neglected Tropical Diseases | DOI:10.1371/journal.pntd.0005310
January 23, 2017 Prevalence of intestinal parasitic infections in the study population according to different diagnostic methods. Whiskers indicate 95% confidence intervals of the observed prevalence. Percentages are based on observations in 303 individuals. (A) Prevalence of helminth infections. Strongyloides stercoralis infection was not determined (ND) in Kato smears, hookworm was not
determined by the Baermann method, while A. lumbricoides, T. trichiura, and S. mansoni were not determined by the Baermann
method or coproculture. Trichuris trichiura was not determined by PCR either, and the composite reference standard (CRS) for this
infection was consequently based on microscopic results only. (B) Prevalence of intestinal protozoan infections. Feces were
examined by both microscopy and PCR for G. intestinalis, and E. histolytica complex spp. (one observation was missing for PCR, and
consequently for the CRS). Only PCR data was used for C. parvum/C. hominis (one observation missing) and for, E. bieneusi and
Encephalitozoon spp. (two observations missing).* While microscopy cannot differentiate between the pathogenic species
Entamoeba histolytica and the nonpathogenic species of the E. histolytica complex, PCR is specific for the pathogenic species (E. histolytica). Fig 2. Prevalence of intestinal parasitic infections in the study population according to different diagnostic methods. Whiskers indicate 95% confidence intervals of the observed prevalence. Percentages are based on observations in 303 individuals. (A) Prevalence of helminth infections. Strongyloides stercoralis infection was not determined (ND) in Kato smears, hookworm was not
determined by the Baermann method, while A. lumbricoides, T. trichiura, and S. mansoni were not determined by the Baermann
method or coproculture. Trichuris trichiura was not determined by PCR either, and the composite reference standard (CRS) for this
infection was consequently based on microscopic results only. (B) Prevalence of intestinal protozoan infections. Feces were
examined by both microscopy and PCR for G. intestinalis, and E. histolytica complex spp. (one observation was missing for PCR, and
consequently for the CRS). Only PCR data was used for C. parvum/C. hominis (one observation missing) and for, E. bieneusi and
Encephalitozoon spp. (two observations missing).* While microscopy cannot differentiate between the pathogenic species
Entamoeba histolytica and the nonpathogenic species of the E. histolytica complex, PCR is specific for the pathogenic species (E. histolytica). population harbored three or more different helminth species (Fig 3). Up to five helminth
infections were found in one individual. The prevalence of infections with pathogenic protozoa is shown in Fig 2B. doi:10.1371/journal.pntd.0005310.g002 Infection prevalence STH infections were widespread with a prevalence of 93%, 56%, 48% and 38% for T. trichiura,
A. lumbricoides, S. stercoralis and hookworm, respectively (Fig 2A). PCR indicated that Ancy-
lostoma spp. was the most abundant hookworm: 25% of the population (75/303) harbored
Ancylostoma spp. while N. americanus was detected in 15% (46/303) of the population. Mixed
Ancylostoma spp. and N. americanus infections were observed in 5% (15/303) of the popula-
tion. The prevalence of S. mansoni was 10.9%. Within the population of Inhamudima, 96% (292/303) of individuals were found to harbor
at least one of the following helminths in their stool sample: S. stercoralis, Ancylostoma spp.,
N. americanus, A. lumbricoides, T. trichiura, S. mansoni, and 49% (147/303) of the total 7 / 18 PLOS Neglected Tropical Diseases | DOI:10.1371/journal.pntd.0005310
January 23, 2017 Diagnosis of Intestinal Parasites in Mozambique Fig 2. Prevalence of intestinal parasitic infections in the study population according to different diagnostic methods. Whiskers indicate 95% confidence intervals of the observed prevalence. Percentages are based on observations in 303 individuals. (A) Prevalence of helminth infections. Strongyloides stercoralis infection was not determined (ND) in Kato smears, hookworm was not
determined by the Baermann method, while A. lumbricoides, T. trichiura, and S. mansoni were not determined by the Baermann
method or coproculture. Trichuris trichiura was not determined by PCR either, and the composite reference standard (CRS) for this
infection was consequently based on microscopic results only. (B) Prevalence of intestinal protozoan infections. Feces were
examined by both microscopy and PCR for G. intestinalis, and E. histolytica complex spp. (one observation was missing for PCR, and
consequently for the CRS). Only PCR data was used for C. parvum/C. hominis (one observation missing) and for, E. bieneusi and
Encephalitozoon spp. (two observations missing).* While microscopy cannot differentiate between the pathogenic species
Entamoeba histolytica and the nonpathogenic species of the E. histolytica complex, PCR is specific for the pathogenic species (E. hi t l ti
) Fig 2. Prevalence of intestinal parasitic infections in the study population according to different diagnostic methods. Whiskers indicate 95% confidence intervals of the observed prevalence. Percentages are based on observations in 303 individuals. (A) Prevalence of helminth infections. Strongyloides stercoralis infection was not determined (ND) in Kato smears, hookworm was not
determined by the Baermann method, while A. lumbricoides, T. trichiura, and S. mansoni were not determined by the Baermann
method or coproculture. Trichuris trichiura was not determined by PCR either, and the composite reference standard (CRS) for this
infection was consequently based on microscopic results only. (B) Prevalence of intestinal protozoan infections. Feces were
examined by both microscopy and PCR for G. intestinalis, and E. histolytica complex spp. (one observation was missing for PCR, and
consequently for the CRS). Only PCR data was used for C. parvum/C. hominis (one observation missing) and for, E. bieneusi and
Encephalitozoon spp. (two observations missing).* While microscopy cannot differentiate between the pathogenic species
Entamoeba histolytica and the nonpathogenic species of the E. histolytica complex, PCR is specific for the pathogenic species (E. histolytica) valence of intestinal parasitic infections in the study population according to different diagnostic methods. Fig 2. Prevalence of intestinal parasitic infections in the study population according to differ Fig 2. The pathogenic
protozoan Cystoisospora belli was not detected in this population. Giardia intestinalis was the 8 / 18 PLOS Neglected Tropical Diseases | DOI:10.1371/journal.pntd.0005310
January 23, 2017 Diagnosis of Intestinal Parasites in Mozambique Fig 3. Number of helminth species found per person. Prevalence of infection is based on the composite
reference standard for S. stercoralis, A. lumbricoides, T. trichiura, and S. mansoni, and on PCR for hookworm
—Ancylostoma spp. and N. americanus (n = 303). doi:10.1371/journal.pntd.0005310.g003 Fig 3. Number of helminth species found per person. Prevalence of infection is based on the composite
reference standard for S. stercoralis, A. lumbricoides, T. trichiura, and S. mansoni, and on PCR for hookworm
—Ancylostoma spp. and N. americanus (n = 303). Fig 3. Number of helminth species found per person. Prevalence of infection is based on the composite
reference standard for S. stercoralis, A. lumbricoides, T. trichiura, and S. mansoni, and on PCR for hookworm
—Ancylostoma spp. and N. americanus (n = 303). doi:10.1371/journal.pntd.0005310.g003 doi:10.1371/journal.pntd.0005310.g003 most common pathogenic protozoan with a prevalence of 37% (113/302). Microsporidia were
found in 9% (28/301) and mainly consisted of E. bieneusi infections (27/28 cases). Cryptospo-
ridium parvum/C. hominis was found by PCR in 2% (6/302) of the study population. All
showed a Ct value higher than 30 (median Ct 37.6) and none of these 6 overlapped with the 3
samples positive in the Ziehl-Neelsen staining (legend f, Table 1). The prevalence of E. histoly-
tica complex spp. infections was 10% (30/303) based on microscopy. However, PCR showed
that only 10% (3/30) of these infections involved E. histolytica, i.e. the pathogenic species. Combining the findings of pathogenic protozoa with the detection of helminths, 98% (294/
300) of the tested inhabitants of Inhamudima were found to be infected with at least one intes-
tinal parasite species. The prevalence of non-pathogenic protozoa varied between 4% and 34% for the different
species, with a prevalence of 34% (102/303) for Entamoeba coli, 26% (80/303) for Endolimax
nana, 21% (63/302) for Blastocystis, 9% (26/303) for Entamoeba hartmanni, 8% (24/303) for
Chilomastix mesnili and a prevalence of 4% (13/303) for Iodamoeba bütschlii. PLOS Neglected Tropical Diseases | DOI:10.1371/journal.pntd.0005310
January 23, 2017 Microscopy versus PCR The sensitivity of PCR for the detection of each of the parasite species tested was higher than
that of any of the microscopic techniques applied (Fig 4). This difference was statistically sig-
nificant for all microscopic methods used for S. stercoralis, hookworm, S. mansoni, and G. intestinalis, and for the direct smear for the detection of A. lumbricoides. For some species, the
sensitivity of the best microscopic technique was manifold lower than that of PCR. For exam-
ple, the estimated sensitivity for the detection of S. mansoni was 12% for Kato smear versus
97% for the detection of Schistosoma DNA in feces via PCR. For G. intestinalis, sensitivity of
FEC was 21% while that of PCR was 99%. In addition, in only one of the three PCR-positive E. histolytica samples, E. histolytica complex spp. cysts could be detected by microscopy (FEC). The sensitivity of PCR for the detection of each of the parasite species tested was higher than
that of any of the microscopic techniques applied (Fig 4). This difference was statistically sig-
nificant for all microscopic methods used for S. stercoralis, hookworm, S. mansoni, and G. intestinalis, and for the direct smear for the detection of A. lumbricoides. For some species, the
sensitivity of the best microscopic technique was manifold lower than that of PCR. For exam-
ple, the estimated sensitivity for the detection of S. mansoni was 12% for Kato smear versus
97% for the detection of Schistosoma DNA in feces via PCR. For G. intestinalis, sensitivity of
FEC was 21% while that of PCR was 99%. In addition, in only one of the three PCR-positive E. histolytica samples, E. histolytica complex spp. cysts could be detected by microscopy (FEC). Fig 5 shows that, for each parasite species, PCR-positive but microscopy-negative samples
had significantly lower DNA loads (i.e. higher Ct values) than PCR-positive samples that were
also microscopy-positive. Although observed less frequently, some microscopy-positive sam-
ples could not be confirmed by PCR. In most of these samples, only few parasites were detected
by microscopy (see Table 2). Fig 5 shows that, for each parasite species, PCR-positive but microscopy-negative samples
had significantly lower DNA loads (i.e. higher Ct values) than PCR-positive samples that were
also microscopy-positive. Although observed less frequently, some microscopy-positive sam-
ples could not be confirmed by PCR. In most of these samples, only few parasites were detected
by microscopy (see Table 2). Diagnosis of Intestinal Parasites in Mozambique Fig 4. Sensitivities of the different diagnostic methods for the detection of intestinal parasitic infections. Whiskers indicate 95%
confidence intervals of observed sensitivities (n = 303). Strongyloides stercoralis infection was not determined (ND) in Kato smears,
hookworm was not determined by the Baermann method, while A. lumbricoides, T. trichiura, and S. mansoni were not determined by the
Baermann method or coproculture. Trichuris trichiura was not determined by PCR, and for this species the sensitivity was therefore based
on microscopic results only. Giardia intestinalis was not determined by Kato smear, the Baermann method or coproculture (one observation
missing). d i 10 1371/j
l
td 0005310 004 Fig 4. Sensitivities of the different diagnostic methods for the detection of intestinal parasitic infections. Whiskers indicate 95%
confidence intervals of observed sensitivities (n = 303). Strongyloides stercoralis infection was not determined (ND) in Kato smears,
hookworm was not determined by the Baermann method, while A. lumbricoides, T. trichiura, and S. mansoni were not determined by the
Baermann method or coproculture. Trichuris trichiura was not determined by PCR, and for this species the sensitivity was therefore based
on microscopic results only. Giardia intestinalis was not determined by Kato smear, the Baermann method or coproculture (one observation
missing). doi:10.1371/journal.pntd.0005310.g004 Comparison of microscopic techniques Diagnostic sensitivity was estimated for the different microscopic techniques and for each of
the parasite species (Fig 4). FEC and Kato smear had the highest sensitivities for the detection
of each of the helminths, except for S. stercoralis. The direct smear was inferior to these two
methods for the detection of S. stercoralis, hookworm, T. trichiura and S. mansoni. Similarly,
the direct smear was inferior to FEC for the detection of G. intestinalis. The Baermann method
and coproculture showed the highest sensitivities for S. stercoralis (48% and 57%, respectively),
while the direct smear and FEC showed equally low sensitivities (18% and 25%, respectively). The sensitivity of coproculture for the detection of S. stercoralis and hookworm (57% and 77%,
respectively) tended to be higher than the sensitivity of the other microscopic techniques (up
to 48% and 69%, respectively). Moreover, the sensitivity of helminth detection increased upon
combination of multiple microscopic methods. 9 / 18 PLOS Neglected Tropical Diseases | DOI:10.1371/journal.pntd.0005310
January 23, 2017 Discussion The present study was initiated because a local hospital noticed many cases of diarrhea in
Inhamudima. Given the adverse living conditions in the study area, intestinal parasites were
suspected to be the cause of these complaints. However, diagnostic methods that were being
used in the hospital at that time were not adequate. Clinical records thus far suggested that
there were only low numbers of intestinal parasite infections in the area. The first aim of the
present study was to obtain a comprehensive overview of the intestinal helminth and proto-
zoan infections that occur in the informal settlement of Inhamudima in Beira. 10 / 18 PLOS Neglected Tropical Diseases | DOI:10.1371/journal.pntd.0005310
January 23, 2017 Diagnosis of Intestinal Parasites in Mozambique Fig 5. Ct values in PCR-positives: microscopy-negative versus -positive samples. Total number of PCR-positives per
species is indicated between brackets. ‘-’ indicates microscopy-negative and ‘+’ microscopy-positive samples. Horizontal lines
indicate median Ct values. Differences in Ct values between microscopy-positive and–negative samples were all significant
p0.007, a microscopy cannot differentiate the two hookworm species. doi:10.1371/journal.pntd.0005310.g005 Fig 5. Ct values in PCR-positives: microscopy-negative versus -positive samples. Total number of PCR-positives per
species is indicated between brackets. ‘-’ indicates microscopy-negative and ‘+’ microscopy-positive samples. Horizontal lines
indicate median Ct values. Differences in Ct values between microscopy-positive and–negative samples were all significant
p0.007, a microscopy cannot differentiate the two hookworm species. doi:10.1371/journal.pntd.0005310.g005 The major strength of the present study is the unprecedentedly large panel of different diag-
nostic techniques used [48]. This diagnostic rigor resulted in high quality data regarding a
uniquely wide spectrum of intestinal parasite species, including helminths as well as protozoa. Prevalences observed here are thus likely to approach the true prevalence of individual para-
sitic infections as well as co-infections [49], in contrast to other prevalence studies where gen-
erally only one microscopic technique is used. This approach led to several important
observations concerning the presence of intestinal parasite infections in the target population,
such as the remarkably high levels of S. stercoralis, and high parasite infection levels in general,
as well as a high degree of polyparasitism. g
g
p yp
According to Scha¨r et al., only 44% of African countries have prevalence data on S. stercora-
lis, and a majority of these studies is based on inadequate diagnostic techniques [50]. In
Mozambique, so far, only Mandomando et al. reported on S. stercoralis. pp
a One case was detected by coproculture as well as the Baermann method. In addition to stool, Baermann medium and coproculture medium were analyzed
by PCR in the 7 Baermann-positives and 3/5 coproculture-positives, respectively. These samples also tested negative.
b One case was detected by FEC Kato smear as well as coproculture Discussion A prevalence of 1.1%
was reported in under-five-year-olds with diarrhea referred to a district hospital in Southern
Mozambique, but only direct smear was used which is ineffective for the detection of this
parasite [51]. As a result, S. stercoralis prevalences were assumed to be low, i.e. 6.2% prevalence
in Mozambique [50]. Our results (prevalence of 48%) indicate that this may be a gross Table 2. Discordance between microscopy and PCR in microscopy-positives. FEC, Formal ether concentration; NA, Not applicable
a One case was detected by coproculture as well as the Baermann method. In addition to stool, Baermann medium and coproculture medium were analyzed
by PCR in the 7 Baermann-positives and 3/5 coproculture-positives, respectively. These samples also tested negative.
b One case was detected by FEC, Kato smear as well as coproculture.
c In addition, 3 S. haematobium eggs were observed
d P
it
b
d t
i
d
i
tit ti
l doi:10.1371/journal.pntd.0005310.t002 ,
;
,
pp
a One case was detected by coproculture as well as the Baermann method. In addition to stool, Baermann medium and coproculture medium were analyzed
by PCR in the 7 Baermann-positives and 3/5 coproculture-positives, respectively. These samples also tested negative.
b One case was detected by FEC, Kato smear as well as coproculture.
c In addition, 3 S. haematobium eggs were observed
d Parasite numbers were determined semi-quantitatively.
doi:10 1371/journal pntd 0005310 t002 Diagnosis of Intestinal Parasites in Mozambique underestimation of the nationwide prevalence and once more illustrate how often strongyloi-
diasis is overlooked due to poor diagnostics. Another important observation was that Ancylostoma spp. was more prevalent than N. americanus (25% versus 15%, respectively). This is in line with the studies described by Gold-
smid [52], as well as a more recent study in pre-school children living in a rural area in Malawi
where Ancylostoma spp. prevailed over N. americanus [53]. Despite of these reports, it is often
assumed that only N. americanus is endemic in East Africa [54]. High levels of intestinal parasitic infections as observed in the present study are not excep-
tional in urban informal settlements. More than 20 years ago, it was already predicted that
ongoing urbanization in the developing world would increase levels of intestinal parasites in
areas like Inhamudima [55]. In such adverse living conditions, widespread environmental con-
tamination is likely to occur for parasites such as A. lumbricoides, T. trichiura, and G. intestina-
lis [55]. The present observations confirm this idea as these three parasites ranked in the top
four of most prevalent parasites in Inhamudima, with a prevalence as high as 93% for T. tri-
chiura. Augusto et al. (2009) also noted a particularly high STH prevalence in (peri-)urban
areas of Mozambique (including Beira, although these were not explicitly reported), and these
prevalences were higher than those reported in older studies [22]. The authors hypothesized
that the increase of STH infections in Mozambican informal settlements was due to rural-
urban migration, low levels of socioeconomic development, and overcrowding together with
the deterioration of water supply and sewage systems after independence [22]. In addition to determining infection levels of intestinal parasites in the inhabitants of Inha-
mudima, this study aimed to compare diagnostic accuracy between five microscopic tech-
niques that are commonly used for the detection of intestinal parasites. The common
microscopic techniques direct smear, FEC, Kato smear, Baermann method, and coproculture
were applied and compared for the detection of parasitic helminths as well as protozoan infec-
tions. The classical broad-spectrum techniques FEC and Kato smear proved useful for the
detection of intestinal helminths: hookworm, T. trichiura and A. lumbricoides. Table 2. Discordance between microscopy and PCR in microscopy-positives. Parasite species
Number of microscopy-positive cases being PCR-negative (number of parasites
observed per technique)
Total number of microscopy-positives
Direct smear
FEC
Kato smear
Baermann
Coproculture
Total
S. stercoralis
1 (1)
0
NA
7 a
5 (6–141) a
12
100
Hookworm
0
1 (2) b
3 (1–47) b
NA
7 (2–1000) b
9
98
A. lumbricoides
2 (1)
5 (1–4)
0
NA
NA
7
161
S. mansoni
0
0
1 (1) c
NA
NA
1
5
G. intestinalis d
0
1 (1–10)
NA
NA
NA
1
24 copy-positive cases being PCR-negative (number of parasites
i
)
Total number of microscopy-positives es
Number of microscopy-positive cases being PCR-negative (number of parasites
observed per technique)
Total num FEC, Formal-ether concentration; NA, Not applicable PLOS Neglected Tropical Diseases | DOI:10.1371/journal.pntd.0005310
January 23, 2017 11 / 18 PLOS Neglected Tropical Diseases | DOI:10.1371/journal.pntd.0005310
January 23, 2017 Diagnosis of Intestinal Parasites in Mozambique results from the five microscopic techniques were combined. These findings are in line with
earlier studies, despite differences in endemicity and geographic locations [44]. For example,
Arndt et al. showed the same helminth PCRs that were used in the present study (in a multi-
plex format [36]) to be more sensitive for the detection of S. stercoralis, hookworm and Schisto-
soma spp. than the combination of direct smear, FEC, and Kato smear [57]. Also Easton et al. recently reported that real-time PCR outperformed Kato-Katz for the detection of soil-trans-
mitted helminths and has the advantage of demonstrating parasite species which are not diag-
nosed by Kato-Katz such as G. intestinalis, E. histolytica and S. stercoralis [58]. Earlier findings
from our group comparing Kato smear and PCR in Kenyan schoolchildren showed that PCR
outperformed the Kato smear for the detection of N. americanus as well as S. mansoni. Even
when three consecutive stool samples were analyzed using Kato smear, while PCR was per-
formed on only one stool sample, PCR detected more cases [41;59]. When focusing on the diagnosis of Schistosoma only, previous studies found PCR to be
more sensitive than microscopy, [41], also when using other Schistosoma-specific targets [60–
64]. It should be noted that S. haematobium was detected in our study populations. This para-
site was seen in six out of 95 examined urine samples (6%). PCR results indicated that Schisto-
soma DNA was absent in stool samples from people with S. haematobium eggs in urine but
without S. mansoni infection according to microscopy. In accordance with the Kenyan study
[41], S. haematobium co-endemicity is thus unlikely to explain the relatively low sensitivity of
microscopy as compared to PCR for the detection of S. mansoni. Yet, we cannot entirely rule
out that “leakage” of S. haematobium DNA may have resulted in occasional false-positive
PCRs. To our knowledge, only one study reported a lower diagnostic accuracy of PCR relative to
microscopy. Knopp et al. showed real-time PCR for the detection of hookworm and of S. ster-
coralis to be less sensitive than microscopy when applied on stool samples collected in a low-
endemicity area in Tanzania [65]. PLOS Neglected Tropical Diseases | DOI:10.1371/journal.pntd.0005310
January 23, 2017 The Kato smear
may be more informative than FEC as it is a quantitative technique, and in accordance with a
recent meta-analysis by Nikolay et al., it tended to detect more STH infections—more hook-
worm, T. trichiura, and A. lumbricoides—than FEC [44]. On the other hand, the Kato smear is
not suitable for the detection of pathogenic intestinal protozoa. In this respect, the two tech-
niques are complementary. In order to also detect S. stercoralis, coproculture and/or the Baer-
mann method should be added to the diagnostic work-up. Consistent with a recent meta-
analysis by Campo Polanco et al. [56], coproculture tended to have a higher sensitivity for the
detection of S. stercoralis than the Baermann method. We demonstrated that coproculture has
the additional advantage that it has a higher sensitivity for the detection of hookworm than the
other techniques, while the Baermann method is suited for the detection of S. stercoralis only. This was also reported by Knopp et al. [48]. The disadvantage of coproculture however, is that
it takes a week to obtain test results while the Baermann method takes several hours, and that
it is more difficult to perform under field conditions [48]. In addition, coproculture harbors
the occupational hazard of infection by L3 larvae. In concordance with earlier reports [44], the
direct smear was inferior to all the other diagnostic tests, for each of the endemic intestinal par-
asite species. This confirms that the direct smear is not at all suitable for the diagnosis of intes-
tinal parasite infections. In practice however, it is widely used in endemic areas, in both
research and clinical settings—including the Beira hospital—resulting in gross underestima-
tions of the spread of intestinal parasites. We aimed to compare the diagnostic accuracy of the different microscopic techniques to
that of real-time PCR. The detection of parasite-specific DNA had a higher sensitivity than
microscopy for S. stercoralis, hookworm, Schistosoma spp., and G. intestinalis, even when PLOS Neglected Tropical Diseases | DOI:10.1371/journal.pntd.0005310
January 23, 2017 12 / 18 Supporting Information
S1 Checklist.
(DOCX)
S1 Database.
(XLSX) S1 Checklist. (DOCX)
S1 Database. (XLSX) Diagnosis of Intestinal Parasites in Mozambique proposed that residual DNA may persist and be detected by PCR after parasite clearance [67],
while others have showed fast DNA clearance after treatment [68;69]. More research is needed
to clarify these issues. proposed that residual DNA may persist and be detected by PCR after parasite clearance [67],
while others have showed fast DNA clearance after treatment [68;69]. More research is needed
to clarify these issues. Acknowledgments We gratefully thank the population of Inhamudima, Beira, Mozambique for participation in
this study, Idelson for washing materials, and Sandra for translating. Also, we would like to
thank the anonymous reviewer who so generously shared his/her encyclopedic parasitology
knowledge. Conclusion We demonstrate that intestinal helminth and protozoan infections and co-infections are wide-
spread in Inhamudima, Beira. We showed that classical techniques such as FEC are useful for
the detection of some intestinal helminths such as hookworm, T. trichiura and A. lumbricoides. However, they lack the sensitivity to reliably characterize the wide range of intestinal parasites
that may coexist in a population or individual. PCR can detect intestinal parasites more accu-
rately but in most endemic areas it is not (yet) possible to perform this technique, at least not
in the more peripheral laboratories. So, until a more field-friendly approach becomes available,
infection levels of intestinal parasites—and polyparasitism—are best approximated by combin-
ing multiple and relatively simple microscopic techniques. Although this study used the same PCR procedures as in the
present study, this inconsistency could still be explained by minor technical differences in the
performance of the DNA extraction or the general PCR set-up, resulting in a reduced test effi-
ciency which becomes more obvious when infection levels are already low. Hence, standardi-
zation of laboratory procedures and the implementation of external quality assessment
schemes are warranted. It is widely recognized that PCR could be particularly useful for the detection of intestinal
parasites in low transmission areas and in post-control settings [48;58]. Here, we show that
PCR can also have added value in high transmission areas, particularly in polyparasitized pop-
ulations. It not only has a higher sensitivity than microscopy for all the intestinal parasites
investigated in this study, but it also has the advantage that it can differentiate between mor-
phologically identical species where microscopy cannot. This is important because infections
caused by species passing morphologically indistinguishable eggs, such as the hookworms, can
have different epidemiology. Moreover, it also detects infections such as microsporidia which
are extremely difficult to diagnose by conventional light microscopy. Although it was not yet
possible to detect T. trichiura by PCR at the time of study, currently available multiplex PCRs
do include this helminth. PCR may thus be a useful tool for evaluation of public health inter-
ventions, for quality control of microscopy procedures as well as for research on the distribu-
tion of intestinal parasitic infections in different endemic settings [46;57]. However, it should
be noted that PCR also has disadvantages. PCR consumables are expensive, and it requires
high-tech equipment which is often not available in laboratories in affected regions. Transfer
of samples to other, better equipped laboratories, may prolong the turnaround time of PCR
results. Secondly, the clinical relevance of submicroscopic intestinal parasitic infections is not
yet fully understood, and therefore remains a matter of debate [16;57;66]. It has also been PLOS Neglected Tropical Diseases | DOI:10.1371/journal.pntd.0005310
January 23, 2017 13 / 18 Conceptualization: AMP. Data curation: LvL LM. Funding acquisition: AMP LvL MCCL RE DHH. Investigation: EATB DHH MCCL RE MM FM. Methodology: AMP LvL JJV. References 1. Guerrant RL, Oria RB, Moore SR, Oria MO, Lima AA. Malnutrition as an enteric infectious disease with
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from the gut after successful treatment. Clin Microbiol Infect 2014 Nov; 20(11):O972–O974. doi: 10. 1111/1469-0691.12626 PMID: 24655130 18 / 18
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As cidades da juventude em Fortaleza
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ABSTRACT A cidade de Fortaleza é apresentada a
partir da experiência juvenil, de suas
organizações, de seus deslocamentos e
de suas formas específicas de apropria-
ção da cidade.Desta observação delineio
a idéia de Experiência Musical e de Des-
locamentos Geo-Estéticos na cidade. As
organizações e experiências juvenis são
tomadas como reveladoras de outros
possíveis e suas movimentações como
manifestação do “novo”urbano contem-
porâneo. A cidade de Fortaleza é apresentada a
partir da experiência juvenil, de suas
organizações, de seus deslocamentos e
de suas formas específicas de apropria-
ção da cidade.Desta observação delineio
a idéia de Experiência Musical e de Des-
locamentos Geo-Estéticos na cidade. As
organizações e experiências juvenis são
tomadas como reveladoras de outros
possíveis e suas movimentações como
manifestação do “novo”urbano contem-
porâneo. The city of Fortaleza is presented through
the juvenile experience of its organiza-
tions, its displacements and its specific
city appropriation ways. It is from this
observation that I take the idea of Musi-
cal Experience and the Geo-esthetic
Displacements of the city. The orga-
nizations and juvenile experiences are
considered to be revealing of other possi-
bilities and their movements, as a man-
ifestation of
the “new” contemporary
urbanity. Palavras-chave:Juventude;cultura;cida-
de; música; hip hop; punk. Keywords:Youth,culture,city,music,hip
hop, punk. Este texto está dividido em cinco partes, em que a juventude e sua atua-
ção na cidade de Fortaleza se esboçam com base na compreensão de sua or-
ganização em dois importantes movimentos: o Movimento Hip-Hop e o Mo-
vimento Punk. Na mesma medida em que esses jovens se organizam e criam
suas organizações, interferem na cidade e nas diversas formas de se organizar
e de viver no final do século XX e início do século XXI. As cidades da juventude em Fortaleza Francisco José Gomes Damasceno1
UECE BAILES E CLUBES: A FORTALEZA DAS AFINIDADES ELETIVAS Freqüentemente pensei nas cartas geográficas como uma grande metá-
fora do conhecimento, como tensão entre compreensibilidade e desorienta-
mento. O que faz o cartógrafo? Transfere para uma folha plana de papel o
‘mundo real’ sob a forma de símbolos e sinais. O que se transcreve é uma ‘di- Revista Brasileira de História. São Paulo, v. 27, nº 53, p. 215-242 - 2007 Francisco José Gomes Damasceno ferença’: a fonte de toda a informação e, portanto, da comunicação. Ainda as-
sim, enquanto algumas dessas diferenças são imediatamente decodificáveis
por qualquer leitor de mapas, outras têm de expressar códigos totalmente no-
vos, de forma tal que até o viajante mais experimentado verá pela primeira
vez o lugar representado, mesmo no caso em que tenha estado ali a vida toda. E como a escala 1:1 não adianta, pode servir no máximo a um eu dilatado do
narcisismo irrefreável, quanto mais a proporção for reduzida — é micrológi-
ca — tanto mais teremos de inventar os códigos a serem transcritos no mapa
para deslocar o leitor. Então talvez descobriremos com esses microcódigos
que o nosso objeto — a comunicação sincrética — já está incorporando, nas
partes terminais de nossos sentidos, enxertos de sensores étnicos, cyber e punk. Mudanças marronizadas. Quilombos patchwork.2 No final dos anos 70 a cidade de Fortaleza fervilhava aos sons de inúme-
ras novidades oriundas da produção da chamada indústria cultural e das cul-
turas de massa. Alguns vindos literalmente ‘de fora’ e outros considerados ‘lo-
cais’e ainda não inseridos no contexto maior de comercialização internacional. Era o caso, por exemplo, do rock, que aportava com força pela musicali-
dade de Led Zeppelin, Black Sabbath, Ramones e Sex Pistols, entre outros. Já
o predomínio do local se matizava nas inúmeras gravações de forró, bem re-
presentados em grupos como Trio Nordestino, Três do Nordeste, Luís Gon-
zaga, Jackson do Pandeiro e tantos outros. Estudos sobre o rock em Fortaleza dão conta de uma trajetória iniciada
ainda na década de 1950 e um predomínio hegemônico do forró, motivo pe-
lo qual se apontaria, até meados dos anos 90, a dificuldade de ‘explosão’ dessa
manifestação em Fortaleza, bem como do surgimento e consolidação de ban-
das e espaços destinados a essas manifestações. Revista Brasileira de História, vol. 27, nº 53 BAILES E CLUBES: A FORTALEZA DAS AFINIDADES ELETIVAS Entretanto, o rock praticado em Fortaleza era ‘regionalizado’, com ban-
das (ou grupos de bailes) como Os Faraós, Os Belgas, Os Diferentes ou, um
pouco depois, O Peso, que seguiam linhas melódicas mais leves e pouco liga-
das às perspectivas mais ‘transgressivas’: Na década de 50, o rock era tocado por grupos de baile — o mais conhecido
era Iranildo e seu conjunto — que animavam as festas de clubes sociais como o
Náutico, Líbano e Maguary ... Na década de 60, com a explosão do rock em todo
o mundo, esses grupos tocavam os sucessos do momento, além de músicas bra-
sileiras, mambo, bolero e rumba. O grande destaque era Os Faraós, banda de
Luizinho que ainda hoje toca em festas ... Outro destaque era Os Belgas, cujo
guitarrista Júlio Sena era o maior sucesso. Tanto um como o outro tinha em seu 216 Revista Brasileira de História, vol. 27, nº 53 As cidades da juventude em Fortaleza repertório, basicamente sucessos dos Beatles e as versões de Renato e seus Blue
Caps. Também vale lembrar d’Os Diferentes, que a exemplo dos demais tocava
música dos outros, mas a diferença estava no fato de cantarem músicas próprias
e fazerem arranjos diferentes para músicas dos outros ... Nos anos 70, destacou-
se Luís Carlos Porto, vocalista da banda O Peso, que talvez seja a única banda
cearense de rock a ter tido projeção nacional ... No final da década surgiu a ban-
da Posh, fazendo um rock mais para o pop.3 Assim, a manifestação nos anos 70 se encontrava na encruzilhada dessas
diferentes formas de apreciar e curtir o rock, parecendo ter havido uma apro-
priação dupla da manifestação: 1) a primeira, do ponto de vista estético-político, com o surgimento de
bandas ‘mais radicais’, com mensagens politizadas e fazendo parte
de um ‘movimento’ que atentava contra os pressupostos anteriores
do próprio rock e da cultura de massas — o punk; e 2) a segunda, do ponto de vista dessas manifestações na própria cidade
de Fortaleza,e de setores de sua sociedade antes relegados ao ‘silêncio’. 2) a segunda, do ponto de vista dessas manifestações na própria cidade
de Fortaleza,e de setores de sua sociedade antes relegados ao ‘silêncio’. junho de 2007 BAILES E CLUBES: A FORTALEZA DAS AFINIDADES ELETIVAS Trata-se de uma complexa articulação de manifestações, intenções, pa-
drões estéticos, éticos e práticas de incorporação musical, que dão origem a
uma forma inusitada de vivência da cidade e da própria música, que sai dos
clubes de elite como o Náutico, Líbano e ou Maguary e passa aos pequenos
clubes de periferia, tais como o Apache Clube, o Mênfis Clube do Antônio
Bezerra, o Keops Clube, o Detroit, ou ainda o Grêmio recreativo do Conjun-
to José Walter, entre tantos outros. É claro que a essa ‘transposição geo-estética’ (ou ‘deslocamento geo-
estético’)4 correspondia uma apropriação de outros setores sociais presentes
na cidade de Fortaleza, e, além disso, marcava de forma definitiva a emergên-
cia de novos atores sócio-históricos: os jovens pobres. Situados nas periferias da então pequena cidade de Fortaleza,5 eles pas-
sam a se manifestar de forma muito mais presente e freqüente, e, além disso,
essa manifestação se dá em um campo particular, próprio, singular e inova-
dor: o campo da arte e do lazer. Assim, os inúmeros bailes que surgem na cidade são mais do que sim-
ples diversão e assumem a conotação de manifestação juvenil, que dentro de
uma trajetória própria, em pouco tempo, guardando a sua característica his-
tórica de transmutação, assumiria uma outra e mais radical forma de se ma-
nifestar, como veremos adiante. 217 Francisco José Gomes Damasceno Os inúmeros bailes geram práticas de sociabilidade, de compreensão es-
tética, ética e musical que são instauradoras de ‘territórios existenciais’ e pos-
suem suas sutilezas e perspectivas próprias. Vejamos como Flor, à época ain-
da menina, passa a experimentar o que ocorria: Eu não sei quando é que a coisa virou movimento punk, porque isso vem de
um outro movimento. Eu tinha 12 ou 13 anos, conheci essa menina, a Guacira,
ela curtia rock e tinha uns festivais de rock que aconteciam no Apache, no Mên-
fis Clube do Antônio Bezerra, no Keops Clube, lá no Detroit, várias casas de su-
búrbio, shows de rock com playback. Era Led Zeppelin, Black Sabbath, Pink Floyd,
Kiss, heavy metal. E aí começou uma reunião aqui na Praça do Ferreira. Eu estu-
dava no Anchieta e gazeava aula, ficava no meio dessa galera porque eu gostava
de banda e curtia essas coisas. Revista Brasileira de História, vol. 27, nº 53 BAILES E CLUBES: A FORTALEZA DAS AFINIDADES ELETIVAS E aí a gente começou a se reunir pra ouvir esses
sons, e tinha concursos de dança...6 Como se pode perceber na lembrança de Flor, iniciava-se na cidade a
produção de pequenas festas nos clubes de subúrbio. Nessas festas, uma das
estratégias para envolver os grupos eram competições de rock ao som dos con-
juntos7 por eles preferidos: Sempre nos finais de semana uma leva de fãs gravitava pelos clubes suburba-
nos atrás de diversão. ‘Competições’ eram travadas entre as diversas turmas. A
turma do Baby do Bairro do Monte Castelo, do Conjunto José Walter, do Par-
que Araxá entre outras, disputava quem agitava mais parecido com seus ídolos
(Robert Plant, Ramones), com direitos a guitarras artesanais, feitas de madeira
ou papelão e até troféus para as turmas vencedoras.8 Além disso, outro aspecto que se deve considerar é a apropriação da ci-
dade de forma itinerante, não fixa, feita de forma ‘errante’, em deslocamentos
pela diversidade de cada bairro ou clube, ou ainda das diversas ‘turmas’ en-
volvidas: Essa coisa do rock não era fixa não, o cara fazia um evento e divulgava, uma
semana num bairro, outra semana em outro totalmente diferente ou distante. Acho que era isso que perpetuava a adrenalina. De repente o cara já tem a adre-
nalina lá em cima, aí faz um evento hoje aqui, no outro final de semana não é
mais aqui, é lá...9 218 Revista Brasileira de História, vol. 27, nº 53 As cidades da juventude em Fortaleza As cidades da juventude em Fortaleza A novidade dessa experiência juvenil que tomava o lazer em suas mãos,
articulava os diversos bairros da cidade, se grupalizava a partir de afinidades,
identificava grupos distantes e/ou rivais, estabelecia uma rede de relações, de
espaços, constituía sua vivência com base em desejos e prazeres dos quais não
se dissociava, e, que em breve se tornariam a referência de suas próprias vidas
de forma completa.10 Estavam constituindo seus espaços: Existe espaço sempre que tomam em conta vetores de direção, quantidade de
velocidade e a variável tempo. O espaço é um cruzamento de móveis. É de certo
modo animado pelo conjunto dos movimentos que aí se desdobram. junho de 2007 BAILES E CLUBES: A FORTALEZA DAS AFINIDADES ELETIVAS O espaço
é o efeito produzido pelas operações que o orientam, o circunstanciam, o tem-
poralizam e o levam a funcionar em unidade polivalente de programas confli-
tuais ou de proximidades contratuais.11 A constituição desses ‘territórios existenciais’ 12 marca de forma peculiar
toda a manifestação juvenil no mundo contemporâneo. Alguns autores têm
intitulado essa característica de elaboração de grupos a partir das afinidades
e diferenças de ‘tribos’,13 numa dupla alusão à historicidade de processos se-
melhantes na antropologia e à constituição de redes no universo contempo-
râneo dito globalizado. Enquanto para os punks essa articulação inicial se dá no final dos anos
70, para os hip-hopers ocorre processo semelhante no início dos anos 80, com
a chegada, primeiro, da dança (break), e depois do canto (rap), e, finalmente,
algum tempo depois, do próprio grafite, considerados os três elementos fun-
damentais do hip-hop. Os bailes nos quais as diversas sonoridades eram experimentadas em par-
tes específicas para cada estilo musical eram uma prática comum na Fortale-
za do final dos anos 70 e dos anos 80. Assim, os amantes de cada tipo de mú-
sica tinham uma parte específica das festas para se deleitarem,e a elas recorriam
nos mais diversos cantos da cidade. Não há ‘oferta’ de bailes específicos, ou seja, de festas com a predominân-
cia de uma única manifestação musical, que só aparece, ou se torna forte, pre-
dominante, em meados dos anos 90. Talvez isso indique o pequeno número
de freqüentadores dos estilos que depois comporiam o underground em For-
taleza, juntando-os numa estratégia de produção, venda e consumo que aten-
dia naquele momento suas expectativas e possibilidades; ou talvez fosse refle-
xo de uma outra compreensão de diversão.O certo é que essa junção contribuía 219 Francisco José Gomes Damasceno na formação dos grupos pelo convívio com a diferença, no nascedouro de
muitas dessas manifestações como é o caso do rock, do punk, do próprio hip-
hop e até do forró, que durante algum tempo freqüentam os mesmos peque-
nos clubes nesses ‘bailes mistos’. Nesse contexto, é interessante lembrar o que afirma Pesavento: a construção do nós identitário pressupõe a existência do outro. O outro é a
concretização da diferença, contraposto como alteridade à identidade que se
anuncia. BAILES E CLUBES: A FORTALEZA DAS AFINIDADES ELETIVAS A visualização, identificação e avaliação classificatória do outro aconte-
ce sob o signo da estrangeiridade, e é pelo distanciamento — contrastivo, anta-
gônico ou de semelhança — que se pode construir uma noção de pertencimen-
to social. Esta lógica de sentido é, pois, relacional e implica construções que
permitem um reconhecimento com sentido. Como diz Ginsburg, todos nós so-
mos em certa medida, estrangeiros com relação a algo ou alguém.14 Assim, essa mistura inicial nos pequenos clubes da periferia da cidade
criou em Fortaleza uma proximidade entre os diversos grupos que posterior-
mente se identificam com este ou aquele estilo musical, e, ao mesmo tempo,
nesse relacional se identificavam entre si como iguais–diferentes, essencial-
mente compondo um mesmo campo: o de oposição à sociedade excludente,
aspecto que posteriormente será incorporado às suas letras, manifestos, mú-
sicas e práticas. Nesses bailes, nos pequenos clubes dos muitos (e à época distantes) bair-
ros da cidade, integrantes dos diversos grupos se encontravam e aprendiam a
se identificar e respeitar: Inter Dance, no Monte Castelo, era um point dos punks, e em 85, 86, 87 o break
estava em ascensão, era de certa forma uma novidade. Lá, o pessoal tinha a festa
de rock, punk, e tinha o pessoal que dançava o break, se congregava lá. Aí, alguns
deles migraram aqui pro Conjunto Ceará...15 Assim, perceba-se: o universo juvenil e o underground eram marcados
por um conjunto de práticas que independiam das musicalidades eleitas. Há
nesse caso um entrelaçamento entre as perspectivas musicais, as visões de
mundo, os interesses mais imediatos e uma aura estética transgressiva que
aportava com essas manifestações musicais e que definiu aos poucos o pró-
prio campo dessas manifestações (punk e hip-hop). Nessas duas manifestações musicais, corpóreas, estéticas, se fundamenta 220 Revista Brasileira de História, vol. 27, nº 53 As cidades da juventude em Fortaleza um conjunto de vínculos articulados pelo gosto, pela afinidade, pela identifica-
ção e identidade comuns e que consubstanciaria nesse período, e nesse proces-
so, os movimentos em suas perspectivas de coletivismo, de crítica e de interven-
ção social, política e cultural, sendo este último, um ‘campo’ entendido como
essencialmente político e se estabelecendo como o lócus de atuação desses jovens. No decorrer dos anos 80, tanto o punk como o hip-hop passam por um in-
tenso processo de articulação interna, de articulações políticas, culturais e so-
ciais, no sentido de se fundarem. BAILES E CLUBES: A FORTALEZA DAS AFINIDADES ELETIVAS Eles se criam, por assim dizer, em si mesmos. Estruturam-se enquanto organizações de cunho libertário e de ação contracul-
tural, numa consistente postura de enfrentamento e negação do mercado de
bens culturais. Assim, o que era apenas festa, lazer, tornou-se movimentação, e
essa movimentação das culturas — como se chamava nos primórdios do mo-
vimento — se institui como movimento sócio-político-cultural, como o en-
tendemos com base nos estudos realizados nos últimos anos.16 OS MOVIMENTOS Hoje existem novos atores, novos movimentos sociais e culturais, tornando
visíveis redes direcionadas a valores referenciados no binômio liberdade e so-
brevivência, em contraposição às condutas de crise da sociedade contemporâ- Foto 1 – Internacional (Inter Dance), no bairro Monte Castelo. Fotografia do autor, 2003. Foto 1 – Internacional (Inter Dance), no bairro Monte Castelo. Fotografia do autor, 2003. 221
junho de 2007 221 junho de 2007 Francisco José Gomes Damasceno nea ... As ações coletivas dos anos 90 abrem o leque do próprio conceito de movi-
mento social, permitindo-nos pensar em sua fragmentariedade/descontinuida-
de como referente de um tempo em que o espaço democrático se relaciona dire-
tamente com a dinâmica entre ação coletiva, cultura política e formação da
consciência social das novas gerações.17 Na medida em que essas musicalidades tomavam corpo na cidade, como
resultado da experiência direta desses jovens com a música e a própria cida-
de, foram tomando feições próprias, articulando-se de forma singular como
resultado dessa fusão entre os elementos dispostos e construídos. Em outras
palavras: foram se tornando ‘movimentos’. Ao que tudo indica, como observa Melucci, nas sociedades contemporâ-
neas, a identidade seria vivenciada como ação e não como situação, sendo
construída no complexo de relações sociais estabelecidas no processo de vida
e dentro dos meios e lugares sociais ocupados. Assim, haveria uma alteração na noção de identidade que, baseada nesse
pressuposto de constituição permanente, auto-reflexiva, e, sobretudo, opera-
da por cada pessoa, precisaria de novos termos: “A mesma palavra identidade
não é mais apropriada para exprimir essa mudança e será necessário falar de
identificação para exprimir o caráter processual, auto-reflexivo e construído
da definição de nós mesmos”.18 Dessa forma, o autor quer dar conta da transitoriedade do contemporâneo,
da multiplicidade de papéis que cada indivíduo assume cotidianamente. Entre-
tanto, no caso dos jovens desses grupos (punk e hip-hop), uma das suas muitas
identidades surgidas no momento de identificação pela música, pela dança, por
manifestações afins, e de uma definição de papéis, acaba tornando-se duradou-
ra; o que ocorre é que a grande maioria acaba assumindo de forma definitiva es-
sas identificações (que em princípio são transitórias). O fato de esses jovens assumirem uma dessas identificações de forma per-
manente não significa que o caráter de autoconstrução, de auto-reflexão, seja
dispensado, mas parece que o processo se dá de forma intensa, dentro do pró-
prio estilo que assumem. Revista Brasileira de História, vol. 27, nº 53 OS MOVIMENTOS Assim, pode-se falar em identidades da mesma for-
ma ou deslocar o conceito de identidade nesse sentido. Se, como afirma Maffesoli,“a sensibilidade coletiva, originária da forma
estética acaba por constituir uma relação ética”,19 então pode-se considerar
que esse conjunto de relações instituídas no campo ético/estético, pode se tor-
nar o centro do complexo processo de identificação/identidade, como de fato
acontece com muito dos entrevistados.20 222 Revista Brasileira de História, vol. 27, nº 53 As cidades da juventude em Fortaleza As cidades da juventude em Fortaleza As cidades da juventude em Fortaleza Dessa forma, constituem uma ‘linguagem’ própria, que articula não só
suas falas, mas também os tipos de música, o uso de determinadas roupas e
adereços, os símbolos, os locais de freqüência, os usos da cidade, ou as cons-
tituições de suas trajetórias da/na cidade, as manifestações do próprio corpo,
os cortes de cabelo, as gírias, os aliados, e também, por que não dizer, os pró-
prios adversários.21 Essa linguagem articulada dentro do cotidiano é manifesta de forma os-
tentatória, com a sutileza de seu uso dirigido apenas aos iniciados, de tal for-
ma que mesmo numa multidão, pode-se passar por eles sem percebê-los, num
jogo do visível/invisível, em alguns casos. Em outros casos, o visual é assumido para chocar, impactar, como acon-
tece com os punks, nessa fase. Entretanto, mais recentemente, essa caracterís-
tica é abandonada, em detrimento de uma postura em que esse aspecto se tor-
na opcional. Oliveira nos dá conta de um congresso ocorrido em João Pessoa,
em que se discutiu e se deliberou o seguinte: “os que ali estavam concluíram
que, o mais importante na sua estética não é a forma ‘X’ ou ‘Y’, mas os princí-
pios que a guiam...”.22 Assim, pode-se inferir que essa linguagem constituída de símbolos e si-
nais, pontos geográficos e espaciais, sentimentos e formas diversas de sentir e
participar, são incorporadas e desenvolvidas com base em uma noção maior
que acaba assumindo proporções cada vez mais significativas tanto em suas
vidas como,em resultado disso,na própria vida social e política: o movimento. junho de 2007 OS MOVIMENTOS É em torno do movimento que tanto punks quanto hip-hopers se insur-
gem no espaço urbano contra a exploração, a mesmice, a guerra, o capitalis-
mo, a anti-cidadania, a falta de condições de vida dignas e justas, e tantas ou-
tras questões que os afligem e incomodam e com o que imaginam estar
produzindo um mundo melhor e reinventando sua cidadania. No início dos anos 90 cria-se na cidade de Fortaleza uma entidade orga-
nizativa dos hip-hopers (MH2O — “Movimento Hip-Hop Organizado do Cea-
rá”) em torno da qual todas as atividades ligadas a essa manifestação se de-
senrolaram a partir de então. No final da mesma década, muitas outras
entidades surgem como ‘rachas’ da primeira. Hoje, uma outra além dela é for-
te e ativa na cidade — O “Movimento Cultura de Rua” ou MCR. Esta entida-
de se coloca como um movimento social organizado, ou pretende se produ-
zir como tal. Nesse mesmo período a entidade MH2O se institui como movimento e
cria um estatuto jurídico, tornando-se uma organização não-governamental
(ONG), o mesmo ocorrendo com o MCR pouco tempo depois. O trabalho 223 Francisco José Gomes Damasceno das duas é muito semelhante: música, dança e arte são utilizadas no engaja-
mento político, social e profissional dos jovens pertencentes ao movimento. Sutileza que diferencia as entidades das demais, já que o trabalho político vem
à frente do musical. Embora o foco inicial do hip-hop em Fortaleza tenha sido o bairro do
Conjunto Ceará, onde até hoje o movimento é muito forte e manifesta-se co-
tidianamente, com a criação das entidades, com o intenso bombardeio das
mídias e a divulgação do estilo, e, principalmente pela atuação do próprio
movimento e de suas diversas entidades, toda a cidade, em seus diversos bair-
ros, já é possuída pelos jovens. De acordo com a nucleação das entidades há uma maior presença de um
ou outro movimento nesse ou naquele bairro. Assim, na Quadra de Santa Ce-
cília, onde reside boa parte dos jovens ligados ao MCR, onde se situa sua ‘se-
de’ e se desenvolvem suas principais atividades e projetos pilotos, tem-se “uma
área do MCR”. O Conjunto Ceará “se constitui em uma área do MH2O”, gra-
ças aos vínculos históricos com esse bairro. Revista Brasileira de História, vol. 27, nº 53 MOVIMENTAÇÕES JUVENIS E TRANSPOSIÇÕES GEO-ESTÉTICAS O fato de os punks não possuírem um território fixo na cidade também me
conduziu a pensar no território como corpo, ainda mais ao perceber que é nas
roupas que o punk estampa seus pensamentos, colocando-se na rua como uma
espécie de bandeira do movimento ... E os critérios dessa seleção não foram os que eu esperava: apego ao lugar, sen-
timento de pertença, de segurança etc., mas sim critérios de praticidade, de pro-
ximidade e de possibilidade. Essas considerações foram tornando a dimensão
territorial do punk em Londrina muito mais confusa, pois era diferente do que
eu havia tomado conhecimento em teoria ... Nesse caso, o território não seria propriamente o bar, mas sim a calçada, e os
limites do território seriam os corpos. As pessoas que transitam pela calçada des-
viam do grupo e lançam olhares desconfiados.24 O que de fato se pode ler nessa movimentação25 é uma apropriação da
cidade de forma diferenciada da que ocorria até então. Primeiro, é interes-
sante lembrar que essa nova forma é feita pelo viés da arte, da cultura e do la-
zer, o que, por si só, já se constitui em uma novidade para essa cidade e sob a
influência direta dela, em outras palavras, em resposta aos desafios colocados
pela própria vida na cidade. Segundo, pelo inusitado do papel que a música
assume nesse processo.26 Há um duplo giratório de apropriação da juventu-
de. Por um lado, do ponto de vista social: trata-se de uma juventude de ‘peri-
feria’ que se manifesta de forma política, social e cultural e insere-se nos mais
variados contextos pela sua arte, pela sua música, fazendo disso sua forma de
intervenção. Do ponto de vista estético, dão uma conotação radical ao rock e ao hip-
hop pela incorporação das condições de vida por eles enfrentadas nesse pro-
cesso de re-invenção de si próprios,como também pela re-invenção da própria
arte por eles praticada.27 A apropriação ‘geo-estética’ da cidade dá-se na medida em que os jovens
utilizando-se da arte de viver — como passo a entender sua arte, produzida
enquanto ética e estética de vida — produzem outras ‘Fortalezas’ para vive-
rem, onde os espaços de convivência se tornam referência de bem viver, rega-
dos a músicas, festas, prazer e trabalho coletivo de construção dessas utopias
juvenis. OS MOVIMENTOS No entanto, suas atuações não se
limitam a esses locais e se desenrolam, como já disse, em toda a cidade, mui-
tas vezes concomitantemente, em relações complexas, nem sempre harmo-
niosas. Com os punks o processo é semelhante, mas a orientação anarquista im-
prime um outro viés: a criação de coletivos de convivência alternativa. O pri-
meiro é criado ainda nos anos 80 (o Núcleo Coletivo de Consciência Libertá-
ria — NCCL), e durou até o final dessa mesma década. Depois disso muitos
outros vieram, e nos anos 90 o mais presente foi o ‘Coletivo Ruptura’. Até re-
centemente a principal articulação se dava em torno da ‘Comuna Libertária’,
uma entidade sediada no bairro de Parangaba e que encaminha a luta de punks
e anarquistas sob essa ótica. As muitas formas de manifestação de seus desejos e necessidades têm
apontado para uma construção alternativa nesses moldes. Além disso, uma
articulação local e global23 se fortalece com vínculos de solidariedade e traba-
lho com jovens e entidades de outros estados, como suas movimentações mais
freqüentes grafitam, cantam e dançam por aí... A música iniciada nos ‘bailinhos’ quebrou as paredes dos clubes de clas-
se média, espalhou-se como um rastro de pólvora, e botou fogo em tudo ao
seu redor. Ao lazer puro e simples, juntou-se um outro com prazer misturado
à política, à arte, à própria vida, como resposta direta aos desafios cotidianos. O canto falado e o grito primal nunca foram tão ouvidos e fortes. 224 Revista Brasileira de História, vol. 27, nº 53 Revista Brasileira de História, vol. 27, nº 53 As cidades da juventude em Fortaleza junho de 2007 MOVIMENTAÇÕES JUVENIS E TRANSPOSIÇÕES GEO-ESTÉTICAS A cidade, constituída por essas outras trajetórias, torna-se ao mesmo
tempo múltipla, e, além de palco, sujeito, com os jovens em questão, de ou- 225 Francisco José Gomes Damasceno tras histórias. O rock e, pouco tempo depois, o hip-hop, apropriados pelos jo-
vens, servem de elementos de ressignificação da cidade e de suas próprias ex-
periências, e a cidade existente, junto com a cidade criada por eles, servem
(ambas) de baliza para sua própria recriação. A reinvenção se dá nesse múlti-
plo onde tudo e todos se insinuam uns sobre os outros. O rock que chegou a Fortaleza, em meados dos anos 50, nos salões dos
clubes de elite ou das camadas médias urbanas e dentro de certos padrões de
socialidade, sendo associado a jovens das famílias mais abastadas, como já o
mostramos, aos poucos se altera, é tomado por outros sujeitos (jovens) e os
toma, re-inventando a si mesmo, como resultado desse processo de tomar e
ser tomado, de inventar e ser inventado, mas também, e, sobretudo, como re-
sultado direto de experiências que se dão com as especificidades do contexto
em questão e do meio (cidade) no qual se dão. Se, no início, o principal clube da cidade e das elites de Fortaleza (Náuti-
co) abrigava o rock, bem como clubes elitizados, embora menos tradicionais
(Maguary e Líbano), clubes estes situados à beira-mar (Náutico) ou em re-
giões centrais e ocupadas por essas elites da cidade, e os grupos de baile ‘arra-
savam’em ritmos ‘frenéticos’com um ‘balanço quente, mas respeitoso’(o mais
conhecido foi “Iranildo e seu conjunto”), logo em seguida o que se operaria
seria muito diferente. Ocorreria uma mudança, dessas regiões das elites para
as periferias, sobretudo das camadas pobres da população, e também desses
clubes para outros, criados no processo de expansão da cidade rumo às di-
versas saídas para o interior.28 Silva esclarece esse modelo de urbanização, que é duramente criticado
pelos jovens punks e hip-hopers em suas letras, em sua arte e intervenção sócio-
histórica: Parte do monstro urbano que conhecemos foi gerado por medidas autoritá-
rias tomadas em gabinetes oficiais, com forte influência de políticos e empresá-
rios. Revista Brasileira de História, vol. 27, nº 53 MOVIMENTAÇÕES JUVENIS E TRANSPOSIÇÕES GEO-ESTÉTICAS A formação de extensas periferias e bolsões de miséria, constituídos quase
que unicamente por conjuntos habitacionais distantes e mal servidos, a opção
pelo transporte individual em detrimento do transporte coletivo, a facilidade de
se implantar novas infra-estruturas que ocasionam a degradação de setores ou
bairros tradicionais, expressam essa forma equivocada de produzir a cidade.29 Nos anos 60 o rock explode mundo afora e a repercussão dessa onda no
Ceará foi inevitável, desta feita misturada a outros ritmos, mas há uma ver-
dadeira proliferação de bandas, como as já apontadas anteriormente — Os 226 Revista Brasileira de História, vol. 27, nº 53 As cidades da juventude em Fortaleza As cidades da juventude em Fortaleza Faraós; Os Belgas tocando basicamente músicas de outros conjuntos como
Beatles e Renato e Seus Blue Caps; e ainda Os Diferentes que tanto tocavam
músicas de outros conjuntos com arranjos próprios, como as próprias com-
posições. Faraós; Os Belgas tocando basicamente músicas de outros conjuntos como
Beatles e Renato e Seus Blue Caps; e ainda Os Diferentes que tanto tocavam
músicas de outros conjuntos com arranjos próprios, como as próprias com-
posições. Nos anos 70 as bandas continuam a sua atuação em bailes e clubes de eli-
te, como os já citados, mas há certo desgaste na fórmula, tanto pela sua exaus-
tão, por certa repetição, como por outros dois fatores: 1) O rock em todo o
mundo sofre mais uma de suas ‘revoluções internas’, desta vez o punk opera
sua obra; e, 2) se inicia um processo, que nos anos 90 se tornaria hegemôni-
co, de construção do forró como ritmo preponderante na cidade, colocado
como “autêntico reflexo de nossa regionalidade”. Mesmo assim, bandas como
a Peso ou a Posh se tornaram bastante conhecidas no cenário local e tiveram
suas contribuições nesse contexto. junho de 2007 MOVIMENTAÇÕES JUVENIS E TRANSPOSIÇÕES GEO-ESTÉTICAS Assim, nos encontramos com os nossos sujeitos-jovens, jovens-sujeitos,
no final dos anos 70, em um cotidiano delicado, refletindo um acentuado cres-
cimento da então pequenina cidade de Fortaleza, graças ao processo de in-
chaço sofrido pela chegada constante de migrantes oriundos do interior do
Estado, que se reflete em espaços compactados, com o surgimento de bairros
e loteamentos, e mesmo de favelas (apesar dos altos investimentos nos con-
juntos habitacionais planejados e construídos a partir de então) sem os ne-
cessários investimentos: lazer, emprego e opções de vida para uma população
marcadamente jovem.30 A semelhança com os aspectos que dão origem às manifestações de jo-
vens punks (no final dos anos 70) e de jovens hip-hopers (no início dos anos
80) não é coincidência. E a chegada de elementos culturais que traduziam es-
sas realidades em outras partes do mundo parece ter sido amalgamada por
esses jovens, incorporada e ter servido de instrumento em seus processos de
apropriação do que se constituía para eles: a vida urbana. Não havia, até então, uma distinção musical, que só viria com o ‘amadu-
recimento’ das manifestações. Apenas algum tempo depois é que alguma di-
ferenciação foi percebida e estabelecida, e fomentou, assim, a formação de
grupos por afinidades musicais e estéticas: “assim que a gente gostava, que tu-
do era rock, mas a diferenciação que a gente não tinha conhecimento; que a
gente foi conhecendo até por um acaso, rolou de conhecer... aí conheci Sex
Pistols, The Clash, The Jam, The Breathers...”.31 Em outras palavras, o rock e o hip-hop (sofrendo a influência do próprio
rock) aportam enquanto musicalidades, agrupam pelo gosto, pela afinidade,
pela eleição do ritmo, pela incorporação estética, até mesmo de elementos co- 227 Francisco José Gomes Damasceno mo a dança e outros de dimensão plástica (como o grafite, ou mesmo o da
constituição dos visuais), e deste ponto servem de elemento de aglutinação
dos jovens identificados com essas manifestações, que passam então a re-
inventá-las a partir de suas próprias experiências, expectativas e desejos. MOVIMENTAÇÕES JUVENIS E TRANSPOSIÇÕES GEO-ESTÉTICAS Esse ponto de partida — a eletividade32 e a comunhão estética e ética —
tem sustentação em um cotidiano de experiências comuns, de perspectivas
de vida semelhantes, e constitui o mais caro no que denominamos de ‘expe-
riência musical’, algo que está na musicalidade, na música propriamente dita,
mas que se constitui diferentemente, apenas a partir do momento em que o
que está nas entrelinhas da pauta musical é absorvido, construído por quem
ouve e faz música. Revista Brasileira de História, vol. 27, nº 53 OS SONS E A DANÇA – UMA OUTRA EXPERIÊNCIA Seu caráter mais importante reside na afirmação da existência de um mun-
do universalmente obrigatório ... um mundo essencialmente diferente do
mundo factual da luta cotidiana pela existência, mas que cada indivíduo pode
realizar ... por si mesmo, ‘do interior’, sem transformação alguma do real ... Na verdade, mesmo quando a arte protesta contra a miséria social e a soli-
dão, esse protesto é reapropriado esteticamente e solto de maneira gratificante,
na experiência imaginária individual, como parte integrante do prazer que a
obra lhe proporciona, e não como crítica real e desejo de mudar o mundo. As-
sim, Marcuse conclui que mesmo ‘a idéia rebelde se torna um acessório para a
justificação’.33 É desta forma que se constitui aos poucos o que denominamos de expe-
riência musical, entre encontros furtivos no centro da cidade, nas praças dos
diversos bairros periféricos da cidade de Fortaleza, em encontros nas casas
para a audição do que ‘vem de fora’, em bailes e festas para a prática pública
da escuta e da dança, no exercício, enfim, da manifestação da forma específi-
ca como se resolve constituí-la. Disto resultam os deslocamentos diferenciados pela cidade em busca de
diversão e arte, em busca dos iguais a si, dos diferentes de si, dos espaços para
as práticas recém-instauradas de escuta, dança e vivência da música, então
novidade na pequena cidade de Fortaleza. Com ela surge toda uma angústia
de ser jovem naquele momento, associada a certa rebeldia presente nas musi-
calidades em questão, veiculadas, sobretudo, em suas letras. 228 Revista Brasileira de História, vol. 27, nº 53 As cidades da juventude em Fortaleza As cidades da juventude em Fortaleza As cidades da juventude em Fortaleza O que ocorre é que ‘música’ deixa de ser apenas uma construção meló-
dica e passa a dar sustentação a suas vidas de forma mais ampla. Passa a cons-
tituir eticamente, se não todas, pelo menos grande parte de suas ações. Orien-
ta para a vida ou pela vida afora, de tal modo que nos casos dos universos
pesquisados nos últimos dez anos (hip-hop e punk rock), todos os jovens en-
volvidos com essas musicalidades e ou movimentos continuam se pautando
por essas perspectivas. A noção de música se amplia, e tem-se uma outra, a de ‘experiência mu-
sical’, que se entende dentro desse complexo quadro de fusão entre experiên-
cias de vida, eletividades afetivas, estéticas e práticas sócio-musicais. junho de 2007 OS SONS E A DANÇA – UMA OUTRA EXPERIÊNCIA E tam-
bém a dimensão de uma arte enquanto prática, saber-fazer específico, e como
tal com uma categoria de agentes ou sujeitos que detêm esse conhecimento.34 Essa arte transformada em experiência musical passa a ser construída de
forma social por seus sujeitos, que atribuem significados a ela, extrapolando
o campo de uma arte fechada em si mesma e se tornando o ponto de articu-
lação entre suas concepções de mundo e suas experiências de vida, dadas no
universo do sensível. Nesse sentido, a dança é outro aspecto da ‘experiência musical’ que deve
ser observado como parte indissociável da musicalidade. O rock trouxe à ce-
na no mundo contemporâneo essa dimensão de forma explícita e irrevogá-
vel, e essa apropriação que, em certa medida, rompia com uma postura de
contemplação estática, dos modelos anteriores, associados à música de câma-
ra, é, talvez, sua marca mais registrada.35 Há uma espécie de dissolução dos sentidos como os conhecemos, de for-
ma que cada um possua uma função específica, empregada quando necessá-
rio. É como se todos os sentidos se tornassem um só e fossem empregados na
mesma tarefa, ao mesmo tempo. A força da música, das letras, do ambiente,
da bebida, da catarse promovida pelos efeitos de tudo isso junto, cria uma
forma de sentir diferente, feita com o corpo como um todo homogêneo e in-
divisível: “Ouvir com o corpo e empregar no ato da escuta não apenas os ou-
vidos, mas a pele toda, que vibra ao contato com o dado sonoro: é sentir em
estado bruto. É misturar o pulsar do som com as batidas do coração, é um
quase não pensar”.36 Da mesma forma que essa dissolução se opera na musicalidade e nas suas
danças, opera-se também uma fusão ética/estética, de tal modo que o ideal
de beleza das manifestações musicais se transfere para a vida, a vida bela é
‘dançada’ aos ritmos de suas músicas, e mesmo compreendida diferentemen-
te do que encontram no desigual quadro social no qual estão inseridos. 229 Francisco José Gomes Damasceno Francisco José Gomes Damasceno Torna-se, então, uma forma política de entendimento e de manifestação
aos poucos burilada em uma interação política e social com outros atores so-
ciais, tais como partidos, sindicatos, coletivos, universidades e grupos. OS SONS E A DANÇA – UMA OUTRA EXPERIÊNCIA A par-
tir disso criam-se os movimentos, caudalosos, com adeptos onde quer que
ocorram, em uma ‘nação imaginada’, ligada pelos sentimentos despertados
pela música e construídos com suas próprias especificidades e sutilezas. As singularidades dos movimentos em Fortaleza estão justamente na cria-
ção de suas entidades em torno de uma manifestação inicialmente musical,
ou, em outras palavras, na constituição de um movimento sócio-político-
cultural em torno do hip-hop e do punk. Portanto, o que antes era música, aos
poucos fundamenta-se e se torna movimento.37 Revista Brasileira de História, vol. 27, nº 53 A INVENÇÃO PELA MÚSICA – DESLOCAMENTOS DA/NA CIDADE Nós vemos, toda Cidade é uma espécie de comunidade, e que toda comuni-
dade é constituída em função de um certo bem (porque é em vista de obter aqui-
lo que lhes aparece como um bem realizar sempre seus atos): daí resulta clara-
mente que se todas as comunidades visam um bem determinado, aquela que está
mais alta de todas e que engloba todas as outras, visa também, mais que as ou-
tras, um bem que é o mais alto de todos. Esta comunidade é aquela que é cha-
mada Cidade, é a comunidade política.38 Assim, a experiência musical desses jovens é um aspecto que deve ser le-
vado em consideração no deslocamento dos clubes de elite para os clubes de
periferia. Trata-se de uma juventude pobre e de periferia se manifestando e
tendo a atenção de setores sociais diversos. Saímos do Maguary,do Líbano e do Náutico,de grupos de baile como “Ira-
nildo e seu conjunto”, e, de uma musicalidade comportada e aceita, para clubes
de periferia como o Apache, o Mênfis Clube do Antônio Bezerra, o Keops Clu-
be, o Detroit, entre tantos outros, produzidos enquanto espaços por jovens po-
bres, muitas vezes radicais, e de periferia, com bandas como Estado Indigente,
Zueira, Desnutrição, e rappers como Titio e Cachorrão, W Man, Poeta Urbano
e ZMC, entre tantos outros. Esse deslocamento que é ao mesmo tempo geográfico, posto que se faz
dos espaços das elites para os espaços populares, tem ao mesmo tempo um
caráter de classe e etário, já que é operado por jovens trabalhadores, ou mes-
mo jovens sem profissão, desempregados e pobres, ou ainda estudantes, por- 230 Revista Brasileira de História, vol. 27, nº 53 As cidades da juventude em Fortaleza tanto, em formação para o mundo do trabalho, habitantes dos bairros mais
pobres de minha cidade. Também tem um caráter estético, já que dentro das duas manifestações
o que ocorre de forma invariável é uma sucessão de transgressões estéticas de
cada uma dessas musicalidades, além de ser uma transgressão também para
o tipo de música (mesmo de rock e de hip-hop) feito em Fortaleza no perío-
do. Fortaleza jamais havia visto algo tão ‘radical’. Além disso, essa dimensão
transgressiva de alguma forma constituiu os seus repertórios de compreen-
são e de articulação estética e posteriormente também política. A INVENÇÃO PELA MÚSICA – DESLOCAMENTOS DA/NA CIDADE Amalgamando todos esses aspectos, consubstanciados nesse deslocamen-
to, estão suas experiências de vida, suas experiências musicais, que dão a esse
movimento um caráter de duplo deslocamento, por um lado estético e por
outro geográfico, ou mesmo geopolítico, daí por que falamos em deslocamen-
tos ‘geo-estéticos’ em Fortaleza. Utilizo a noção de ‘deslocamento geo-estético’ 39 como uma fusão do es-
paço geográfico e da manifestação estética e que dá sentido a esses espaços,
configurando assim, com base na idéia de que o espaço é o lugar praticado,40
uma outra dimensão: a dimensão da arte de viver, de viver com as referências
de beleza por eles instituídas, e, assim, elegendo as trajetórias a serem estabe-
lecidas dentro do universo urbano, que redimensiona o espaço a partir da ar-
te (música, dança) e instaura a cidade na qual vivem, dentro das muitas cida-
des existentes na cidade de Fortaleza. junho de 2007 EXPERIÊNCIAS MUSICAIS, DESLOCAMENTOS
GEO-ESTÉTICOS: AS CIDADES E OS JOVENS As leituras construídas pelos jovens em suas letras e manifestos guardam
características peculiares. Vejamos especificamente e de forma rápida o caso
de algumas noções dos jovens punks e hip-hopers. Em primeiro lugar, como a
sociedade que emerge de suas letras nos é re-apresentada; em seguida, a no-
ção que têm de si próprios. Emerge de suas letras uma cidade identificada com a divisão social, ou
uma ‘sociedade’ constituída a partir de suas experiências na/da cidade. Assim
se caracteriza uma cidade/sociedade marcadamente repressora, desigual, di-
vidida, exploradora e injusta. É entendida como a máxima manifestação or-
ganizacional humana e, portanto, o que se representa é uma humanidade di-
vidida e à beira de um tipo qualquer de colapso. 231 Francisco José Gomes Damasceno Francisco José Gomes Damasceno Divididos em classes, os raps e punk rocks são construídos com a inten-
ção de serem a fala das minorias sem voz, dos injustiçados, marginais, prosti-
tutas, mendigos, meninos de rua, trabalhadores, e, sobretudo, das periferias,
do povo pobre, operários e desvalidos.41 pois nós somos a voz da periferia / o grito do gueto / o argumento da pobreza /
pra lutar-brigar...42 Ossos quebrados / Mente vazia / Olhos arrancados / Intestino apodrecido / Re-
trato operário / Pobre indigente / Largado jogado / Vítimas inocentes / Largado
jogado / Estado esquecido / De putrefação / De imperialismo / Diferença social
/ Grito de dor / Invasão militar / Só carnificina / Não há sobrevivência / Minha
vida esquecida / Não há sobrevivência / Sua vida apodrecida.43 A sociedade para eles é a ‘encarnação’ dessas diferenças, e, portanto, a sua
manutenção é necessária para quem delas se beneficia. Identificados como os
inimigos, os ricos são o principal alvo: a sociedade fecha as portas na cara de quem nada tem / pois as portas são fecha-
das / grades seladas / pois é isso que convém antes de você / cometer o delito /
delito ou ato de sobreviver...44 Em outro rap se mostra concretamente como essa sociedade de ricos se
fecha: Em outro rap se mostra concretamente como essa sociedade de ricos se
fecha: De que vale o Beach Park se eu não posso entrar? / de que me serve o Iguatemi
se eu não tenho ‘grana’ pra gastar? / carro importado pra quê se eu não posso
comprar? EXPERIÊNCIAS MUSICAIS, DESLOCAMENTOS
GEO-ESTÉTICOS: AS CIDADES E OS JOVENS 45 A inclinação desses jovens à esquerda (não única) a partir da criação de
seus movimentos e de articulações com partidos políticos, sindicatos, ONGs,
coletivos e outras instituições revela que a cidadania não é uma construção meramente voltada e construída pelo políti-
co, embora possa vir a ser desta forma enunciada. A questão social aparece co-
mo um problema posto pelo desenvolvimento das cidades, e uma das formas do
seu enfrentamento é o deflagrar da construção da cidadania.46 A aparição no universo citadino é uma intervenção de conquista dos di-
reitos elementares negados, e as suas letras, os seus manifestos podem ser en- 232 Revista Brasileira de História, vol. 27, nº 53 As cidades da juventude em Fortaleza tendidos como a fala de sujeitos sociais em ebulição na dividida cidade de ri-
cos e pobres, na Fortaleza de muitos caminhos possíveis e instituição de seus
desejos de cidades outras. Eles se apresentam aos poucos, em indícios sutis do lugar social de suas
falas. Isso pode ser observado em um dos primeiros raps do movimento a fa-
zer sucesso, em toda a cidade, o “Rap da Dora” da dupla Titio e Cachorrão. Nele o trabalho é identificado ao sofrimento — leia-se o trabalho que não re-
compensa — criando a necessidade de espaços onde o sofrimento deve ser
substituído pela alegria e pela vida. EXPERIÊNCIAS MUSICAIS, DESLOCAMENTOS
GEO-ESTÉTICOS: AS CIDADES E OS JOVENS Expressa de forma ambígua a Dora:“boto a mão na cabeça é tão bom co-
mo me sinto / pois nesses dias não tem que trabalhar fico em casa a se ajeitar
/ pois nesses dias quer também se divertir...” — observe-se que o sujeito ‘eu’
elíptico e depois indefinido pelo uso da partícula ‘se’, provocando uma auto-
mática identificação/confusão autores/Dora — como se por sua boca eles fa-
lassem, mostrando como se sentem estes jovens que, imersos no mundo do
trabalho, na tensão nele estabelecida, encontram nos bailes, nos momentos
de diversão e lazer, uma das poucas alegrias que têm.47 Também nos punk rocks a condição operária é sentida em manifestos,
documentos, músicas: A vida de operário é um tormento / A vida de operário é um sacrifício / Pra vi-
ver (2x) / Sem razão (2x) / Escravo moderno nos campos / Escravo moderno nas
fábricas / Pra viver (2x) / Sem razão (2x) / Viver pelas leis dos patrões / Conde-
nados à pena de morte / Pra viver (2x) / Sem razão (2x) 48 Eis que se revelam suas faces: jovens trabalhadores da cidade. Pequenos
operários, desempregados, mão-de-obra pouco qualificada e barata, que na
cidade através de sua arte se produz enquanto grupo e aponta os sérios pro-
blemas que os cercam e demandam solução imediata. EXPERIÊNCIAS MUSICAIS, DESLOCAMENTOS
GEO-ESTÉTICOS: AS CIDADES E OS JOVENS A cidade apartada aparece em vários momentos, como neste trecho: quem vê Fortaleza como no resto do Brasil / vê estupro, assassinato, corrupção
desenfreada / temos brigas de gangues / assaltos a mão armada, traficantes de
maconha e polícia bem safada / os garotos roubando lá na praça da estação / e
no passeio público tem até prostituição / sem esquecer dos mendigos lá do cen-
tro da cidade / e dos apatoladores que existem aos milhares / temos mortes no
trânsito e muitos viciados / motoristas imprudentes e hospitais superlotados /
temos roubo de carro e cheques sem fundos / pode crer na capital nós temos de 233 junho de 2007 junho de 2007 Francisco José Gomes Damasceno tudo / o nosso Ceará com todos seus problemas / é o rap do Conscientes do Sis-
tema (3x) / aqui também tem desemprego aumentando a violência / e escolas
do governo numa tremenda decadência / temos rádio e TV / revista e jornal /
essa merda que ilude e nos fazem passar mal / distorcendo as notícias isso eles
fazem muito bem / transformando em marginais muitas pessoas de bem / essa é
a dura realidade quem tem grana mata e rouba e ainda fica em liberdade / ao
contrário do pobre que não tem nada e é fodido / só por causa da aparência leva
nome de bandido / temos o Iguatemi e o Shopping Aldeota / a rota dos burgue-
ses que nos vêem como idiotas / temos fome e miséria e até racionamento / gen-
te inocente passando o maior tormento / “eu sou a nata do lixo eu sou o luxo da
aldeia eu sou do Ceará” (sampler) / o nosso Ceará com todos seus problemas / é
o rap do Conscientes do Sistema (3x) 49 Outra letra bastante significativa de suas idéias sobre si e sobre a cidade
é “Nativo Urbano”, da banda Estado Indigente: Mesmo que eu morasse / No mundo sozinho / Mesmo que eu morasse / Em
mundo perfeito / Tocava hardcore pra vida / Com prazer (2x) / E nada me im-
pediria / Consciência, capacidade e prazer / Virtudes de poucas viventes / No ha-
bitar sonolento da vida / Almeja sonho impotente / Desmotiva pela inconsciên-
cia / De outros / Paralelo, direto, nostálgico. Revista Brasileira de História, vol. 27, nº 53 EXPERIÊNCIAS MUSICAIS, DESLOCAMENTOS
GEO-ESTÉTICOS: AS CIDADES E OS JOVENS A consciência de si e dos outros, a possibilidade de intervenção direta,
operada pelo sujeito consciente, apesar da identificação da vida aos bailes, re-
vela certa tristeza, oriunda decerto de suas origens, ou dos meios nos quais se
manifestam: as ruas.50 O espaço da rua, longe de ser o espaço de ‘deambulação’ como é signifi-
cado para os rockers,51 constitui-se em espaço de vivências, de violência, de
amores, de lutas, de revolta, torna-se o palco de suas vidas, o local onde vi-
vendo, buscam inspiração para suas letras, ou ainda onde vivem as histórias
que suas letras contam. Locais perigosos e instáveis, onde se corre o risco de viver. Seus habitan-
tes, longe da calma das casas, da segurança das famílias, da limpeza e higiene
dos lares normais, são múltiplos e dividem a agitação, o vai-e-vem de tran-
seuntes, a insegurança da luta pela sobrevivência, a sujeira causada pelas aglo-
merações etc. É o espaço da violência, da morte, da dor, da fome, da miséria, dos des-
validos, dos marginalizados, dos menores abandonados, delinqüentes, prosti- 234 Revista Brasileira de História, vol. 27, nº 53 As cidades da juventude em Fortaleza tutas, pobres e desocupados. Tipos como o ‘vagabundo’, que, como nos mos-
tram B.Girl e Rainha,“vive pelas ruas do centro da cidade”: tutas, pobres e desocupados. Tipos como o ‘vagabundo’, que, como nos mos-
tram B.Girl e Rainha,“vive pelas ruas do centro da cidade”: aonde a cada esquina um assalto acontece ... marginais andam a solta, tromba-
dinhas cheiram cola / se não preferem o roubo muitos deles pedem esmola / são
jogadores nas ruas pra ganhar algum dinheiro / mas as ruas lhes ensinam o ver-
dadeiro desacerto...52 se os meus filhos estão nas ruas / suas vidas aventuram lances, mortes,
extermínio ... Rap e Hip-Hop nas calçadas da cidade / o Break violento e
forte... (grifo meu)53 se os meus filhos estão nas ruas / suas vidas aventuram lances, mortes,
extermínio ... Rap e Hip-Hop nas calçadas da cidade / o Break violento e
forte... (grifo meu)53 As ruas tomam conta desses seres / menores carentes ninguém quer saber deles
... aqui trombadinhas aumentam a cada dia / menores nas ruas, crianças perdi-
das / filhos das ruas, quem vai tirá-los da rua? / quem os fará viver? / ... e você...? EXPERIÊNCIAS MUSICAIS, DESLOCAMENTOS
GEO-ESTÉTICOS: AS CIDADES E OS JOVENS / mas vocês são todos iguais / em suas casas tão em paz / longe das ruas, longe
da sujeira, longe da vida imunda / eu aqui e você na sua / e os menores carentes
estão todos nas ruas / filhos da rua / filhos da rua / sem-tetos dormindo nas pra-
ças / ... matar ou morrer faz parte da vida / da vida na verdade não são inocen-
tes são vítimas conseqüentes / crianças delinqüentes / ... prostituir o próprio
corpo em troca de quase nada ... / flanelas na mão pedindo um trocado / sem
esperanças, sem família, nas ruas jogados / não existe para eles sonho algum /
vivem o presente sem futuro nenhum...54 No percurso pelas trilhas abertas na cidade, na instauração das muitas
cidades, se estabelecem marcações, se estabelecem territórios no andamento
da vida. Eles instituem os seus marcos, ou pequenos portos no mar da cidade. Depois de algum tempo, as turmas punks passaram a se encontrar na re-
gião do centro da cidade, na Praça do Ferreira, depois na Praça José de Alen-
car; os rappers em locais específicos nos bairros, como o Pólo de Lazer do
Conjunto Ceará, que se constituíram em points, onde eram ‘trocadas as idéias’
e traçadas as trajetórias de cada final de semana: Eu estudava ainda, vinha à Praça José de Alencar, que de certa forma sempre
foi um point, aqui em frente ao teatro todas as sextas tinha um grupo tocando
MPB, música daqui mesmo. Aí, os mesmos roqueiros que pintavam lá no Grê-
mio do José Walter se reuniam todas as sextas aqui nessa praça ou então na an- 235 junho de 2007 junho de 2007 Francisco José Gomes Damasceno tiga Praça do Ferreira, e aí a gente foi se entrosando. Quando não queria ir pra
casa, ia pro Monte Castelo, a gente virava a noite, ficava ouvindo som...55 Nos points se decidia a programação do final de semana, marcavam-se
os encontros, escolhiam-se os melhores programas, além, é claro, da tradicio-
nal ‘troca de idéias’: Nessa época eu não tinha muito tempo, porque eu trabalhava durante o dia e
estudava à noite, mas foi uma das épocas que eu vacilei mais com o estudo. Eu
deixava de ir pro colégio pra pintar nos points às quartas e sextas, ali na Praça
do Ferreira. EXPERIÊNCIAS MUSICAIS, DESLOCAMENTOS
GEO-ESTÉTICOS: AS CIDADES E OS JOVENS E tinha um dos primeiros clubes com sons de bandas, só Ramortes
que tocava na época, o Davis Clube, lá na pista da Barra. Que lá o cara aloprava
todas as quartas, sextas, sábados e domingos, quando a banda não tocava era
som de fita...56 O point nunca era o fim de uma trajetória e ou de uma jornada, mas sem-
pre o seu ponto inicial, o local de encontro, o ponto de referência em torno
do qual todos aqueles que queriam podiam se encontrar. O fim da jornada,
quando não havia um baile em jogo, era a experiência comum de ouvir mú-
sica, de trocar idéias e depois fazer as próprias músicas, aprender como tecer
sua arte: a gente se reunia ali, e ia pra casa dum, pra casa do outro, do João Wilsom, aí fi-
cava lá, ouvindo o som, trocando idéia, fazendo música... Era isso quase todo
dia. Nos finais de semana, sempre tinha os locais pra gente ir, uma banda que ia
tocar... Descia pro Davis Clube, que quase todo dia tinha som lá...57 O point, no entanto, era mais do que um local. Em inúmeros momentos
andei, divaguei, pontuei, trilhei as cidades dos desejos dos jovens. Nosso des-
locamento em bando causava medo e reações diversas nas pessoas pois a caminhada vai adquirindo uma autonomia de point, nesse durante con-
versamos, um espera o outro ou se adianta para falar com alguém que está na
frente. Poderíamos talvez não chegar. Big me diz rindo: ‘acho que a gente está
andando por andar’.58 Ruas e praças, clubes, centros comunitários, casas e universidades, sedes
de sindicatos, movimentação na cidade e pela cidade que suas experiências di-
retas instituem, criam como espaço de vida. As apropriações feitas revelam ou-
tras possibilidades, outras formas de perceber o mundo e também de criá-lo. 236 Revista Brasileira de História, vol. 27, nº 53 As cidades da juventude em Fortaleza As cidades da juventude em Fortaleza Talvez caminhem esses jovens por caminhos que não vemos, mesmo quan-
do os seus deslocamentos se fazem nas ruas que vemos, nas praças nas quais
sentamos, conversamos... talvez cantem esses jovens realidades que não conse-
guimos sequer imaginar, compreender.... 1 Pesquisador ligado ao Grupo de Práticas Urbanas do Mestrado Acadêmico em História
— Mahis, e do Labvida — Laboratório de Direitos Humanos, Cidadania e Ética do Mes-
trado em Políticas Públicas também da UECE. 2 CANEVACCI, Massimo. Conclusão em forma-de-mapa. In.: Sincretismos: uma explora-
ção das hibridações culturais. São Paulo: Studio Nobel; Instituto Cultural Ítalo Brasilei-
ro–Instituto Italiano de Cultura, 1996. p.91. 3 VIEIRA, Roberto César. Pedras que não rolam criam limo: rock cearense — consumo e
mercado. Monografia apresentada ao curso de Comunicação Social, Universidade Federal
do Ceará. Fortaleza, 1994. p.57-8. EXPERIÊNCIAS MUSICAIS, DESLOCAMENTOS
GEO-ESTÉTICOS: AS CIDADES E OS JOVENS talvez essas outras realidades, cidades,
estejam no limiar de suas experiências e reflitam essa ambigüidade, pois: Enquanto o adulto vive ainda sob o impacto de um modelo de sociedade que
se decompõe, o jovem já vive em um mundo radicalmente novo, cujas catego-
rias de inteligibilidade ele ajuda a construir. Interrogar essas categorias permite
não somente uma melhor compreensão do universo de referências de um grupo
etário particular, mas também da nova sociedade transformada pela mutação.59 Foto 2 – Davis Clube (extinto). Fotografia do autor, 2003. Foto 2 – Davis Clube (extinto). Fotografia do autor, 2003. NOTAS Muitas outras no-
tícias dão a dimensão das carências da cidade, como por exemplo: “Comunidades carentes
reclamam das péssimas condições de vida”, Tribuna do Ceará, 21 jan. 1980, p.7; ou ainda:
“Passagem cara faz o povo andar a pé”, Diário do Nordeste, 24. dez. 1981, capa, onde se
anunciava o presente de Natal dos fortalezenses, um aumento das passagens de ônibus. Algum tempo depois, em reação explícita a essa situação o movimento punk decreta uma
campanha pela gratuidade do transporte coletivo em Fortaleza, com o ‘desça por trás’. Cf. “Fora aumentos, chega de exploração, coletivo grátis já!”, nota/panfleto, Fortaleza, s.d. Di-
ziam eles em determinado momento: “Chega! Chega!... Tá na hora de dizer um basta a tu-
do isso. Só vão aprender quando a população se revoltar. Por isto estamos propondo que
os trabalhadores reajam diante dessa situação. Vamos subir por trás, pular a catraca, con-
versar entre nós e vamos lutar pelo transporte coletivo gratuito. Já estamos pagando caro
demais em impostos e ainda sermos obrigados a enriquecer mais e mais os bolsos desses
safados, enquanto o povo morre de fome? Não! Chega! NCCL — Núcleo Coletivo de Cons-
ciência Libertária e MAP — Movimento Anarco-Punk”. 6 Entrevista realizada por este pesquisador, com a ex-integrante do movimento punk, à
época chamada Flor Punk, na Praça dos Leões, centro da cidade de Fortaleza, em 6 mar. 2003. p.2-3. 6 Entrevista realizada por este pesquisador, com a ex-integrante do movimento punk, à
época chamada Flor Punk, na Praça dos Leões, centro da cidade de Fortaleza, em 6 mar. 2003. p.2-3. 7 Carmo nos informa que, nesse período, o termo ‘bandas’não era empregado. Cf. CARMO,
Paulo Sérgio do. Culturas da rebeldia: a juventude em questão. São Paulo: Ed. Senac, 2001. 279p. 8 XIMENES, Amaudson. A música underground em Fortaleza: resistência ou crise de iden-
tidade? Monografia apresentada ao curso de Ciências Sociais, Universidade Estadual do
Ceará. Fortaleza, 1998. p.34. É interessante frisar que Ximenes é guitarrista da banda Obs-
kure, e fundador da Associação Cearense do Rock (ACR). Roqueiro ‘de primeira hora’, vi-
venciou esse período e alguns dos acontecimentos que narra, tendo em dado momento
desenvolvido alguns trabalhos com os punks em Fortaleza. Foi entrevistado no sentido de
delinear a ação dos grupos punks, e de identificar as relações estabelecidas nesse momento
de surgimento do punk na cidade. 9 Entrevista realizada por este pesquisador com ‘Magoo’, em 28 jan. NOTAS NOTAS 1 Pesquisador ligado ao Grupo de Práticas Urbanas do Mestrado Acadêmico em História
— Mahis, e do Labvida — Laboratório de Direitos Humanos, Cidadania e Ética do Mes-
trado em Políticas Públicas também da UECE. 2 CANEVACCI, Massimo. Conclusão em forma-de-mapa. In.: Sincretismos: uma explora-
ção das hibridações culturais. São Paulo: Studio Nobel; Instituto Cultural Ítalo Brasilei-
ro–Instituto Italiano de Cultura, 1996. p.91. 3 VIEIRA, Roberto César. Pedras que não rolam criam limo: rock cearense — consumo e
mercado. Monografia apresentada ao curso de Comunicação Social, Universidade Federal
do Ceará. Fortaleza, 1994. p.57-8. 3 VIEIRA, Roberto César. Pedras que não rolam criam limo: rock cearense — consumo e
mercado. Monografia apresentada ao curso de Comunicação Social, Universidade Federal
do Ceará. Fortaleza, 1994. p.57-8. 237 junho de 2007 Francisco José Gomes Damasceno 4 Mais à frente comentarei a noção de deslocamento geo-estético. 5 A matéria do Diário do Nordeste, 16 jan. 1982, p.7, dava conta das dimensões da cidade:
“Com um milhão e trezentas mil pessoas, Fortaleza só tem sete creches”. Muitas outras no-
tícias dão a dimensão das carências da cidade, como por exemplo: “Comunidades carentes
reclamam das péssimas condições de vida”, Tribuna do Ceará, 21 jan. 1980, p.7; ou ainda:
“Passagem cara faz o povo andar a pé”, Diário do Nordeste, 24. dez. 1981, capa, onde se
anunciava o presente de Natal dos fortalezenses, um aumento das passagens de ônibus. Algum tempo depois, em reação explícita a essa situação o movimento punk decreta uma
campanha pela gratuidade do transporte coletivo em Fortaleza, com o ‘desça por trás’. Cf. “Fora aumentos, chega de exploração, coletivo grátis já!”, nota/panfleto, Fortaleza, s.d. Di-
ziam eles em determinado momento: “Chega! Chega!... Tá na hora de dizer um basta a tu-
do isso. Só vão aprender quando a população se revoltar. Por isto estamos propondo que
os trabalhadores reajam diante dessa situação. Vamos subir por trás, pular a catraca, con-
versar entre nós e vamos lutar pelo transporte coletivo gratuito. Já estamos pagando caro
demais em impostos e ainda sermos obrigados a enriquecer mais e mais os bolsos desses
safados, enquanto o povo morre de fome? Não! Chega! NCCL — Núcleo Coletivo de Cons-
ciência Libertária e MAP — Movimento Anarco-Punk”. 5 A matéria do Diário do Nordeste, 16 jan. 1982, p.7, dava conta das dimensões da cidade:
“Com um milhão e trezentas mil pessoas, Fortaleza só tem sete creches”. Revista Brasileira de História, vol. 27, nº 53 NOTAS 18 Melucci, citado em CARRANO, P. C. R. Juventude — as identidades são múltiplas. Re-
vista Movimento, São Paulo: DP&A, n.1, maio 2000. O mesmo Melucci (1997) propõe em
outro artigo que se deveria falar em redes conflituosas, em vez de em movimentos, dado o
caráter de produção de formas culturais. Apesar de interessante, no caso dos movimentos
aqui estudados não se aplica, posto que os significados construídos por esses jovens pas-
sam pela noção de movimento. 18 Melucci, citado em CARRANO, P. C. R. Juventude — as identidades são múltiplas. Re-
vista Movimento, São Paulo: DP&A, n.1, maio 2000. O mesmo Melucci (1997) propõe em
outro artigo que se deveria falar em redes conflituosas, em vez de em movimentos, dado o
caráter de produção de formas culturais. Apesar de interessante, no caso dos movimentos
aqui estudados não se aplica, posto que os significados construídos por esses jovens pas-
sam pela noção de movimento. NOTAS 2003, em sua residên-
cia na cidade de Caucaia, região metropolitana de Fortaleza. p.1. Magoo foi um dos prin-
cipais expoentes do punk durante os anos 80 e 90, distanciou-se do movimento organiza-
do, mas continua com a banda Estado Indigente, que formou ainda no final dos anos 80. 10 A priorização total do estilo ocorre com a maioria dos entrevistados da pesquisa realiza-
da com punks e hip-hopers na cidade de Fortaleza. 11 CERTEAU, Michel de. A invenção do cotidiano. Artes de Fazer. Petrópolis (RJ): Vozes,
1999. p.202. 12 Cf. JANOTTI JR., Jéder S. Heavy metal: o universo tribal e o espaço dos sonhos. Disser-
tação (Mestrado) — Depto. de Comunicação Social, programa de mestrado em Multi- 238 Revista Brasileira de História, vol. 27, nº 53 As cidades da juventude em Fortaleza meios, Unicamp. Campinas, 1994. Segundo ele através “do vestuário, dos shows e dos lo-
cais de encontro do grupo, os jovens procuram construir ‘territórios existenciais’ que pos-
sibilitem exercitar sua subjetividade situada além dos espaços normatizados (tais como fa-
mília, escola e trabalho)”. meios, Unicamp. Campinas, 1994. Segundo ele através “do vestuário, dos shows e dos lo-
cais de encontro do grupo, os jovens procuram construir ‘territórios existenciais’ que pos-
sibilitem exercitar sua subjetividade situada além dos espaços normatizados (tais como fa-
mília, escola e trabalho)”. 13 MAFFESOLI, Michel. O tempo das tribos: o declínio do individualismo nas sociedades
de massa. Rio de Janeiro: Forense-Universitária, 1987. 14 PESAVENTO, Sandra Jatahy. Uma outra cidade: o mundo dos excluídos no final do sé-
culo XIX. São Paulo: Cia. Ed. Nacional, 2001. p.11-2. 15 Entrevista realizada por este pesquisador com o rapper Sátiro Silvestre, no dia 10 ago. 2003, no bairro do Conjunto Ceará, em Fortaleza, p.1. Atual integrante da coordenação
do MH2O e um dos primeiros dançarinos de break da cidade, foi um dos fundadores da
Striking gangue de break nos anos 80. 16 Cf. DAMASCENO, Francisco José Gomes. O movimento hip-hop organizado do Ceará /
MH2O-Ce (1990-1995) Dissertação (Mestrado) — Pontifícia Universidade Católica de
São Paulo. São Paulo, 1997. 333p.; e DAMASCENO, Francisco José Gomes. Sutil diferença:
o movimento punk e o movimento hip-hop em Fortaleza — grupos mistos no universo ci-
tadino contemporâneo. Tese (Doutorado) — PUC-SP, São Paulo, 2004. 511p. 17 SOUZA, Janice Tirelli Ponte de. Reinvenções da utopia: A militância política de jovens
nos anos 90. São Paulo: Hacker, 1999. p.93-4. junho de 2007 19 MAFFESOLI, Michel. O tempo das tribos..., cit., p.27. 19 MAFFESOLI, Michel. O tempo das tribos..., cit., p.27. 20 É interessante observar que todos os entrevistados desta pesquisa têm o estilo no centro
de suas atividades, sejam elas profissionais, sociais, políticas etc. Há a priorização total do
estilo, que assume a dimensão de ordenador das relações. Neste caso, o termo ‘identidade’
se aplica perfeitamente. 21 De um modo geral, cada entrevistado tem sempre uma história de como ocorreu consi-
go, ou mesmo com outros do grupo. 22 OLIVEIRA, Vantiê Clínio de Carvalho. O movimento anarco-punk (A identidade e a au-
tonomia nas produções e nas vivências de uma tribo urbana juvenil). Dissertação (Mes-
trado) — Programa de Pós-Graduação em Ciências Sociais, UFRN. Natal, 2003. p.47. Ape-
sar de o estudo versar sobre a cidade de Natal,a referência é feita com relação a um congresso
dos anarco-punks ocorrido na cidade de João Pessoa. 23 “No interior desses remoinhos flutuantes e plurais de panoramas glocal, emerge com for-
ça a produção, a difusão e o consumo de sincretismos culturais. Essa palavra nova, fruto
de recíprocas contaminações entre global e local, foi forjada justamente na tentativa de 239 Francisco José Gomes Damasceno captar a complexidade dos processos atuais. Nela foi incorporado o sentido irrequieto do
sincretismo. O sincretismo é glocal” (grifos meus). CANEVACCI, op. cit., p.25. captar a complexidade dos processos atuais. Nela foi incorporado o sentido irrequieto do
sincretismo. O sincretismo é glocal” (grifos meus). CANEVACCI, op. cit., p.25. 24 TURRA NETO, Nécio. Enterrado vivo: identidade punk e território em Londrina. São
Paulo: Ed. Unesp, 2004. p.100, 104, 117. 25 ‘Movimentação’ aqui se entende por ‘tornar-se movimento’ e, ao mesmo tempo, como
ação dinâmica dos jovens em fazer, criar, inventar-se enquanto processo complexo de rela-
ções sociais, políticas, culturais, artísticas etc. 26 É interessante frisar que música, aqui, se configura basicamente pelo rock e demais ma-
nifestações underground, que tendo o rock como referência se posicionam como elemento
de transgressão de determinados setores, sobretudo da juventude contemporânea, assu-
mindo o caráter de instrumento de crítica social e política. 27 Refiro-me aqui a arte como prática, como defendido por SHUSTERMAN, Richard. Vi-
vendo a arte: o pensamento pragmatista e a estética popular. São Paulo: Ed. 34, 1998. Revista Brasileira de História, vol. 27, nº 53 19 MAFFESOLI, Michel. O tempo das tribos..., cit., p.27. Vivendo a arte…, cit., p.68-9. 34 A idéia da arte enquanto prática sócio-histórica, desenvolvida por Shusterman, foi uma
das que mais me auxiliaram na compreensão dos fenômenos da arte e da cultura juvenil. 35 Alguns autores sugerem essa prática como uma metáfora da vida cotidiana urbana con-
temporânea. Aqui nos interessa apenas anotar a dança como parte indissociável da expe-
riência musical. 35 Alguns autores sugerem essa prática como uma metáfora da vida cotidiana urbana con-
temporânea. Aqui nos interessa apenas anotar a dança como parte indissociável da expe-
riência musical. 36 MORAES, Jota de. O que é música. São Paulo: Nova Cultural; Brasiliense, 1986. 37 Cf. DAMASCENO, cit., 2004. A constituição dos movimentos punk e hip-hop como mo-
vimentos juvenis sociais contemporâneos é desenvolvida nesse trabalho. 37 Cf. DAMASCENO, cit., 2004. A constituição dos movimentos punk e hip-hop como mo-
vimentos juvenis sociais contemporâneos é desenvolvida nesse trabalho. 38 Aristóteles, cit. em PESAVENTO, Sandra Jatahy. A vitória de Antígona sob o signo de Ba-
bel, a cidade brasileira dessacralizada. In: PESAVENTO, Sandra Jatahy (Org.) Escrita, lin-
guagem, objetos: leituras de história cultural. Bauru (SP): Edusc, 2004. p.168. 38 Aristóteles, cit. em PESAVENTO, Sandra Jatahy. A vitória de Antígona sob o signo de Ba-
bel, a cidade brasileira dessacralizada. In: PESAVENTO, Sandra Jatahy (Org.) Escrita, lin-
guagem, objetos: leituras de história cultural. Bauru (SP): Edusc, 2004. p.168. 39 Faço assim uma apropriação livre de conceitos como o de ‘geopolítica’, por exemplo, e,
dilato seu sentido, reinvento o seu uso e o avalio sob a ótica de que a manifestação juvenil
contemporânea é essencialmente cultural e nisso reside a articulação entre cultura e polí-
tica para esses sujeitos: uma intervenção cultural, social e artística na cidade, que é marca-
damente política pela crítica e pelas práticas que estabelecem. 40 Cf. CERTEAU, Michel de. A invenção do cotidiano..., cit. 40 Cf. CERTEAU, Michel de. A invenção do cotidiano..., cit. 41 Essa é uma pretensão assumida por eles próprios, e colocada e reexposta constantemen-
te. Sente-se isso nas entrevistas realizadas com eles no Ceará, e essa também é uma carac-
terística mais geral do rap. Mano Brown, líder dos Racionais MCs declara (Folha de S. Pau-
lo, Ilustrada, 7 maio 1994, “A vez do Rap”.): “Não sou porta-voz do movimento hip-hop,
mas da periferia talvez!”. Cf. 19 MAFFESOLI, Michel. O tempo das tribos..., cit., p.27. 28 “Nossa cidade teve seu processo de metropolização e expansão da malha urbana direcio-
nado pelas suas principais vias rádio-concêntricas, herança de seus antigos caminhos co-
mo o do Soure, antigo nome de Caucaia, que provocou o adensamento urbano na porção
oeste da cidade”. SILVA, José Borzacchiello da. Nas trilhas da cidade. Fortaleza: Museu do
Ceará; Secretaria de Cultura e Desporto do Ceará, 2001. (Coleção Outras Histórias). p.70. 29 Ibidem, p.103. 30 Cf. SILVA, José Borzacchiello da. Movimentos sociais populares em Fortaleza: uma abor-
dagem geográfica. Tese (Doutorado) — Departamento de Geografia, Universidade de São
Paulo. São Paulo, 1986. Os inúmeros migrantes expulsos do interior e sem a certeza de
qualidade de vida em terras distantes fazem de Fortaleza um pólo atrativo, não só pela sua
proximidade. Este dado evidencia a enorme influência do ‘sertão’ ou do ‘campo’ na cidade
de Fortaleza e em suas manifestações culturais. 31 Magoo, cit., p.1. 31 Magoo, cit., p.1. 32 São muitas as narrativas que citam esse aspecto, de tal modo que se pode avaliar o papel
da eletividade como fundamental: Sátiro Silvestre, um rapper, ex-breaker, e desde o princí-
pio ligado ao hip-hop, em tom de brincadeira, alude ao tempo que já tem de ‘movimento’:
“Era uma vez... (risos) cara meu contato com o hip-hop foi... desde os 12 anos, faz tem-
po... mas foi aqui na comunidade mesmo, nas festas que tinha, que eu via a galera dançan-
do break, a rapaziada que antecedeu a gente, que era o Lula, não tinha nada organizado,
mas o pessoal que dançava lá no Inter ... Inter dance, no Monte Castelo, era um point on-
de os punks, e no tempo... 85, 86, 87 o break tava em ascensão, era de certa forma uma no-
vidade, aí lá o pessoal tinha a festa de rock, punk, e tinha o pessoal que dançava o break se
congregava lá, aí eles migraram alguns aqui pro Conjunto Ceará, aí foi quando eu vi o Lu-
la fazendo o Moinho de Vento eu endoidei, ‘meu irmão, como é que esse cara faz isso?’. A
partir daí, fui chegando na roda e me engajei, a a partir daí foi o 1o contato mesmo com o
hip-hop... (Entrevista realizada com Sátiro em 10 ago. 2003, no bairro do Conjunto Ceará
em Fortaleza). 240 Revista Brasileira de História, vol. 27, nº 53 As cidades da juventude em Fortaleza 33 SHUSTERMAN, Richard. 19 MAFFESOLI, Michel. O tempo das tribos..., cit., p.27. ainda as matérias “Tiroteio na Periferia”, em O Globo, 30
jun.1994; “Rap é a língua oficial da periferia” Estado de S. Paulo, 27 nov. 1992, e ainda
“Rap’eriferia” no jornal O Povo, caderno Tribos, 15 maio 1994. 42 “Mais do que nunca a Fortaleza é DEF” — O grupo Ataque Frontal era composto pelos
rappers ZMC e Ligado. Ambos participam do movimento, aproximadamente desde 1993. ZMC é um dos membros mais engajados com setores diversos das ‘esquerdas’ como o Mo-
vimento Estudantil e Popular. Depois de trabalhar como lavador de carros à noite no esta-
cionamento de casas noturnas da cidade, tem trabalhado como prestador de serviços na
Câmara de Vereadores de Fortaleza. Ligado, além de seu vizinho na favela da Quadra, no
coração da Aldeota, sem profissão definida, trabalha como vendedor de pequenas lojas de
grifes locais e como servente de pedreiro, quando não faz pequenos biscates. 44 “Sem Título” — Realidade em Cadeia. A pergunta insistentemente repetida nesse rap é
“Quem são os mandantes, quem são?” e “Olhe pra cima e veja então, quem é que mata sem
arma na mão / me diga agora aonde devo ir? Me diga agora mano? Me diga agora!”
45 “Mais do que nunca a Fortaleza é DEF” — Ataque Frontal (ZMC e Ligado). 46 PESAVENTO Sandra cit p 15 241 junho de 2007 junho de 2007 Francisco José Gomes Damasceno 47 É interessante a reflexão feita por STAM, Robert. Bakhtin: da teoria literária à cultura de
massa. São Paulo: Ática, 1992. p.75-6: “Porém os mesmos mecanismos dialógicos básicos
operam também dentro da chamada ‘cultura popular’. Veja-se, por exemplo, o fenômeno
do hip-hop nos Estados Unidos, que inter-relaciona os universos culturais do rap, do gra-
fite e da dança break. O rap, forma de música popular que utiliza os efeitos pirotécnicos
executados em toca-discos combinados com um discurso verbal agressivo e ritmado — a
própria palavra rap significa conversar, dialogar —, pode ser considerado uma esperta vi-
são ‘de rua’ das teorias bakhtinianas sobre o dialogismo. Criado por adolescentes negros e
hispânicos da classe operária ou lúmpen,o rap é intensamente,exuberantemente dialógico”. 48 “Tortura”
Estado Indigente 48 “Tortura” — Estado Indigente. Revista Brasileira de História, vol. 27, nº 53 48 “Tortura” — Estado Indigente. 49 “Ceará e seus problemas” — Conscientes do Sistema. Este rap dá título ao primeiro dis-
co inteiramente de rap do Ceará. O grupo era ligado ao MH2O, de onde saiu para fazer
carreira solo. Recentemente se desmembrou em dois, ficando uma parte ainda com o no-
me de Conscientes do Sistema, e o outro passando a chamar-se de Consciência Armada,
nome de uma das composições desse CD. 50 Ambas as manifestações se consideram “culturas de rua”. 51 “O cotidiano que se apreende no rock é o da deambulação. Trata-se da negação de um
cotidiano tradicional e da afirmação de um cotidiano de rua. O lugar do dia-a-dia deixa
de ser a casa para ser a cidade. Uma cidade com suas ruas e esquinas, com pistas de carro
em alta velocidade, dos gigantescos anúncios luminosos, da multiplicidade de informa-
ções, da multidão nas calçadas, da intensa movimentação nos bares, dos encontros furti-
vos e, sobretudo, do anonimato. Seus personagens não têm nomes...”. GUERREIRO, Al-
merinda Sales. Retratos de uma tribo urbana: rock brasileiro. Dissertação (Mestrado) —
Faculdade de Filosofia, Letras e Ciências Humanas, Depto. de Antropologia, USP. São Pau-
lo, 1991. p.83. 52 “O Poder da Palavra” — Rap Força. 52 “O Poder da Palavra” — Rap Força. 53 “Sangue Negro” — Conexão Racial. 54 “Filhos da Rua” — Flip Rap. 54 “Filhos da Rua” — Flip Rap. 55 Entrevista realizada por este pesquisador com Eliane ‘Zueira’ em 12 fev. 2003 na Praça
José de Alencar, Fortaleza. p.2. 55 Entrevista realizada por este pesquisador com Eliane ‘Zueira’ em 12 fev. 2003 na Praça
José de Alencar, Fortaleza. p.2. 56 Flor Punk, cit., p.3. 57 Entrevista realizada por este pesquisador com o punk Lezado, em 20 fev. 2003, em sua
residência no bairro do Conjunto Ceará, Fortaleza. p.1. 57 Entrevista realizada por este pesquisador com o punk Lezado, em 20 fev. 2003, em sua
residência no bairro do Conjunto Ceará, Fortaleza. p.1. 58 CAIAFA, Janice. Movimento Punk na Cidade: a invasão dos bandos sub. Rio de Janeiro:
Jorge Zahar, 1985. p.49. 59 PERALVA, Angeline. O jovem como modelo cultural. Juventude/Contemporaneidade, Re-
vista Brasileira de Educação, São Paulo: Anped, n.5-6, 1997. p.23. 242 Revista Brasileira de História, vol. 27, nº 53 Revista Brasileira de História, vol. 27, nº 53
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Pairmate-dependent pup retrieval as parental behavior in male mice
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ORIGINAL RESEARCH ARTICLE
published: 11 July 2014
doi: 10.3389/fnins.2014.00186
Pairmate-dependent pup retrieval as parental behavior in
male mice
Mingkun Liang 1,2 , Jing Zhong 1,2 , Hong-Xiang Liu 1 , Olga Lopatina 1 , Ryusuke Nakada 2 ,
Agnes-Mikiko Yamauchi 2 and Haruhiro Higashida 1*
1
2
Department of Basic Research on Social Recognition and Memory, Research Center for Child Mental Development, Kanazawa University, Kanazawa, Japan
Department of Biophysical Genetics, Graduate School of Medical Sciences, Kanazawa University, Kanazawa, Japan
Edited by:
Sonoko Ogawa, University of
Tsukuba, Japan
Reviewed by:
Aldo Lucion, Universidade Federal
do Rio Grande do Sul, Brazil
Tomoko Soga, Monash University,
Malaysia
*Correspondence:
Haruhiro Higashida, Department of
Basic Research on Social
Recognition and Memory, Research
Center for Child Mental
Development, Kanazawa University,
13-1 Takara-machi,
Kanazawa 920-8640, Japan
e-mail: haruhiro@
med.kanazawa-u.ac.jp
Appropriate parental care by fathers can greatly facilitate healthy human family life.
However, much less is known about paternal behavior in animals compared to those
regarding maternal behavior. Previously, we reported that male ICR strain laboratory mice,
although not spontaneously parental, can be induced to display maternal-like parental care
(pup retrieval) when separated from their pups by signals from the pairmate dam (Liu et al.,
2013). This parental behavior by the ICR sires, which are not genetically biparental, is novel
and has been designated as pairmate-dependent paternal behavior. However, the factors
critical for this paternal behavior are unclear. Here, we report that the pairmate-dependent
paternal retrieval behavior is observed especially in the ICR strain and not in C57BL/6 or
BALB/c mice. An ICR sire displays retrieval behavior only toward his biological pups. A sire
co-housed with an unrelated non-pairing dam in a new environment, under which 38-kHz
ultrasonic vocalizations are not detected, does not show parenting behavior. It is important
for sires to establish their own home territory (cage) by continuous housing and testing
to display retrieval behavior. These results indicated that the ICR sires display distinct
paternity, including father-child social interaction, and shed light on parental behavior,
although further analyses of paternal care at the neuroendocrinological and neurocircuitry
levels are required.
Keywords: parental behavior, paternal care, pup retrieval behavior, paternity, mouse
INTRODUCTION
According to Schor and others, “a stable, well-functioning family
that consists of two parents and children is potentially the most
secure, supportive, and nurturing environment in which children
may be raised” (Schor and American Academy of Pediatrics
Task Force on the Family, 2003; Fortunato and Archetti, 2010;
Benbassat and Priel, 2012). Thus, the role of a father in the home
is highly significant, and currently, the physical absence of the
father in the home is seen as a major problem facing families
worldwide (Feinberg, 2002; Fleming et al., 2002; Amato, 2005;
Benbassat and Priel, 2012; Morrongiello et al., 2013; Bornovalova
et al., 2014). This raises questions regarding which factors determine paternal care and how they are maintained. This may be
addressed by behavioral studies and neuroendocrinological analysis of oxytocin, stress hormones, sex hormones, or epigenetic
mechanisms (Ogawa et al., 1998; Pfaff et al., 1999; Nunes et al.,
2001; Gammie, 2005; Jin et al., 2007; Bridges, 2008; Nishimori
et al., 2008; Lee et al., 2009; Neumann, 2009; Chourbaji et al.,
2011; Douglas, 2011; Morgan and Bale, 2011; Hashimoto et al.,
2012; Higashida et al., 2012a,b; Parhar et al., 2012; Soga et al.,
2012; Bambico et al., 2013; Salmina et al., 2013; Morrison et al.,
2014).
Although a number of animal models have been used in experimental studies of parental care (Reburn and Wynne-Edwards,
1999; Carter et al., 2009; de Jong et al., 2009; McGraw and Young,
2010; Ozawa et al., 2010; Kuroda et al., 2011; Mogi et al., 2011;
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Saltzman and Maestripieri, 2011; Lambert et al., 2013; Tachikawa
et al., 2013; Yoshida et al., 2013), given its value for genetic
studies, a mouse model of paternal behavior may be especially
useful (Hager and Johnstone, 2003; Jin et al., 2007; Liu et al.,
2013). While some strains of the laboratory mouse Mus musculus become biparental (Wright and Brown, 2000; Chourbaji et al.,
2011), a phenomenon called sensitization (Rosenblatt, 1967;
Rosenblatt et al., 1996), little information is available regarding the factors that specifically induce male parental behavior
(Gubernick and Alberts, 1987, 1989; Lonstein and De Vries, 2000;
Kentner et al., 2010; Leuner et al., 2010).
Previously, we reported that the outbred ICR strain is uniparental and is a good model for studies of parental behavior
(Jin et al., 2007; Liu et al., 2008, 2013; Higashida et al., 2012a),
because these mice actively reproduce offspring and exhibit easily
monitored pup retrieval after separation (Fujimoto et al., 2013;
Liu et al., 2013), which is a reliable indicator of parental behavior (Gammie, 2005; Wynne-Edwards and Timonin, 2007; Yoshida
et al., 2013). We demonstrated that male ICR mice display robust
parental care, which is induced by signaling from the pairmate
dam, after separation from the pups (Liu et al., 2013). We demonstrated that this signaling is mediated through as yet unidentified
olfactory pheromonal cues and auditory 38-kHz ultrasonic vocalization (USV) cues (Liu et al., 2013), that the male response can
be modified hormonally via oxytocin (Akther et al., 2013), that
CD38 in the nucleus accumbens is critical (Akther et al., 2013),
July 2014 | Volume 8 | Article 186 | 1
Liang et al.
and that the central cholinergic system is involved (Fujimoto
et al., 2013). However, the factors influencing singly isolated sires
in which there is no direct communicative interaction between
dams and sires remain unclear.
In the present study, to simplify fatherhood evaluation, we
used an all-or-nothing type of pup retrieval behavior by calculating the percentage of sires that displayed retrieval behavior (Liu
et al., 2013). We investigated paternal behavior in terms of the
types of conditions that can induce or maintain paternal retrieval
behavior by sires when the males are isolated before the offspring
are delivered by pregnant mates, and the males are held separately
to prevent them being sires by physically separating them from
other family members for 3 days. Then, family ties are formed
with or without mate information. In other experiments, we
examined isolation from pups under different housing conditions
in which either pairmate dam and pup olfactory information is
present or excluded.
MATERIALS AND METHODS
ANIMALS
Male and female Slc:ICR, C57BL/6, and BALB/c mice were
obtained from Japan SLC, Inc. (Hamamatsu, Japan) via a local
distributor (Sankyo Laboratory Service Corporation, Toyama,
Japan). The ICR mice were originally obtained from Charles
River Laboratories in 1965 and since then bred in Japan with the
alternative name Swiss CD1. The offspring of these mice were
born in our laboratory colony, weaned at 21–28 days of age,
and housed in same-sex groups of 3–5 animals until pairing (Liu
et al., 2013). The animals were paired and kept in our laboratory
under standard conditions (24◦ C; 12-h light/dark cycle, lights on
at 08:00) with food and water ad libitum. The mice were housed
together continuously in standard mouse maternity cages. The
experiments were performed in accordance with the Guidelines
for the Care and Use of Laboratory Animals of Kanazawa
University.
BEHAVIORAL TESTING
Virgin males and females were paired at 45–55 d. A single male
and a single female were continuously housed together in a standard mouse maternity cage from the mating period until the
delivery of pups. In some experiments, the males were separated
in new cages 1 day before parturition to prevent formation of
family relationships and kept in the new cages for 3 days. Then,
the males were allowed to meet their pups with or without pairmates from day 3 to day 5. All family units composed of a new sire
(first-time father), dam, and their first litter were experimentally
naïve.
One male parent was placed for 10 min in the original cage or
new cage alone or with his pairmate (separation environment).
Five pups were randomly selected from the litter and placed individually at a site remote from the nest in the original cage. The
sires were returned to the original home cage or a new cage in the
presence of their five biological or foster pups to assess parental
behavior. Parental retrieval behavior (percentage of sires exhibiting retrieval) was examined for 10 min following reunion. The
behavioral tests were performed in a randomly mixed sequence
of experimental groups. Experiments were usually performed at
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Mate-dependent paternal behavior
10:00–15:00. We defined retrieval as positive if the sires carried all
5 pups to the original nesting place or within two thirds of the
distance between the nest and the place at which the pups had
been placed (Liu et al., 2013). We also observed other parental
behaviors (grooming, crouching, and huddling) as defined by
Gubernick and Alberts (1987, 1989). The animals in this and
subsequent experiments were tested only once.
MEASUREMENT OF USVs
Experiments were carried out in a soundproof chamber measuring 600 × 500 × 500 mm (model MC-050/VA; Muromachi Kikai,
Tokyo, Japan). USVs were detected with a condenser microphone (Type 7016; Aco, Tokyo, Japan) and a preamplifier (type
4116; Aco) designed for sound pressure level (SPL) measurements
between 20 Hz and 90 kHz. A 4-kHz band-pass filter was used
to minimize background noise during recordings; however, most
WAV files still contained a considerable amount of “non-USV”
signal. Extraneous noise was identified and removed from the
sonograms as far as possible. When a rater found an ultrasound
signal that was difficult to interpret, the call was evaluated by a
minimum of one additional trained observer and identification
required a consensus by all raters. Each sonogram was then evaluated with a series of automated parameters. The microphone
was placed 50 cm above the cage in a soundproof chamber and
connected to an amplifier (model UMA-2; Muromachi Kikai).
Acoustic signals were transmitted to a vocalization analyzer system (model MK-1500; Muromachi Kikai) with functions such
as an analog-to-digital converter (192 kHz), frequency filters, a
digital fast-Fourier-transform analyzer, and signal input—output
terminals. Input signals were visualized on SpectraLAB (Sound
Technology Inc., State College, PA) in the analyzer system on a
personal computer. USVs were recorded as WAVE files and analyzed; the number of calls, frequency, and wave width (>40 ms)
were measured using a USV monitor (Muromachi Kikai).
STATISTICAL ANALYSIS
The data were calculated as the means or the means ± s.e.m.
Two-tailed Fisher’s exact probability test was used for single comparisons of retrieval behaviors. The remaining data were analyzed
by two-tailed Student t-test.
RESULTS
It has been reported that parental behavior in mice is dependent
on the strain (Wright and Brown, 2000). Therefore, we first examined and compared parent–pup family units in three strains, i.e.,
ICR, C57BL/6, and BALB/c mice, under various experimental settings. The data are summarized in Table 1. Maternal nurturing
behavior was observed in dams of all three strains, in a strainnonspecific fashion, except for the low rate of retrieval by the
BALB/c dams. In contrast, paternal behavior was variable between
the strains. No retrieval behavior was observed by BALB/c sires
(n = 15). C57BL/6 sires displayed retrieval during reunion after
single-separation in new cages (approximately 40%, n = 15).
However, isolation together with the partner in new cages did
not potentiate but rather decreased this rate to 13.3% (n = 15).
This parental behavior suggests that C57BL/6 males display mateindependent paternal behavior. Interestingly, 38-kHz USVs were
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Liang et al.
Mate-dependent paternal behavior
Table 1 | Parental behaviors in three strains of mice.
Behavior
Dam
Sire
Mouse strain
ICR (n = 15)
C57BL/6 (n = 15)
BALB/c (n = 15)
Retrieval
100% fast, rhythmic
100% fast, rhythmic
60% slow, interrupted
Crouching
Over all pups
Over not all pups
Over all pups
Grooming
Rare
Rare
Rare
Nest building
Sometimes
Sometimes
Sometimes
Retrieval by separation
10%
40%
0%
After co-housing pairmates
60%
10%
0%
Fast (<4 min)
Very slow
–
Smooth
Intermittent
–
Over not all pups
Not often
–
Crouching
Grooming
Rare
Rare
–
Nest building
Not often
Rare
–
Number of pups per litter
∼15
∼5
∼7
Survival ratio
∼100%
60–70%
∼100%
USVs
>70 calls/2 min
<20 calls/2 min
>80 calls/2 min
Communication from
dams to sires with
38 kHz USVs
Detected
Not detected
Not detected
Pattern of paternal
care
Mate-dependent
Mate-independent
None
Pup
not recorded from any dam–sire pairs of C57BL/6 and BALB/c
strains separated in new cages for 10 min. These results indicated that pairmate-dependent care is specific to the ICR strain.
Therefore, in the following experiments, we examined various
critical conditions under which ICR strain males did or did not
show paternal behavior.
RETRIEVAL BEHAVIOR BY SIRES SEPARATED ALONE IN HOME CAGES
The experimental paradigms for each experiment are shown
schematically in each figure. In Figure 1, we first reproduced
our previous results (Liu et al., 2013). Male and female ICR
strain mice were paired and housed together continuously in a
standard mouse maternity cage (Figure 1A). The mice were left
undisturbed during the first 3 days after the birth of their pups
(Figure 1B), during which they displayed distinct paternal and
maternal behaviors as described previously (Liu et al., 2013).
The sire and dam nursed the pups. This involved nest-building,
pup retrieval, licking, and huddling over the pups and lactating. However, as described in the Methods section, we mainly
analyzed the male’s retrieval behavior, as a parental role, in the
following experiments.
The sire in the first family was left alone in the vacated cage
during the period of separation (Figure 1C), whereas the pups
and dam were removed and placed in a new cage (Figure 1D) separated from the family cage. After 10 min, the five selected pups
of the sire (biological offspring) were returned to the nursing cage
in a remote area away from the nest, where the sire was present
(Figure 1E). The sire retrieved the offspring over 10 min (86% of
the sires, n = 15; Figures 1F,S).
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If the non-biological (foster) pups (Figure 1N) of the third
family (Figures 1M,N) were introduced into the vacated home
cage with the second sire (Figures 1I,K) in the second family
(Figures 1G,H), instead of the biological pups (Figures 1H,J),
33% of the 15 sires displayed pup retrieval (Figures 1L,S; twotailed Fisher’s exact probability test between sires toward biological (F) and non-biological (L) pups, P < 0.01).
When a sire from the third family (Figure 1N) was placed
and isolated for 10 min in the home cage of the second family (Figure 1O), the third sire did not retrieve any of the foster
(second family’s) pups (Figures 1R,S; n = 15, two-tailed Fisher’s
exact probability test between unrelated sires (R) and sires with
non-biological (L) or biological (F) pups, P < 0.05 and P <
0.0001, respectively). These results suggested that paternal pup
retrieval behavior in the home cage is maintained by biological
family cues of their mate dams and remaining pups.
RETRIEVAL BEHAVIOR BY SIRES AFTER SEPARATION IN NEW CAGES
Male parental care in Figure 1 may have been induced by the
fact that the males were left in the nursing environment during
parent–pup separation. To select out pup information during isolation, we used the co-housing paradigm presented in Figure 2.
We examined whether sires developed paternal behavior following time spent with the family. Pup retrieval increased on a daily
basis after parturition, while dams displayed a higher retrieval
ratio from the first day of parturition than the sires (Table 2).
The sires alone (Figure 2D) or together with the mate dams
(Figure 2H) were placed in a new cage for 10 min, whereas
the pups alone (Figure 2G) or together with dams (Figure 2C)
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Liang et al.
FIGURE 1 | Parental retrieval test in ICR mice for biological and
non-biological pups. Schematic representations of the parental care test in
three mated pairs (A,G,M). After cohabiting with their pups as a family for 3
days from postnatal day 1 (P1) until postnatal day 3 (P3) (B,H,N), the sires were
separated in the home cage (C,I,O) from the pups and pairmates (D,J,P) for
10 min. The sires were then reunited with five biological (E) or non-biological
(K) pups. Subsequent pup retrieval behavior over a 10-min period was then
observed (F,L). The third sire (M,N) was placed in the home cage (O) of
were left in the home cage. Then, the sires were returned to
the home cages in which five pups remained (Figures 2E,I).
The male’s retrieval behavior was undiminished when housed
with the pairmate (66%, n = 30; Figures 2J,U) but was strongly
reduced when housed alone (24%, n = 41; Figures 2F,U). As
expected, a high level of sire care was displayed after isolation
in the new environment together with mate dams and pups (as
the whole family (Figures 2K,L) (66%, n = 15; Figure 2U): twotailed Fisher’s exact probability test between sires separated alone
(F) and together (J) or as a whole family (N), P < 0.001, equally.
The latter was specifically associated with co-habitation with
the pairmate dam during the separation period (Figure 2H),
because negligible retrieval behavior was apparent if the sire was
housed with the dam of another brood (Figures 2O–T; 20%,
n = 15); two-tailed Fisher’s exact probability test shows no significant difference between sires separated together with unrelated
dams (T) and alone (F); and separated together (J), P < 0.01;
and separated as a whole family (N), P < 0.05, Figure 2U). Thus,
it appears that the mate dam provides some signal(s) during
the separation period to induce parental behavior in the sire, in
agreement with the results reported previously (Liu et al., 2013).
Whereas parental care by the dam is independent of the presence of the male or the housing environment, that by the male
is strongly dependent on cues from the pairmate dam and/or
home cage.
We recorded USVs (with >40 ms in wave width) to determine
their role as one form of critical interactive information in this
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Mate-dependent paternal behavior
another family (H), and retrieval was tested for non-biological (another family’s)
pups in an unrelated cage (Q,R). The numbers of positive mice/number of
mice tested are shown in parentheses. The number of sires displaying retrieval
behavior out of sires tested was expressed as a percentage (S). N = 15 for
each experiment. Two-tailed Fisher’s exact probability test: between sires
toward biological (F) and non-biological (L) pups or unrelated sires (R),
∗∗ P < 0.01 and ∗∗∗∗ P < 0.0001, respectively; and between sires tested toward
non-biological pups (L) and unrelated sires (R), ∗ P < 0.05.
paradigm. We detected 38-kHz USVs identical to those reported
previously (Liu et al., 2013) under isolation conditions in new
cages for 10 min between sires and mate dams at a frequency of
25.9 ± 4.8 calls/10 min (n = 8, Table 3; P < 0.01 from other values, two-tailed Student t-test). No identical 38-kHz USVs were
recorded between sires and unrelated dams. Instead, 30–80-kHz
USVs were recorded infrequently at 40.7 ± 26.7 calls/10 min (n =
11) between unfamiliar couples. These 30–80-kHz USVs were
emitted when a sire was co-housed with a virgin female at 313.6 ±
64.9 calls/10 min (n = 11, P < 0.001 from two other values, twotailed Student t-test). These data clearly support the suggestion
that paternal retrieval is essentially triggered by the pairmate’s
38-kHz USVs.
RETRIEVAL BY ISOLATED BEFORE PAIRMATE PARTURITION
The retrieval behavior displayed by males may have been induced
by family formation in the nursing cage environment. To
assess this possibility, data were obtained from parting males
(Figures 3, 4) that remained with the paired pregnant females
1 day before parturition of their first litter and were then separated into a new cage (Figures 3A–C). The males were then
isolated alone for 3 days (Figure 3E). When sire paternal retrieval
was examined immediately on day 3 in the sire home cage
(Figure 3K), 21.4% of sires with no prior contact with their biological pups and pairmate dam, i.e., the paternity unformed state
(Figure 3E), displayed retrieval behavior (n = 42; Figure 3N).
Next, when the isolated males were relocated in the home cage
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Liang et al.
Mate-dependent paternal behavior
FIGURE 2 | Paternal retrieval test in ICR mice isolated in new cages.
Paired couples were kept in rearing cages from mating to postnatal day
3 (P3) (A,B,O,P). In (C,D), the pups and the mating dam were left in
their home cages (C), and the sire alone was placed in a new holding
cage (D). In (G,H), the pups were kept in the original cage, and the
parents were placed in a new cage (H). In (K,L), the whole family was
moved to a new cage (L). In (Q,R), the sire was kept during the
separation period (Q) with a non-mating dam of another family (O,P).
After isolation for 10 min in each cage, pup retrieval behavior over a
10-min period was observed in each case (E,I,M,S). The number of sires
displaying retrieval behavior was scored (F,J,N,T). The numbers of
positive mice/number of mice tested are shown in parentheses and
expressed as percentages (U). Two-tailed Fisher’s exact probability test:
between sires separated alone (F) and together (J) or as a whole family
(N), ∗∗∗ P < 0.001 equally; between sires separated together (J) and as a
whole family (N), ∗∗ P < 0.01; between sires separated alone (F) and
co-housed with unrelated dams (T), not significant (n.s.); sires separated
as a whole family (N) and co-housed with unrelated dams (T), ∗ P < 0.05.
Table 2 | Percentages of sire’s or dam’s exhibiting retrieval behavior
Table 3 | Number of USVs recorded from cages of sires co-housed
during the postpartum period.
with different types of females for 10 min.
Postnatal day
Percentage of exhibiting
of pups
Type*
Number of USVs
retrieval behavior
By sires
(n)
(calls/10 min)
By dams
38-kHz
30–80-kHz
1
14 (15)
55 (20)
With pairmate dam
25.9 ± 4.8**
0
(8)
2
40 (20)
90* (20)
With unrelated dam
0
40.7 ± 26.7
(11)
With virgin female
0
313.6 ± 64.9***
3
65** (20)
90* (20)
4
70** (17)
85 (20)
5
65** (20)
75 (16)
USVs (with >40 ms in duration) were recorded in n pairs.
*Judging from the previous results (Liu et al., 2013), 38-kHz USVs appear to be
Number of mice tested are shown in parentheses. *, **Significantly different
from day 1, *P < 0.05 and **P < 0.01, respectively, two-tailed Fisher’s exact
emitted from pairmate dams and 30–80-kHz USVs from sires.
**P < 0.01 or ***P = 0.001, from pairmate dams, unrelated dams or virgin
probability test.
females, respectively, two-tailed Student’s t-test.
and stayed with the family (pups and pairmate dam) for 3 days
(Figure 3F), the rate of retrieval in their home cage was only 4%
(n = 25; Figures 3G–J). Although the sire lived together with the
family for 3 days, such treatment made no contribution to the formation of paternity (two values in Figure 3O were equally very
low; no significance, two-tailed Fisher’s exact probability test).
To further analyze the relevance of family interaction during the stay as a whole family on postnatal days 3–5 (P3–P5)
(Figure 3), we used the short-term pup exposure method (twice
for 3 h for a total 6 h a day; Figure 4) to acquire or learn the
process of paternity for the family. Males were isolated in new
cages prior to parturition (Figures 4B,C) and kept in the cages for
2 days (Figures 4D,E). Then, pairmate dams and pups were relocated to the male’s cage, and the whole family was kept there for
3 days (Figure 4F). Retrieval behavior was displayed by 8 (62%)
of 13 sires (Figures 4G–J). The high level of retrieval appears to
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July 2014 | Volume 8 | Article 186 | 5
Liang et al.
FIGURE 3 | Paternal retrieval test in ICR mice isolated prior parturition
from the mating pair and then united as a whole family. A paired
couple was kept in a rearing cage from mating (A) to 1 day before
parturition, and the female and male were then kept in a home cage (B) or
in a new cage (C). The next day, the female delivered her pups (D) and
remained until postnatal day 2 (P2). The male was kept continuously in the
new cage until P2 (E). From P3 to P5, the sire was introduced to the
have been caused by continuously living in new cages that had
been established as the male’s territory.
In this suitable condition, we examined whether the presence
of the dam was necessary for parental behavior by the isolated
males. From P3 to P5, the pups and dam were kept together in
their original home cages (Figure 4K), but the pups were temporarily transferred to the sire’s cage twice for 3 h (a total of
6 h) per day (Figure 4L), and the males were otherwise alone
for the rest of the day (18 h; Figure 4M). These sires showed
retrieval behavior at a very high rate (17 (85%) of 20 sires tested;
Figures 4O–Q). In both cases, the sires displayed a very high frequency of retrieval after living as the whole family or only with
pups shortly in new cages that had, nevertheless, been established as the territory and established nest of the male, although
no significant differences were observed between two types of
sire (J and Q in Figure 4R; not significant, two-tailed Fisher’s
exact probability test). Furthermore, these results indicated that
direct interaction with the mate dam is not necessary if the home
territory is established by the sires.
Finally, we further examined the impact of territorial information on male retrieval behavior. Family cues were learned
by individual sires in a manner identical to that shown in
Figure 4 (Figures 5A–F) during P3–P5, but in this case, via
Frontiers in Neuroscience | Neuroendocrine Science
Mate-dependent paternal behavior
family cage with the dam and pups (F). In another experiment, pup
retrieval behavior over a 10-min period was examined for sires at P2 (K–N)
or at P5 (G–J). The number of sires displaying retrieval behavior was
scored (J,N). The numbers of positive mice/number of mice tested are
shown in parentheses and expressed as percentages (O). Note that two
values in O are equally very low: no significance (n.s.) between (J) and
(N), two-tailed Fisher’s exact probability test.
short exposure by transferring of their biological pups with their
dams in new cages to the nursing cage with the sires. Then,
retrieval behavior was examined under two housing conditions:
in the sire’s home cage in which the sire had stayed continuously
(Figures 5F,G,I–L), or in a new cage (to the sires) in which the
mate dams and pups had been staying (Figures 5G,H,M–P). In
the home cages, 10 (50%) of 20 sires showed retrieval (Figure 5L),
whereas only 3 (15%) of 20 sires in new cages displayed retrieval
behavior (P < 0.05 between testing in old (L) and new (P) cages
shown in Figure 5Q, two-tailed Fisher’s exact probability test). In
both cages, nests were established by the sire and dam. However,
the new cages established by the sires’ mate dams were quite new
to the sires, even if the cages were fully filled with the mate dam’s
olfactory information.
DISCUSSION
The studies described here were performed to test several
hypotheses that had not been explored previously (Akther et al.,
2013; Fujimoto et al., 2013; Liu et al., 2013), pertaining to the various conditions responsible for parental behaviors other than the
communicative interaction between sires and dams. Four findings
are of particular interest: (1) among the mouse strains tested, the
mate-dependent paternal retrieval behavior was observed only in
July 2014 | Volume 8 | Article 186 | 6
Liang et al.
FIGURE 4 | Paternal retrieval test in mice isolated prior parturition from
the mating pair and then united as a whole family or with pups only.
A paired couple was kept in a rearing cage from mating (A) to 1 day
before parturition. The female was kept in a home cage (B) and delivered
her pups (D) and remained until postnatal day 2 (P2) (D). The male was
kept in a new cage before meeting the pups (C) and kept until P2 (E). The
dam and pups were introduced in the sire’s own (new) cage and stayed as
a whole family until P5 (F). Instead of the whole family, in another
experiment, only pups in home cages with their dams (K) were transferred
the ICR strain (Table 1), and acquisition of such paternal behavior increased slowly following parturition of the dam (Table 2);
(2) the ICR sires displayed parental retrieval behavior only for
their own biological pups (Figure 1); (3) interaction between the
sires and unrelated non-mating dams is not effective (Figure 2)
and does not involve 38-kHz USVs (Table 3); (4) it is important
for the sire to establish its home cage (territory) by continuous
housing to display parental retrieval behavior (Figures 3–5).
After separation from pups in the home or new cages with the
sires alone or together with the pairmate dam, the sires displayed
retrieval behavior, as shown in Figures 2J,N, in agreement with
previous reports (Liu et al., 2013). We designated this behavior
of the sire as mate-dependent parental behavior. In the present
study, this particular behavior was specific to the ICR strain and
was not observed in two other laboratory strains, i.e., C57BL/6
and BALB/c mice. Therefore, the ICR strain’s mate-dependent
retrieval is not a general behavior observed equally in all mice
but is strain-specific. However, this does not reduce the value of
our findings because the observed paternal behavior is unique.
Furthermore, when considering human society, human males are
not completely and genetically predisposed to display parental
behavior. In this context, the behavior of the ICR strain may be
www.frontiersin.org
Mate-dependent paternal behavior
twice for 3 h (total 6 h) a day to the sire’s cage (L). During the rest of the
time from P3 to P5, the sire stayed alone (M), and pups were located
with the dam (K). Pup retrieval behavior over a period of 10 min was
examined (G–J and N–Q, respectively). The number of sires displaying
retrieval behavior was scored (J,Q). The numbers of positive mice/number
of mice tested are shown in parentheses, and the numbers of sires
displaying retrieval were expressed as percentages (R). Note that the
retrieval rate in two cases (J,Q) was high enough to have no significance
(n.s.), two-tailed Fisher’s exact probability test.
a more suitable and novel model for investigating paternal behavior, comparing the genetically determined paternity, observed in
animals such as voles or California mice (de Jong et al., 2009;
Ahern et al., 2011).
The ICR sires displayed parental retrieval behavior only for
their biological pups, indicating that they can discriminate
between their biological and non-biological offspring. This discrimination likely depends on odor or USV (Kuroda et al., 2007,
2011). The characteristic 38-kHz USVs were not recorded during
co-housing of ICR sires with non-mate dams, suggesting that the
sires can distinguish the mate from non-mate dams or that the
dams can distinguish the mate from non-mate sires. These results
strongly support our suggestion that 38-kHz USVs are critical and
have context for sires to induce retrieval behavior.
In these experiments, we examined the olfactory information of pups and cages (homes) for the sires prior to separation
from the mate dam and their offspring. In habituation as a
family, the presence of the mate was not completely essential.
Interestingly, we estimated that the territory information is much
more important to sires than the pheromones in the cages once
they had established their home cage. Surprisingly, when the
sires were continuously housed in their newly established home
July 2014 | Volume 8 | Article 186 | 7
Liang et al.
FIGURE 5 | Paternal retrieval test in ICR mice isolated prior parturition
from the mating pair and then united with pups only. A paired couple
was kept in a rearing cage from mating to 1 day before parturition (A). The
male was kept in the old cage (B) before meeting the pups and kept until P2
(D). The female was kept in a new cage (C), delivered her pups, and
remained until postnatal day 2 (E). The pups were transferred twice for 3 h
(total 6 h) a day (G) from the dam’s (new) cage (H) during P3 to P5. During
cages, they displayed paternal retrieval. In sharp contrast, if the
cage was new to the sire, even though the dam’s and sire’s olfactory information was there, the sire failed to display retrieval
behavior. These observations suggested that territory establishment is critical to maintaining paternity (Wright and Brown,
2000).
Pup retrieval as a parental behavior is rare among laboratory
mice that are not genetically monogamous (Wright and Brown,
2000; Kalueff et al., 2007). We found conditions in which the ICR
sires retrieved their pups related to their family structure. This
unique ability of the ICR sires will contribute to the increased
survival rate after reproduction and to the high level of social
attachment and interaction. We have recently reported that central cholinergic cellular signaling (Fujimoto et al., 2013) and
CD38 and oxytocin signaling in the nucleus accumbens (NAcc)
(Akther et al., 2013) are critical for the expression of paternal care
of the ICR mice. We also demonstrated the modulatory roles of
the mPOA and VP on parental behavior in rodents (Akther et al.,
2014). These published findings suggest that the neural circuitry
mediating paternal behavior includes the mPOA, VTA, NAcc, and
VP, and may be similar to those that mediate maternal behavior as proposed by Numan and others (Numan et al., 2005; Lee
and Brown, 2007; Wynne-Edwards and Timonin, 2007; Numan
and Stolzenberg, 2009). In addition, it is particularly interesting to test if mPOA galanin neurons regulate mate-dependent
parental behavior in the ICR strain (Wu et al., 2014). Further neuroendocrinological and neurocircuitry analyses in ICR mice will
be useful for understanding disorders with social impairment,
such as autism spectrum disorders and schizophrenia (Insel, 2010;
Munesue et al., 2010; Riebold et al., 2011; Feldman et al., 2012;
Salmina et al., 2013).
Frontiers in Neuroscience | Neuroendocrine Science
Mate-dependent paternal behavior
the rest of the time, the sires stayed alone in the home cages (F) and the
dams were with the pups (H). Pup retrieval behavior over a period of 10 min
was examined at P5 (I–L and M–P, respectively). The number of sires
displaying retrieval behavior was scored (L,P), and the numbers of positive
mice/number of mice tested are shown in parentheses. Pup retrieval was
expressed as percentages (Q; ∗ P < 0.05 between old (L) and new (P) cages,
two-tailed Fisher’s exact probability test).
AUTHOR CONTRIBUTIONS
Haruhiro Higashida designed experiments. Mingkun Liang,
Jing Zhong, Hong-Xiang Liu, Olga Lopatina, Ryusuke Nakada,
Agnes-Mikiko Yamauchi, and Haruhiro Higashida performed
animal experiments. Haruhiro Higashida and Mingkun Liang
wrote the manuscript.
ACKNOWLEDGMENTS
This work was supported in part by the Core Research for
Evolutional Science and Technology (CREST) from the Japan
Science and Technology Agency and by the Strategic Research
Program for Brain Sciences from Ministry of Education, Culture,
Sports, Science, and Technology, Japan.
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Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be
construed as a potential conflict of interest.
Received: 24 February 2014; accepted: 11 June 2014; published online: 11 July 2014.
Citation: Liang M, Zhong J, Liu H-X, Lopatina O, Nakada R, Yamauchi A-M and
Higashida H (2014) Pairmate-dependent pup retrieval as parental behavior in male
mice. Front. Neurosci. 8:186. doi: 10.3389/fnins.2014.00186
This article was submitted to Neuroendocrine Science, a section of the journal Frontiers
in Neuroscience.
Copyright © 2014 Liang, Zhong, Liu, Lopatina, Nakada, Yamauchi and Higashida.
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Received: 24/11/2019. Accepted: 15/12/2019
Copyright © 2019 Алла Борисовна Лихачева. Published by Vilnius University Press. This is an Open Access article distributed
under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in
any medium, provided the original author and source are credited. Immigration Discourse and the Prospects of Russian Language in Lithuania Summary. In contrast to the ongoing emigration, immigration to Lithuania cannot be called intensive,
and this topic as a whole remains on the periphery of public and academic attention. This article offers
a description of the situation of immigration from the perspective of sociolinguistics. In particular, an
analysis of the discourse of today’s Russian-speaking immigration to Lithuania reveals the linguistic
attitudes of immigrants with a native Russian language or their own, their adaptation strategies, family
language planning, and, ultimately, makes it possible to formulate general conclusions about the pros-
pects of preserving / not preserving Russian-speaking community in the host country. The paper summarizes interview materials with immigrants from Russia and some post-Soviet coun-
tries posted on the Lithuanian Internet, as well as recorded during this study. Key words: Russian-speaking immigrants; immigration discourse; language attitudes; interviews; the
language group vitality Contents lists available at Vilnius University Press
Slavistica Vilnensis
ISSN 2351-6895 eISSN 2424-6115
2019, vol. 64(2), pp. 128–142
DOI: https://doi.org/10.15388/SlavViln.2019.64(2).26 Contents lists available at Vilnius University Press
Slavistica Vilnensis
ISSN 2351-6895 eISSN 2424-6115
2019, vol. 64(2), pp. 128–142
DOI: https://doi.org/10.15388/SlavViln.2019.64(2).26 Contents lists available at Vilnius University Press Slavistica Vilnensis
2019, vol. 64(2), pp. 128–142 ISSN 2351-6895 eISSN 2424-6115
DOI: https://doi.org/10.15388/SlavViln.2019.64(2).26 ISSN 2351-6895 eISSN 2424-6115
DOI: https://doi.org/10.15388/SlavViln.2019.64(2).26 Алла Борисовна Лихачева Алла Борисовна Лихачева Вильнюсский университет (Литва)
E-mail: ala.lichaciova@flf.vu.lt Вильнюсский университет (Литва)
E-mail: ala.lichaciova@flf.vu.lt Вильнюсский университет (Литва)
E-mail: ala.lichaciova@flf.vu.lt Аннотация. В отличие от непрекращающейся эмиграции, иммиграцию в Литву нельзя назвать
интенсивной, и эта тема в целом остается на периферии общественного и академического вни-
мания. В данной статье предлагается описание ситуации иммиграции с позиций социолингви-
стики. В частности, анализ дискурса сегодняшней русскоязычной иммиграции в Литву позволя-
ет выявить языковые установки иммигрантов с родным русским языком или владеющих им, их
адаптационные стратегии, семейное языковое планирование, а в конечном итоге — сформули-
ровать общие выводы о перспективах сохранения/несохранения русскоязычия в принимающей
стране. В работе обобщаются материалы интервью с иммигрантами из России и некоторых постсовет-
ских стран, выложенных в литовском Интернете, а также материалы интервью, записанных в
ходе данного исследования. Ключевые слова: русскоязычные иммигранты; дискурс иммиграции; языковые установки; ин-
тервью; витальность языковой группы Received: 24/11/2019. Accepted: 15/12/2019 Imigracijos diskursas ir rusakalbiškumo perspektyvos Lietuvoje Santrauka. Kitaip nei neslūgstama emigracija, imigracija į Lietuvą nėra intensyvi, tad ši tema išlieka
visuomenės ir akademinės bendruomenės dėmesio periferijoje. Šiame straipsnyje yra siūlomas imigra-
cijos situacijos aprašymas iš sociolingvistikos pozicijų. Konkrečiai – šiuolaikinės rusakalbės imigraci- 128 Статьи. Алла Борисовна Лихачева. jos diskursas leidžia išsiaiškinti imigrantų su gimtąja ar gerai mokama rusų kalba kalbines nuostatas,
jų adaptacijos strategijas, šeimos kalbinį planavimą, o galiausiai – suformuluoti bendras išvadas apie
rusų kalbos gyvybingumo perspektyvas priimančioje šalyje. Straipsnyje apibendrinama medžiaga iš įdėtų internete, taip pat šiam darbui specialiai užrašytų interviu
su imigrantais iš Rusijos ir kai kurių kitų posovietinių šalių. Reikšminiai žodžiai: rusakalbiai imigrantai; imigracijos diskursas; kalbinės nuostatos; interviu; kal
binės grupės gyvybingumas Проблемы, обусловленные активными миграционными процессами,
все больше привлекают внимание ученых во всем мире. Одна из основ
ных тем, исследуемых представителями социальных и гуманитарных
наук, работающими в области критического анализа дискурса, связана
с имиджем иммигрантов в принимающих странах. Выявляются типич
ные приемы описания иммигрантов в политическом, научном, медий
ном, правоохранительном дискурсах. Как показывают исследования, вне
зависимости от страны, о которой идет речь, в разных типах дискурса
процессы иммиграции и их участники в основном получают негативные
характеристики (см. об этом [Матицына 2018; Бардин 2016; Richardson,
Wodak 2013; Tereškinas 2009; Sipavičienė, Gaidys, Jeršovas 2010; Collins
2007; Van der Valk 2000] и др.). Миграционная ситуация “глазами” самих
переселенцев, их самоощущение и способы адаптации в новой стране
становятся объектом анализа значительно реже; как правило, это работы
социологического, правового, экономического характера (напр.: [Зимова
2017; Bartkevičienė 2015; Kuznecovienė 2009; Li Xue 2007]). В Литве центральной темой медийных и научных публикаций по
следних лет является интенсивная эмиграция литовцев. В частности,
появилась целая серия работ социолингвистической направленности, из
учающих языковую жизнь и идентичность представителей эмигрантских
сообществ в Великобритании, США, Ирландии, Норвегии и других стра
нах традиционно активного “исхода” литовцев (таковы, например, кол
лективные монографии о языках и идентичности эмигрантов [Ramonienė
2015; Ramonienė 2019]). В Литве центральной темой медийных и научных публикаций по
следних лет является интенсивная эмиграция литовцев. В частности,
появилась целая серия работ социолингвистической направленности, из
учающих языковую жизнь и идентичность представителей эмигрантских
сообществ в Великобритании, США, Ирландии, Норвегии и других стра
нах традиционно активного “исхода” литовцев (таковы, например, кол
лективные монографии о языках и идентичности эмигрантов [Ramonienė
2015; Ramonienė 2019]). Imigracijos diskursas ir rusakalbiškumo perspektyvos Lietuvoje Сегодняшняя иммиграция в Литву освещается в основном средствами
массовой информации: на телевидении и в Интернете периодически по
являются репортажи и интервью с иностранцами, приезжающими в Лит
ву на учебу, желающими получить временное разрешение на работу, по
литэмигрантами и др. (см. материалы delfi.lt, 15min.lt, Žinių radijas, LRT
и др.). Академические исследования, связанные с “новыми” иммигран
тами в Литву, немногочисленны, в частности, можно назвать работы со
циологов [Petrušauskaitė, Batuchina 2015; Žibas 2014, 2012; Beresnevičiūtė,
Leončikas, Žibas 2009]. В этих работах затрагивается, но не является цен 129 ISSN 2351-6895 eISSN 2424-6115 Slavistica Vilnensis тральной, и основная для настоящей статьи тема языкового существова
ния иммигрантов в принимающем обществе. Вначале некоторые официальные данные. По определению литовско
го подразделения Европейской сети миграции (EMN) [Migracija skaičiais],
иммиграцией называется прибытие в страну с целью проживания в ней
дольше одного года; понятие иммиграции применимо не только к ино
странцам, планирующим проживание в Литве более года, но и к возвра
щающимся из эмиграции литовцам. Иностранцами считаются лица, не
имеющие гражданства Литвы. С 2004 по 2016 г. в среднем 80% потока
иммигрантов составляли возвращающиеся литовцы. В 2017 г. эта тенден
ция изменилась: литовцы и иностранцы составляют по 50% въезжающих
в Литву на проживание. В 2017 г. большую часть иностранцев, живущих
в Литве, составляли граждане России (26 %), Украины (24 %), Белару
си (19 %). В течение первых месяцев 2018 г. большинство иностранцев,
получивших разрешение на временное проживание в Литве, состави
ли украинцы (56,2 %), белорусы (23 %) и россияне (5,4 %) [Migracija
skaičiais]. Таким образом, картина иммиграции остается неизменной: по ко
личеству новых иностранцев в Литве лидируют славянские соседи, да
лее следуют жители некоторых бывших советских републик. В связи с
устойчивостью этой тенденции представляется актуальным выявление
установок иммигрирующих в Литву граждан относительно использова
ния русского языка как родного или привычного для них как жителей
постсоветского пространства лингва франка, а также относительно ис
пользования в их языковом существовании международного английского
и готовности к овладению литовским как государственным языком стра
ны иммиграции. По мнению ученых, “отбор данных для анализа дискурса может осу
ществляться несколькими способами в зависимости от функций текстов. В случае если сами тексты являются объектами изучения, исследователь
идентифицирует все тексты соответствующего типа и тематики и анали
зирует всю совокупность или определенную выборку текстов. Однако,
если тексты изучаются в качестве более широкой репрезентации соци
альных явлений или процессов, <…> предлагается начинать с индиви
дов, с поиска текста или текстов, артикулируемых в социальных группах
и возникающих в определенной ситуации. Imigracijos diskursas ir rusakalbiškumo perspektyvos Lietuvoje Точно так же, как в первом
случае, исследователь может анализировать всю совокупность текстов
или конктретную выборку” (S. Titscher (2002) – цит. по: Tereškinas 2009,
303). Для данной статьи применялся второй тип отбора текстов, посколь
ку всех участвовавших или упоминавшихся в исследовании людей без
условно можно отнести к социальной группе иммигрантов, к субъектам 130 Статьи. Алла Борисовна Лихачева. Статьи. Алла Борисовна Лихачева. Дискурс иммиграции и перспективы русскоязычия в Литве р
Дискурс иммиграции и перспективы русскоязычия в Литве ситуации иммиграции, а следовательно — к непосредственным и посто
янным участникам дискурса иммиграции (о таких понятиях анализа дис
курса как ситуации и их участники см. [van Dijk 1987, 161–163, 191]). Причем следует отметить, что все они становятся участниками несколь
ких ситуативных разновидностей, или типов дискурса (о типах дискурса
см. [Карасик 2004, 231-240]): институционального — в общении с пред
ставителями государственных учреждений, частных фирм; академиче
ского — актуального для студентов университетов; медицинского — в
поликлиниках и больницах; делового — в рабочих коллективах; быто
вого — в повседневном общении с соседями, продавцами, прохожими,
новыми друзьями и знакомыми, и др. Общее представление о жизни недавно прибывших в Литву имми
грантов дают материалы социальной группы “Жизнь в Литве” [facebook. com/groups/porusski.lt/]. Как пишут ее администраторы, все желающие
приглашаются обмениваться информацией о событиях, происходящих в
Литве, описывать свой опыт проживания в Литве, задавать вопросы и
отвечать на них, а также просто общаться на разные темы по-русски, т.к. группа объединяет людей, для которых русский является родным или хо
рошо усвоенным языком. Там же приводятся ссылки на короткие видео
сюжеты, снятые самими иммигрантами, об их условиях жизни и работы,
экскурсиях по Вильнюсу и Литве, их рассказы о временных коммуника
тивных трудностях и дальнейших жизненных планах. Два из таких видеосюжетов, снятых в 2017 [youtube...Vu1dQ] и 2018 г. [youtube…TKkU] в виде интервью с иммигрантами из России, анализи
руются в настоящей работе. Также используется видеозапись обсужде
ния пребывания в Литве и дальнейших планов с иммигрантами из пост
советских стран, состоявшегося в мае 2019 г. в студии русской редакции
DELFI [delfi.lt/video]. Анализируются и материалы пяти интервью с
иммигрантами из Азербайджана, записанных и проанализированных в
дипломной работе, защищенной в Вильнюсском университете в 2016 г.1
Для информации о сегодняшней языковой жизни бывших иммигрантов
мы обратились также к сделанным автором дипломной работы записям
двух интервью с азербайджанцами позднесоветской волны иммиграции,
получившими литовское гражданство. Кроме того, анализируются 4 ин
тервью, записанные в 2019 г. 1 Дипломная работа студентки магистерской программы “Русистика” Гульнар Агаевой
“Культурно-языковая адаптация иммигрантов, проживающих в Литве (случай иммигрантов из
Азербайджана)” была написана под моим руководством и защищена в Вильнюсском универси
тете в 2016 г. Imigracijos diskursas ir rusakalbiškumo perspektyvos Lietuvoje специально для данного исследования: одно
из них — с сотрудницей миграционной службы, занимающейся приемом
и адаптацией иммигрантов, и по одному — с иммигрантами из Беларуси, 131 ISSN 2351-6895 eISSN 2424-6115 Slavistica Vilnensis Украины и России, которые в настоящее время живут в Вильнюсе. Общая
продолжительность звучащего текста проанализированных интервью —
около 5 часов. Для всех респондентов русский язык является родным или
хорошо усвоенным языком. Следует отметить, что за каждым рассказом можно увидеть не только
обстоятельства переезда конкретных людей или конкретной семьи, но и
их опыт вербализации возникающих проблем и принимаемых решений в
общении и дискуссиях с другими представителями местного иммигрант
ского сообщества. Поэтому собранный материал фактически можно рас
сматривать как отражение общей ситуации с постсоветскими иммигран
тами в Литве. С респондентами обсуждались причины выезда из страны
проживания, соответствия/несоответствия ожиданий иммигрантов и
реальной жизни в Литве, опыта общения с государственными и други
ми официальными институциями, языковой специфики их сегодняшней
жизни, ощущения Литвы как толерантной/интолерантной страны и под. Вопросы, касающиеся языкового существования иммигрантов, в ходе
интервью не выделялись как основные для данной работы. В формулировке социологов, “социальные процессы, происходящие
в этнических группах, являются не только частью личного опыта от
дельных индивидов, но и результатом развития общества” [Kasatkina,
Leončikas 2003, 9]. Это утверждение актуально как при анализе причин
эмиграции жителей конкретной страны (в частности, литовцев), так и
при изучении стремлений и адаптационных интенций приезжающих в
страну на проживание, в нашем случае — “новых” иммигрантов в Литву. Обобщить результаты анализа текстов интервью можно следующим
образом: 1. В первую очередь очевидно, что тема языка актуальна не только для
филолога-социолингвиста, изучающего дискурс иммиграции как “сово
купность тематически соотнесенных текстов” [Чернявская 2001, 16] и
анализирующего языковые аспекты данного социального процесса. Это
центральная тема в судьбах самих иммигрантов, в рассказах об обстоя
тельствах их переезда и адаптации в новой стране. В рассуждениях о по
иске работы, о дальнейших планах для себя и своих детей, информанты
постоянно возвращались к роли языка/языков на новом жизненном этапе. В беседе с работницей миграционной службы обсуждался вопрос об
иммигрантах из постсоветских стран. По ее наблюдениям, среди них
есть русские, но есть и представители других национальностей: бело
русы, украинцы, чеченцы, таджики, азербайджанцы, армяне, грузины,
евреи. Их всех можно назвать русскоязычной или относительно рус
скоязычной иммиграцией, и для них существование русского языка в
бывшей советской республике чрезвычайно важно — они оценивают 132 Статьи. Алла Борисовна Лихачева. Imigracijos diskursas ir rusakalbiškumo perspektyvos Lietuvoje р
Дискурс иммиграции и перспективы русскоязычия в Литве русский язык как значительную помощь и опору на начальном этапе
адаптации в чужой стране. Вот иллюстрации этого мнения из интервью. Женщина-программист, в 2016 г. переехавшая из Москвы вместе с
10-летней тогда дочерью, рассказывает: До решения перебраться в Литву я бывала здесь у знакомой, тоже бывшей мо Д р
р
р
у
у
,
сквички, и поняла, что здесь (в Вильнюсе) достаточно много русских и пото
му ассимиляция будет достаточно легкой, для ребенка в первую очередь (РЖ44,
2019)2. сквички, и поняла, что здесь (в Вильнюсе) достаточно много русских и пото
му ассимиляция будет достаточно легкой, для ребенка в первую очередь (РЖ44,
2019)2. Из рассказа женщины из Азербайджана, в 2010 г. вышедшей замуж за
гражданина Литвы: Я приехала в страну, которую вообще не знала. Пугало сначала то, что не знаю
языка, но здесь многие говорят на русском, поэтому мне очень повезло (AЖ29,
2016). Я приехала в страну, которую вообще не знала. Пугало сначала то, что не знаю
языка, но здесь многие говорят на русском, поэтому мне очень повезло (AЖ29,
2016). 2. Многие иммигранты владеют русским и английским языками,
причем первый язык они воспринимают как традиционный язык меж
национального общения, все еще используемый на бывшем советском
пространстве, а второй — как актуальный, но пока не всеми освоенный
глобальный язык-посредник. Показателен фрагмент из интервью с 48-летним иммигрантом из
Минска, в детстве не раз бывавшим в Вильнюсе вместе с родителями, а 4
года назад открывшим здесь собственный автосервис: Здесь, как было в советское время, так и осталось: все говорят по-литовски и
по-русски. Поэтому Вильнюс для меня — типичный постсоветский город в язы
ковом отношении. Фактически мне литовский здесь не очень-то и нужен, можно
без него обойтись. Если уж кто-то не ответит по-русски, могу обратиться
по-английски (БМ48, 2019). Своим опытом адаптации делятся студентки из Азербайджана, живу
щие в Каунасе: Не было очень сложно установить связь с литовцами, они доброжелательны, во
многом мне помогают и отлично знают английский язык. С другими иностран
цами на самом деле и не общаюсь, только с литовцами и с людьми своей нацио
нальности. С близкими и родными общаюсь на своем родном языке, а также и на
русском (АЖ20, 2016); Чаще всего пользуюсь английским и русским. На английском языке ведётся об
учение, русский же язык помогает мне “выжить” (АЖ27, 2016). 2 В скобках после фрагментов интервью заглавные буквы указывают на страну эмиграции
и пол информанта, сообщается его возраст, если он известен, и год записи интервью. 2 В скобках после фрагментов интервью заглавные буквы указывают на страну эмиграции
и пол информанта, сообщается его возраст, если он известен, и год записи интервью. Из интервью с бывшей москвичкой, хорошо владеющей английским и
изучавшей литовский язык на начальных курсах для иммигрантов: Из интервью с бывшей москвичкой, хорошо владеющей английским и
изучавшей литовский язык на начальных курсах для иммигрантов: Все (в государственных учреждениях, банках, поликлиниках) сразу предлагают говорить по-русски, всюду без проблем, как только слышат мой литовский. Но я, говорить по-русски, всюду без проблем, как только слышат мой литовский. Но я,
правда, начинаю с того, что прошу прощения за свой плохой литовский. Говорю
им по-литовски: “Gal jūs suprantat rusiškai arba angliškai?” (‘Может, вы понима
ете по-русски или по-английски?’), и они сразу предлагают общаться по-русски
(РЖ44, 2019). правда, начинаю с того, что прошу прощения за свой плохой литовский. Говорю
им по-литовски: “Gal jūs suprantat rusiškai arba angliškai?” (‘Может, вы понима
ете по-русски или по-английски?’), и они сразу предлагают общаться по-русски
(РЖ44, 2019). 3. О намерении учить литовский язык говорят все русскоязычные
иммигранты, отмечая не столько насущную необходимость этого в се
годняшнем Вильнюсе и даже в традиционно литовскоязычном Каунасе
(особенно при владении английским), сколько свое желание проявить
уважение и симпатию к принявшей их стране. Из интервью со студентом из Азербайджана, живущим в Каунасе око
ло пяти лет к моменту записи интервью и владеющим, кроме азербайд
жанского, турецким, английским и русским языками: Как только приехал в Литву, поначалу думал, что литовским языком не буду ин
тересоваться. Со временем узнал, что в университете есть курсы литовского
языка, поэтому решил познакомиться с Литвой поближе, узнав её язык. Я счи
таю, что я неплохо владею литовским. Изучать начал его три года назад, так
что разговариваю и понимаю (АМ23, 2016). Еще одно мнение — украинца, в 2015 г. приехавшего учиться в маги
стратуре по приглашению Вильнюсского университета и оставшегося в
Вильнюсе работать экспертом по вопросам дискриминации: Когда я ехал в Литву, совершенно не собирался учить литовский язык. Тем более
что мне вообще трудно даются языки. В университете можно было обойтись
английским и даже русским, потому что программа была для иностранцев. Но
параллельно предлагался двухнедельный вводный курс литовского языка. Ну и,
если уж начал здесь жить, подумал, что нужно хоть как-то знать язык. В пер
вую очередь — это уважение к стране, ведь язык — это культурный код, а во-
вторых — элементарное бытовое удобство, если идешь по улице и понимаешь
по витринам, что здесь чинят компьютеры, а здесь продается мед… Я и сейчас
хотел бы учить литовский, если бы это было бесплатно. Конечно, по характеру
работы, я мог бы здесь жить и общаться только по-украински и по-русски. Но
ведь язык дает тебе больше возможностей (УМ30, 2019). 4. Imigracijos diskursas ir rusakalbiškumo perspektyvos Lietuvoje 133 ISSN 2351-6895 eISSN 2424-6115 Slavistica Vilnensis Из интервью с бывшей москвичкой, хорошо владеющей английским и
изучавшей литовский язык на начальных курсах для иммигрантов: Согласно данным интервью, нередко толчком к изучению государ
ственного языка для иммигрантов становится ощущение родственности
литовской культуры их национальной культуре. Сотрудница службы ми
грации также обращает внимание на то, что многие иммигранты, особен 134 Статьи. Алла Борисовна Лихачева. р
Дискурс иммиграции и перспективы русскоязычия в Литве но граждане России, Беларуси, Украины отмечают, что приезжают жить в
Литву, поскольку литовцы им эмоционально, ментально близки. Например, в одном из включенных в наш анализ видеоинтервью мо
лодая пара из Екатеринбурга, вместе с маленьким ребенком приехавшая
в Литву по рабочей визе и живущая здесь полтора года, рассказывает, что
изучает литовский язык не только на курсах и с помощью аудиосамоу
чителей, но и постоянно слушая песни в исполнении популярных литов
ских исполнителей: Литовский очень классный язык… Для нас звучание литовского языка — оно
очень крутое… Мы песни слушаем на литовском, мы вообще балдеем от раз
ных песен… Песни на литовском — это прямо отдельная наша любовь… Нам
нравится, что у этой страны (Литвы) своя богатая история и очень интерес
ная культура, нравится то, что эта культура переплетается с традиционной
русской культурой и историей. У нас действительно очень много общего (РМЖ,
2018). Аналогичное мнение высказывают и иммигранты более старшего
возраста: Аналогичное мнение высказывают и иммигранты более старшего
возраста: Я приехал по делам бизнеса. У меня работа всегда была связана с заграницей. До
этого 17 лет работал в Голландии, пробовал начать работу в Польше — там не
понравилось. Три с половиной года жил в Москве. Но здесь, в Вильнюсе, мне удоб
нее, спокойнее. Для белорусов литовский менталитет гораздо ближе, чем рус
ский… Я еще до переезда сюда начал самостоятельно изучать литовский язык
по аудиосамоучителю. Теперь уже по работе все понимаю по-литовски. Сказать
не все могу. Но в магазинах говорю по-литовски, я не комплексую по поводу своего
уровня (БМ48, 2019). Я приехал по делам бизнеса. У меня работа всегда была связана с заграницей. До
этого 17 лет работал в Голландии, пробовал начать работу в Польше — там не
понравилось. Три с половиной года жил в Москве. Но здесь, в Вильнюсе, мне удоб
нее, спокойнее. Для белорусов литовский менталитет гораздо ближе, чем рус
ский… Я еще до переезда сюда начал самостоятельно изучать литовский язык
по аудиосамоучителю. Теперь уже по работе все понимаю по-литовски. Сказать
не все могу. Но в магазинах говорю по-литовски, я не комплексую по поводу своего
уровня (БМ48, 2019). Сотрудница службы миграции также рассказала о политэмигрантах
из Таджикистана, утверждающих, что, выбирая между Литвой и Герма
нией, останавливаются на Литве из-за более традиционного и близкого
им отношения литовцев к воспитанию детей. 5. Можно заметить, что литовский язык изучается иммигрантами не
столько из-за его потенциальной полезности в жизни вообще, сколько из
благодарности за проявления толерантности и усилия государства и его
жителей облегчить адаптацию иммигрантов. Из интервью с бывшей москвичкой: Из интервью с бывшей москвичкой: Знание языка — это знак уважения к стране, которая нас приняла… Мне не
хотелось бы, чтобы сюда приезжали люди (из России), которые не хотят учить
язык. Мне важно, чтобы к нам, иммигрантам, не возникало отрицательного от
ношения местных жителей. Это то, чего я не встречала за три года ни разу со
стороны литовцев (РЖ44, 2019). 135 ISSN 2351-6895 eISSN 2424-6115 Slavistica Vilnensis На доброжелательность жителей Литвы к русскоязычным иммигран
там обращают внимание и две молодые пары, выложившие свои видео
интервью в Интернете. Супруги из Екатеринбурга отмечают: За полтора года ни разу не было какого-то там презрительного отношения или
агрессии из-за того, что мы русские или мы не понимаем по-литовски, не гово
рим по-литовски или плохо говорим. Никогда такого нет, и все литовцы, наобо
рот, с очень большим интересом… когда узнают, что мы не русские Литвы, а
приехавшие. Аналогичное мнение высказывают и иммигранты более старшего
возраста: Если у нас всё наладится, мы вер
немся на родину. Но, может быть, ребята наши (о детях и внуках) тут останут
ся. Моя внучка уже ходит в первый класс, и она уже пошла в литовскую школу. Мы
хотим научить ее культуре литовского государства, литовских людей (ТМ, 2019). Мы не говорим, что навсегда останемся тут. Если у нас всё наладится, мы вер
немся на родину. Но, может быть, ребята наши (о детях и внуках) тут останут
ся. Моя внучка уже ходит в первый класс, и она уже пошла в литовскую школу. Мы
хотим научить ее культуре литовского государства, литовских людей (ТМ, 2019). Еще одно характерное замечание относительно школы для дочери
прозвучало в интервью с российской иммигранткой, покинувшей страну,
когда, по ее словам, однозначно стало ясно, что из России надо уезжать: Я выбрала для дочери русскую школу, потому что хотела, чтобы она не поте
ряла язык. Но я жалею об этом решении. О том, что именно русская школа, я
жалею. Я думаю, что в литовской она сразу приобрела бы язык. Да, русский она
бы потеряла, и это была бы большая проблема (РЖ44, 2019). 7. В социолингвистическом плане иммигрантский дискурс предостав
ляет возможность увидеть перспективы сохранения русскоязычия в Лит
ве. С одной стороны, иммигранты воспринимают как норму использо
вание государственного языка в официальных сферах общения, и ста
раются следовать этой норме даже при наличии собственной минималь
ной языковой компетенции. Распространение русского языка в столице и
крупных городах они считают уходящим явлением: — Как вы думаете, долго ли еще хотя бы в Вильнюсе можно будет жить, не зная
литовского языка? — Ну, поменяются поколения, молодежь уже не понимает по-
русски. Вторым языком станет английский, а русский язык постепенно уйдет. — А приезжие? Или их тут капля в море? — Я думаю, не то чтобы капля в море,
а что не они заказывают музыку (РЖ44, 2019). — Как вы думаете, долго ли еще хотя бы в Вильнюсе можно будет жить, не зная
литовского языка? — Ну, поменяются поколения, молодежь уже не понимает по-
русски. Вторым языком станет английский, а русский язык постепенно уйдет. — А приезжие? Или их тут капля в море? — Я думаю, не то чтобы капля в море,
а что не они заказывают музыку (РЖ44, 2019). Аналогичное мнение высказывают и иммигранты более старшего
возраста: Их наоборот это сильно интересует, и они задают много вопросов. Но их уровень вопросов реально адекватный, в отличие от того, что спрашива
ют у нас в России про каких-то там литовских фашистов… Никакой русофобии,
даже уже немного скучно развенчивать этот миф… (РМЖ, 2018). Схожее мнение высказывают иммигранты, три месяца прожившие в
Литве, а до этого молодой человек успел пожить в Украине, Израиле,
Канаде и России, а девушка — в Украине и России: Вид на жительство мы получили и пойдем обязательно изучать литовский язык. Мы считаем, что, если находишься в какой-то стране, у тебя есть какие-то
обязанности перед ней, ты не можешь как-то халатно относиться… Если они
(иммигранты) приезжают из России, нужно просто забыть, что им там гово
рили в телевизоре (про русофобию). Просто всё классно, люди открытые, очень
спокойно, безопасно… А то такие истории, что вас там выгонят, не дадут вам
ничего, нахамят… Они (литовцы) очень классные! У них все предусмотрено, они
тебе звонят на дом, если чего-то не хватает из документов: приходи, пожалуй
ста, в какое время удобно… В кабинетах (о службе миграции) мы видели игруш
ки… ну нормальный такой подход… (РМЖ, 2017). 6. Как сообщает сотрудница миграционной службы, несмотря на име
ющийся у иммигрантов начальный энтузиазм в овладении государствен
ным языком, приоритетом для них неизбежно становится работа, и люди,
имеющие высшее образование и не планирующие получить его в Литве,
устраиваются на работу туда, где хорошее знание государственного язы
ка не требуется, например, если документация ведется на английском, а в
общении преобладает русский язык. Кроме того, по словам сотрудницы службы миграции, большинство
иммигрантов с родным или хорошо усвоенным русским языком выбира
ют для детей детские сады и школы с русским языком обучения. Из рассказа старшеклассницы-азербайджанки, пять лет назад при
ехавшей с родителями в Шяуляй и заканчивающей русскую школу: Чаще всего я пользуюсь азербайджанским и русским. Изучать (литовский язык)
я начала, как только приехала в Литву, потому что знала, что надо будет сда
вать экзамен, чтобы получить аттестат. В школе брала дополнительные уроки
литовского языка (АЖ18, 2016). 136 Статьи. Алла Борисовна Лихачева. р
Дискурс иммиграции и перспективы русскоязычия в Литве Школы с государственным языком обучения, по данным службы ми
грации, чаще выбирают переселившиеся в Литву украинцы литовского
происхождения, некоторые иммигранты из России, называющие в каче
стве причины переезда политическую ситуацию в их стране, политэми
гранты из Таджикистана. Например, участник видеобеседы в студии DELFI из Таджикистана, с
2015 г. живущий в Вильнюсе, рассказывает: Мы не говорим, что навсегда останемся тут. Аналогичное мнение высказывают и иммигранты более старшего
возраста: С другой стороны, даже те иммигранты, которые приехали в Литву
относительно давно — в позднее советское время, и для которых русский
язык не является родным, продолжают активно его использовать: Как только приехал сюда, не так тяжело все было, как казалось, проблема была
в том, что не знаю литовский язык. Со временем понял, что для начала хватит
и русского языка, до сих пор чаще всего пользуюсь русским языком. <…> В семье
общаемся только на азербайджанском языке, а в общественных местах, с дру
зьями и соседями — на русском (АМ51, 2016). 137 ISSN 2351-6895 eISSN 2424-6115 Slavistica Vilnensis Источники В студии DELFI: cтанут ли иммигранты из Украины, Беларуси, России и дру-
гих стран национальными меньшинствами Литвы? URL: https://www.delfi.lt/video/
laidos/zinios-rusu-kalba/v-studii-delfi-ctanut-li-immigranty-iz-ukrainy-belarusi-rossii-i-
drugih-stran-nacionalnymi-menshinstvami-litvy.d?id=81255171 (21 11 2019). Форум Жизнь в Литве. URL: https://www.facebook.com/groups/porusski.lt/ (21 10
2019). Личный опыт: Иммиграция в Литву из России по Blue Card. URL: https://www. youtube.com/watch?v=OSaw44qTKkU (10 11 2019). О Литве, переезде, националистах и еде. URL: https://www.youtube.com/
watch?v=zOwJx0Vu1dQ (10 11 2019). О Литве, переезде, националистах и еде. URL: https://www.youtube.com/
watch?v=zOwJx0Vu1dQ (10 11 2019). Migracija skaičiais – European Migration Network. URL: http://123.emn.lt/ (10 10
2019). Для позднесоветской волны иммигрантов русский язык был и оста
ется существенным компонентом их языковой жизни и жизни их семей: Дискурс иммиграции и перспективы русскоязычия в Литве Статьи. Алла Борисовна Лихачева. р
Дискурс иммиграции и перспективы русскоязычия в Литве принятыми как выход для Литвы — такой же, как и для многих других
стран. Иммигранты, говорящие по-русски, ощущают, что их присутствие
в Литве не воспринимается как угроза в политическом или в языковом
отношении. Они видят себя субъектами, чей вклад в литовскую экономи-
ку, демографию и даже в количественные показатели носителей литов-
ского языка должен быть поддержан литовским обществом. Для позднесоветской волны иммигрантов русский язык был и оста
ется существенным компонентом их языковой жизни и жизни их семей: Для позднесоветской волны иммигрантов русский язык был и оста
ется существенным компонентом их языковой жизни и жизни их семей: Когда приехала в Литву, говорить начала на русском, поэтому детей отправила
в русский садик. Это было удобно для меня, потому что на тот момент я не
знала литовского языка. Затем дети пошли в русскую школу. Между собой дети
общались в детстве на русском и азербайджанском языке, когда выросли, они
выучили литовский язык. Сейчас дети уже большие, с нами, родителями, гово
рят на азербайджанском, между собой — чаще всего на русском (АЖ50, 2016); Дети у нас пока маленькие. Дочь учится во втором классе в русской школе, сын
ходит в русский садик. Выбрали именно русскую школу и садик, потому что нам
самим легче общаться на русском, чем на литовском языке. У детей много друзей
во дворе, и они общаются на русском (АМ51, 2016). Согласно критериям [Bourhis, Landry 2008], витальность языковой
группы (the language group vitality) напрямую зависит от языкового по
ведения ее членов, частоты использования ими этнического или хорошо
усвоенного, общего для членов группы языка, поддержания этого язы
ка, в частности, в семейном и близком общении. “Использование языка
меньшинства не прекратится до тех пор, пока не изменятся групповые
нормы использования языка” [Fase, Jaspaert & Kroon 1992, 7]. С учетом
этих факторов можно утверждать, что русскоязычие на определенных
территориях Литвы в ближайшие годы сохранится и может даже не
сколько усилить свои позиции за счет новой волны иммигрантов раз
ных национальностей (о тесной зависимости количественных показате
лей языковой группы и шансов сохранения ее общего языка см. [Clyne
1992]), для которых русский язык является основным языком общения во
многих жизненных ситуациях, в том числе, как показывают материалы
интервью, в институциональном и других видах внесемейного дискурса. Важным показателем устойчивого положения языковой группы яв
ляется также положительное восприятие и оценка языка его носителя
ми, живущими в иноязычных условиях [Allard, Landry 1992]. В дискур
се постсоветской русскоязычной иммиграции переезд в Литву, с одной
стороны, рисуется как переселение в Европу, а с другой — как переезд
в более близкое и понятное, чем западноевропейское, культурное, сим
волическое (в том числе и за счет присутствия здесь русского языка)
пространство. При всем уважении к принимающей стране и ее языку
иммигранты не предполагают выбора между литовским и русским, но
планируют обязательное дополнительное овладение литовским языком
при условии дальнейшей жизни в Литве. Продолжающаяся эмиграция и относительно небольшое количество
возвращающихся литовцев имплицирует ожидания иммигрантов быть 138 Статьи. Алла Борисовна Лихачева. Литература Jaspaert and S. Kroon (Eds.). Maintenance and Loss of Minority Languages. Amsterdam: John Benjamins Publishing Company, 17–36. Minority Languages. Amsterdam: John Benjamins Publishing Company, 17–36. COLLINS, J., 2007. Immigrants as Victims of Crime and Criminal Justice Discourse in COLLINS, J., 2007. Immigrants as Victims of Crime and Criminal Justice Discourse in
Australia. International Review of Victimology, Vol. 14, 1, 51–79. COLLINS, J., 2007. Immigrants as Victims of Crime and Criminal Justice Discourse in Australia. International Review of Victimology, Vol. 14, 1, 51–79. FASE, W., JASPAER, K. & KROON, S. 1992. Maintenance and Loss of Minority Lan-
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guages: Introductory Remarks. In: W. Fase, K. Jaspaert and S. Kroon (Eds.). Maintenance
and Loss of Minority Languages. Amsterdam: John Benjamins Publishing Company, 3–13. guages: Introductory Remarks. In: W. Fase, K. Jaspaert and S. Kroon (Eds.). Maintenance
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behaviour. International Journal of the Sociology of Language, 108, 15–42.i LI XUE, 2007. Portrait of an Integration Process. Difficulties encountered and
resources relied on for newcomers in their first 4 years in Canada. Evidence from three
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geroi ili ugroza? Vestnik Omskogo gosudarstvennogo pedagogicheskogo universiteta. Gumanitarnye issledovanija, № 3 (20), 84–88. geroi ili ugroza? Vestnik Omskogo gosudarstvennogo pedagogicheskogo universiteta. Gumanitarnye issledovanija, № 3 (20), 84–88. PETRUŠAUSKAITĖ, V., BATUCHINA, A., 2015. Migrant workers from non-EU
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Arguments in Political Leaflets of Austrian and British Far-Right Parties. In: R. Wodak gl
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(Ed.). Critical Discourse Analysis. Vol. IV. Applications, Interdisciplinary Perspectives a New Trends. Los Angeles – London – New Delhi – Singapore – Washington DC, 245–274. SIPAVIČIENĖ, A., GAIDYS, V., JERŠOVAS, M., 2010. Lietuvos gyventojų požiūris į
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publikacijos/failai/1427788343_8TMOLietuvos%20gyventoju%20poziuris%20i%20
imigracija%20ir%20darbo%20imigrantus.pdf (03 11 2019).i TEREŠKINAS, A., 2009. Imigracijos diskursai JAV ir Airijos spaudoje. Filosofija. Sociologija, t. 20, nr. 4, 302–309. VAN DER VALK, I., 2000. Parliamentary discourse on immigration and nationality in
France. In: R. Wodak & T.A. van Dijk (Eds.). Racism at the top: parliamentary discourses
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& S.J. Samuels, (Eds.) Comprehending Oral and Written Language. New York: Academic
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Azii v Rossii. Migracija i social’no-ekonomicheskoe razvitie. T. 2, №1, 19–28. Azii v Rossii. Migracija i social’no-ekonomicheskoe razvitie. T. 2, №1, 19–28. ŽIBAS, K., 2012. Baltarusiai, rusai ir ukrainiečiai Lietuvoje: nuo imigrantų iki ateities
piliečių. Etniškumo studijos, nr. 1/2, 145–177. ŽIBAS, K., 2014. Turkų ir kinų imigrantai Lietuvoje. Vilnius: Lietuvos socialinių
tyrimų centras. Алла Лихачева, доктор гуманитарных наук, профессор кафедры русской филологии
Вильнюсского университета Alla Likhachiova, PhD (Humanities), Professor of the Russian Department, Vilnius University
Ala Lichačiova, humanitarinių mokslų daktarė, Vilniaus universiteto Rusų filologijos katedros
profesorė Alla Likhachiova, PhD (Humanities), Professor of the Russian Department, Vilnius University
Ala Lichačiova, humanitarinių mokslų daktarė, Vilniaus universiteto Rusų filologijos katedros
profesorė Ala Lichačiova, humanitarinių mokslų daktarė, Vilniaus universiteto Rusų filologijos katedros
profesorė 142
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1 Programa de Pós-graduação
Ciências da Saúde,
Universidade Federal de
Goiás, Goiânia, Brasil.
2 Instituto de Patologia
Tropical e Saúde Pública,
Universidade Federal de
Goiás, Goiânia, Brasil.
3 Faculdade de Medicina,
Universidade Federal de
Goiás, Goiânia, Brasil.
4 Faculdade de Farmácia,
Universidade Federal de
Goiás, Goiânia, Brasil. Correspondência
S. C. Fuini
Programa de Pós-graduação
Ciências da Saúde,
Universidade Federal de Goiás.
Rua 17-A, 1419, Setor
Aeroporto, Goiânia, GO
74075-160, Brasil.
silvana.fuini@gmail.com Qualidade de vida dos indivíduos expostos ao
césio-137, em Goiânia, Goiás, Brasil Quality of life in individuals exposed to
cesium-137 in Goiânia, Goiás State, Brazil Calidad de vida de las personas expuestas al
cesio-137, en Goiânia, Goiás, Brasil Silvana Cruz Fuini 1
Rafael Souto 2
Geraldo Francisco do Amaral 3
Rita Goreti Amaral 4 Silvana Cruz Fuini 1
Rafael Souto 2
Geraldo Francisco do Amaral 3
Rita Goreti Amaral 4 Qualidade de Vida; Césio; Impacto Psicossocial;
Acidentes 1301
ARTIGO ARTICLE 1301
ARTIGO ARTICLE 1301
ARTIGO ARTICLE Qualidade de vida dos indivíduos expostos ao
césio-137, em Goiânia, Goiás, Brasil
Quality of life in individuals exposed to
cesium-137 in Goiânia, Goiás State, Brazil
Calidad de vida de las personas expuestas al
cesio-137, en Goiânia, Goiás, Brasil Quality of Life; Cesium; Psychosocial Impact;
Accidents Introdução nesta direção 10,11. Sendo assim, a avaliação da
qualidade de vida do paciente é reconhecida co-
mo uma importante área do conhecimento cien-
tífico, em razão do seu conceito se interpor ao de
saúde. Tal avaliação tem a vantagem de incluir
aspectos subjetivos geralmente não abordados
por outros critérios de avaliação 12. O acidente radioativo ocorrido em setembro de
1987, na cidade de Goiânia, Estado de Goiás, Bra-
sil, trouxe problemas sérios que perduram até
os dias de hoje 1. Esse evento foi provocado por
meio da ruptura de um aparelho radioterápico
abandonado em uma clínica médica desativada,
e posteriormente agravado pelo manuseio incor-
reto da cápsula que continha o césio-137 (isóto-
po radioativo) por pessoas leigas no assunto 2. O interesse pelo conhecimento da qualidade
de vida dos radioacidentados configura-se como
uma preocupação constante da população do
Estado de Goiás e especialmente do corpo clíni-
co do C.A.R.A., sobretudo, devido à recorrência
no atendimento psicossocial e à busca frequente
pelos serviços médicos e por respostas às dúvi-
das que ainda persistem. Na época, em torno de 112 mil pessoas foram
envolvidas no acidente, cerca de uma centena
diretamente e outras centenas indiretamente,
incluindo familiares, vizinhos e agentes públi-
cos 2,3. Desde então é realizado regularmente por
uma unidade de saúde específica, atualmente
denominada Centro de Assistência aos Radioaci-
dentados (C.A.R.A.) 3,4, que faz parte da estrutura
da Secretaria de Saúde de Goiás, o monitoramen-
to das pessoas envolvidas. Até os dias de hoje,
os resultados do monitoramento não apontam
dados estatisticamente significantes para morbi-
mortalidade associada aos efeitos da radiação
ionizante. Entretanto, pesquisadores recomen-
dam o prosseguimento dos estudos, tendo em
vista os efeitos tardios decorrentes do acidente
radioativo 1,5. O referido fato corrobora com estudos que
consideram o impacto na saúde mental como o
maior problema de saúde pública desencadeado
por acidentes dessa natureza e que, muitas ve-
zes, esses transtornos vêm acompanhado de um
grande número de queixas somáticas inespecífi-
cas 8,9,13,14. Nesse aspecto, é reconhecido pelo cor-
po clínico do C.A.R.A. que desde a ocorrência do
acidente radioativo com o césio-137 foi dada ên-
fase aos aspectos físicos das vítimas do acidente,
em detrimento ao gerenciamento dos problemas
sociais e psicológicos 15. Assim, este trabalho teve como objetivo ava-
liar a qualidade de vida dos indivíduos expos-
tos ao césio-137, durante acidente ocorrido em
Goiânia, e sua associação com fatores sociode-
mográficos. Abstract Estudo observacional transversal realizado em
Goiânia, Goiás, Brasil, em uma amostra de 56%
de 111 pessoas, objetivando avaliar a qualida-
de de vida dos indivíduos expostos ao césio-137
e sua associação com fatores sociodemográfi-
cos. Utilizou-se o WHOQOL-BREF e o Sistema
de Monitoramento dos Radioacidentados. Os
participantes foram divididos segundo critérios
internacionais em: Grupo I – radiodermites e/
ou dosimetria citogenética acima de 20 rads
(n = 33); e Grupo II – dosimetria citogenética ≤ 20
rads (n = 29), totalizando 62 sujeitos. Dentre os
domínios do WHOQOL-BREF, o meio ambiente
apresentou a média de escores mais alta (59,88;
DP = 20,39) e o psicológico a média mais baixa
(53,02; DP = 17,98). As associações entre os do-
mínios físico, psicológico e relações sociais foram
significativas para a variável idade. Não houve
diferença entre grupos. A associação entre os fa-
tores sociodemográficos e qualidade de vida não
foi significativa. Os radioacidentados sofrem
considerável impacto na qualidade de vida, com
persistência de problemas psicossociais, espe-
cialmente para aqueles com mais de 41 anos. This cross-sectional observational study in
Goiânia, Goiás State, Brazil in a sample of 56%
of 111 individuals exposed to cesium-137 aimed
to evaluate their quality of life and associations
with socio-demographic factors. The study used
the WHOQOL-BREF and the Monitoring System
for Radiation Victims. Participants were divided
according to international criteria: Group I – ra-
diation dermatitis and / or cytogenetic dosimetry
above 20 rads (n = 33) and Group II - cytogenetic
dosimetry ≤ 20 rads (n = 29), totaling 62 subjects. Among the WHOQOL-BREF domains, environ-
ment showed the highest mean scores (59.88,
SD = 20.39) and psychological the lowest (53.02,
SD = 17.98). Associations between the physical,
psychological, and social domains were signifi-
cant for the age variable. There was no difference
between groups. The association between socio-
demographic factors and quality of life was not
significant. Radiation victims suffer consider-
able impact on quality of life, with persistent
psychosocial problems, especially among those
older than 41 years. Correspondência
S. C. Fuini
Programa de Pós-graduação
Ciências da Saúde,
Universidade Federal de Goiás. Rua 17-A, 1419, Setor
Aeroporto, Goiânia, GO
74075-160, Brasil. silvana.fuini@gmail.com Quality of Life; Cesium; Psychosocial Impact;
Accidents Quality of Life; Cesium; Psychosocial Impact;
Accidents Qualidade de Vida; Césio; Impacto Psicossocial;
Acidentes Cad. Saúde Pública, Rio de Janeiro, 29(7):1301-1310, jul, 2013 Fuini SC et al. 1302 Introdução Dessa forma, os diversos aspectos relacio-
nados à saúde e fatores psicossociais devem ser
investigados nas populações potencialmente
expostas à radioatividade, sobretudo, tomando-
se em conta o trauma, o medo generalizado e o
estresse inerentes aos efeitos desse tipo de even-
to 6. Estudos apontam que aproximadamente
75% dos indivíduos expostos a contaminações
por acidentes nucleares apresentam alguma for-
ma de sintomas psicológicos, desde a incapaci-
dade de dormir à dificuldade de concentração
e isolamento social. Entre aqueles com maior
risco de efeitos significativos encontram-se as
crianças, mulheres (grávidas e mães de crianças
pequenas), idosos e pessoas com um histórico
de transtorno psiquiátrico 7,8. Além disso, os indi-
víduos expostos têm uma alta taxa de transtorno
do estresse pós-traumático (TEPT), o que cola-
bora para a percepção de que alguns aspectos re-
lacionados à qualidade de vida estão afetados 9. Cad. Saúde Pública, Rio de Janeiro, 29(7):1301-1310, jul, 2013 Métodos Trata-se de um estudo observacional transver-
sal realizado no período de janeiro a agosto de
2011, cuja população-alvo foi composta por
indivíduos expostos à radiação pelo césio-137,
na cidade de Goiânia, devidamente cadastrados
no C.A.R.A., unidade da Secretaria de Estado da
Saúde de Goiás. Desde a época do acidente radioativo, os pa-
cientes cadastrados no C.A.R.A. foram categori-
zados por grupos (I, II e III) de acordo com as
normas da International Atomic Energy Agency
(IAEA), levando-se em conta critérios de classi-
ficação conforme a gravidade das lesões cutâne-
as e da intensidade de contaminação interna e
externa. O Grupo I é composto por indivíduos
com radiodermites e/ou dosimetria citogenética
acima de 20rads. O II é formado por indivíduos
com dosimetria citogenética ≤ 20rads. O Grupo
III é constituído por agentes públicos, voluntá-
rios, familiares das vítimas e vizinhos 2,3. Nesse contexto, a qualidade de vida é entendi-
da a partir de novos paradigmas que têm influen-
ciado as práticas do setor saúde, levando-se em
conta que o processo saúde/doença é complexo
e abrange aspectos econômicos, socioculturais,
experiência pessoal e estilos de vida. De acordo
com esses novos aspectos, a melhoria da quali-
dade de vida passa a ser um dos resultados espe-
rados pelas práticas assistenciais promovidas por
todas as iniciativas de saúde que movem esforços Foram incluídos os sujeitos dos Grupos I e II
com mais de 18 anos. Excluiu-se os indivíduos QUALIDADE DE VIDA DOS INDIVÍDUOS EXPOSTOS AO CÉSIO-137
1303 software desenvolvido para informatizar e orga-
nizar o monitoramento e acompanhamento das
vítimas do acidente 3. As variáveis analisadas fo-
ram idade, sexo, grau de escolaridade e religião. Para a análise tomou-se por base duas faixas etá-
rias (18 a 40 anos e 41 ou mais), de acordo com o
protocolo clínico/laboratorial de monitoramen-
to desses indivíduos, definido pela IAEA, que di-
vide os mesmos nestas duas faixas etárias para a
realização dos procedimentos estabelecidos. com prejuízo cognitivo e os integrantes do Grupo
III, em razão de que a maioria destes não foi mo-
nitorada na época do acidente, portanto há pou-
cos registros da dosimetria correspondente ao
nível de exposição ao césio-137 3. Ainda, o Grupo
III encontra-se em processo de cadastramento
no C.A.R.A., na medida em que consegue o re-
conhecimento (seja administrativo ou judicial)
de seu direito de receber os benefícios (pensões)
concedidos por força de lei 3. Métodos A análise estatística foi realizada pelo pro-
grama SPSS for Windows, versão 15.0 (SPSS Inc.,
Chicago, Estados Unidos). Foi empregada a sin-
taxe WHOQOL-BREF no SPSS para avaliação dos
escores do instrumento de qualidade de vida e
posteriores correlações, baseando-se no coefi-
ciente de Pearson. Para a análise da comparação
das variáveis sociodemográficas e os domínios
do WHOQOL-BREF foi utilizado o teste t de Stu-
dent e Anova para os dados normais. Para verifi-
car a frequência das variáveis sexo e idade com
os domínios do WHOQOL-BREF foi utilizado o
teste qui-quadrado. Considerou-se como nível
de significância o valor de 5% (p < 0,05). Inicialmente, os indivíduos dos Grupos I e
II foram convidados por carta-convite e conta-
tos telefônicos para participarem do estudo. Do
total de 111 sujeitos que atendiam aos critérios
de inclusão, apenas 62 aceitaram participar da
pesquisa (Grupo I n = 33; Grupo II n = 29). Após
essa etapa, o questionário foi autoaplicado sob
a orientação da pesquisadora responsável, em
encontros realizados nas residências dos sujeitos
ou nas dependências do C.A.R.A. O instrumento de coleta de dados para ava-
liar a qualidade de vida foi o questionário World
Health Organization Quality of Life (WHOQOL-
BREF), desenvolvido pela Organização Mundial
da Saúde (OMS) e validado no Brasil por Fleck et
al. 10. Contém 26 perguntas, sendo duas questões
gerais de qualidade de vida – a primeira refere-se
à autopercepção da qualidade de vida e a ou-
tra sobre a satisfação com a saúde. As demais 24
questões são distribuídas em quatro domínios:
físico, psicológico, relações sociais e meio am-
biente. Cada domínio é representado por facetas
e suas perguntas foram formuladas para uma
escala de respostas do tipo Likert (1 a 5), que é
um tipo de escala de resposta psicométrica usa-
da habitualmente em questionários, mais uti-
lizada em pesquisas de opinião. Nessa escala é
solicitado aos entrevistados que indiquem o seu
grau de concordância ou discordância com uma
informação que está sendo medida. Atribui-se
valores numéricos e/ou sinais às respostas para
refletir a força e a direção da reação do entrevis-
tado à declaração. As declarações de concordân-
cia devem receber valores positivos ou altos, e as
declarações das quais discordam devem receber
valores negativos ou baixos. A pontuação total
da atitude de cada respondente é dada pela so-
matória das pontuações obtidas para cada afir-
mação 16,17. Métodos Este trabalho foi aprovado pelo Comitê de
Ética em Pesquisa Dr. Henrique Santillo da Secre-
taria de Estado da Saúde de Goiás, sob protocolo
no 0016.0.177.000-10. Cad. Saúde Pública, Rio de Janeiro, 29(7):1301-1310, jul, 2013 Resultados Aspecto social
Grupo não investigado
Grupo investigado
Grupo I (n = 29)
Grupo II (n = 20)
Valor
de p
Grupo I (n = 33)
Grupo II (n = 29)
Valor
de p
n
%
n
%
n
%
n
%
Sexo
Masculino
18
62,1
9
45,0
18
54,5
14
48,3
Feminino
11
37,9
11
55,0
0,187
15
45,5
15
51,7
0,799 *
Idade (anos)
18 |–| 40
11
37,9
13
65,0
14
42,4
17
58,6
> 41
18
62,1
7
35,0
0,058
19
57,6
12
46,4
0,309 **
Escolaridade
Ensino Fundamental
17
58,6
7
35,0
9
36,0
15
51,7
Ensino Médio
8
27,6
12
60,0
11
44,0
12
41,4
Ensino Superior
4
13,8
1
5,0
0,071
5
20,0
2
6,9
0,047 *
Religião
Não possui
3
10,3
1
5,0
6
18,2
2
6,9
Católico
5
17,2
6
30,0
18
54,5
19
65,5
Espírita
6
20,7
13
65,0
1
3,0
1
3,4
Evangélico
15
51,7
0
0,0
0,001
8
24,2
7
24,1
0,606 *
* Teste qui-quadrado;
** Teste exato de Fisher. meio ambiente indicou a média de escores mais
alta e o domínio psicológico a menor pontuação. Quando analisamos os dois grupos de forma ge-
ral, verificou-se que o domínio psicológico obte-
ve a menor média e o domínio meio ambiente a
maior média. II apresentaram os maiores escores na primeira e
os menores na segunda questão, como se segue:
Grupo I com 61,36 e 50,00 e o Grupo II com 62,93
e 56,90, respectivamente. II apresentaram os maiores escores na primeira e
os menores na segunda questão, como se segue:
Grupo I com 61,36 e 50,00 e o Grupo II com 62,93
e 56,90, respectivamente. Dentre o universo de indivíduos que avalia-
ram sua qualidade de vida de modo geral como
boa/muito boa e ruim/muito ruim, consideran-
do a média nos domínios maior que 70 e menor
que 30, destaca-se que no Grupo I cerca de 50%
eram homens e 50% mulheres, também apre-
sentaram o mesmo porcentual nas duas faixas
etárias (18-40 anos e 41 ou mais). Não houve
predominância de algum grau de escolaridade e
religião. Já no Grupo II a maioria era de homens
e com idades entre 18 a 40 anos. Também, as va-
riáveis escolaridade e religião encontram-se pul-
verizadas. Resultados A população analisada foi composta por 62 sujei-
tos, correspondendo a 56% do universo de radio-
acidentados cadastrados no C.A.R.A, distribuídos
por Grupos I e II. Do total de 111 indivíduos, 49
se negaram a participar da pesquisa, como pode
ser observado na Tabela 1. Verificou-se que na
comparação da distribuição de todas as variáveis
analisadas no grupo não investigado houve dife-
rença significativa na variável religião (p = 0,001)
e no grupo investigado na variável escolaridade
(0,047). Os resultados sobre a percepção da qualidade
de vida relacionada às duas questões genéricas
do WHOQOL-BREF apresentaram na primeira
questão – em que os indivíduos avaliaram a pró-
pria qualidade de vida – 44,9% como “boa”; 6,5%
“muito boa”; e 9,5% “ruim” e “muito ruim”. Des-
ses, 39,9% responderam como “nem ruim, nem
boa”. Quanto à segunda questão, que mensura
a satisfação dos sujeitos em relação à própria
saúde, 35,4% disseram estar “satisfeitos”; 11,4%
“muito satisfeitos”; e 37,7% consideravam-se
“insatisfeitos” e “muito insatisfeitos”. O restante,
16,4%, disse estar “nem satisfeito, nem insatisfei-
to”. Ao separar os grupos, observou-se na média
geral de cada questão que tanto o Grupo I como o O valor mínimo dos escores de cada domínio
do WHOQOL-BREF é zero e o máximo é 100. O
escore de cada domínio é obtido em uma escala
positiva, isto é, quanto mais alto o escore, melhor
a qualidade de vida naquele domínio 11,18. Para obtenção das informações sociodemo-
gráficas utilizou-se o banco de dados do Siste-
ma de Monitoramento dos Radioacidentados
(SISRAD) disponível no C.A.R.A. O SISRAD é um Fuini SC et al. 1304 Distribuição das variáveis de acordo com cada aspecto social por Grupos (I e II) na população de radioacidentados, nos grupos não investigado e
investigado. Goiânia, Goiás, Brasil, 2011. Distribuição das variáveis de acordo com cada aspecto social por Grupos (I e II) na população de radioacidentados, nos grupos não investigado e
investigado. Goiânia, Goiás, Brasil, 2011. Cad. Saúde Pública, Rio de Janeiro, 29(7):1301-1310, jul, 2013 Resultados Nesses dois grupos, as menores mé-
dias (< 30) concentraram-se nos domínios físico
e psicológico e as maiores (> 70) nos domínios
relações sociais e meio ambiente, com predomi-
nância neste último. A Tabela 3 mostra a comparação dos dados
gerais separados por duas faixas etárias de 18 a 40
anos e com mais de 41. Os resultados apresenta-
ram diferença para a variável idade referente aos
domínios físico, psicológico e relações sociais
(p < 0,05). Somente o domínio meio ambiente
não foi estatisticamente significativo (p > 0,05). A Tabela 3 mostra, ainda, que não houve di-
ferença (p > 0,05) na variável sexo entre os domí-
nios do WHOQOL-BREF. Apesar de não haver di-
ferença, as mulheres apresentaram escores mais
baixos em comparação aos homens em todos os
domínios. Ao analisar a variável idade separada por gru-
po, foi verificada diferença no Grupo I nas duas
faixas etárias, referentes aos domínios psicológi-
co (p = 0,017) e relações sociais (p = 0,004). Quan-
to à variável sexo separada por grupo, constatou-
se que não foi significante em todos os domínios,
cujos escores ficaram assim representados nos
Grupos I e II, respectivamente: domínio físico
52,86/53,81; domínio psicológico 50,56/53,06; A Tabela 2 mostra os valores da distribuição
dos índices de qualidade de vida em cada do-
mínio do WHOQOL-BREF, indicando que não
houve diferença. Para o Grupo I, o domínio meio
ambiente apresentou a maior média de escores,
enquanto que a menor média foi para o domínio
relações sociais. Quanto ao Grupo II, o domínio QUALIDADE DE VIDA DOS INDIVÍDUOS EXPOSTOS AO CÉSIO-137
1305 Tabela 2 Média e Desvio Padrão (DP) dos domínios do WHOQOL-BREF de acordo com o Grupo (I e II) e no geral, na amostra de
indivíduos expostos ao césio-137. Goiânia, Goiás, Brasil, 2011. Resultados Domínios WHOQOL-BREF
n
Média
DP
Mínimo *
Máximo **
Valor de p
Físico
Grupo I
33
53,68
16,06
17,86
82,14
Grupo II
29
55,91
17,45
25,00
92,86
Total
62
54,72
16,62
17,86
92,86
0,602
Psicológico
Grupo I
33
50,88
17,61
12,50
87,50
Grupo II
29
55,46
18,40
25,00
87,50
Total
62
53,02
17,98
12,50
87,50
0,322
Relações sociais
Grupo I
33
49,75
21,60
0,00
83,33
Grupo II
29
58,05
17,74
16,67
83,33
Total
62
53,63
20,17
0,00
83,33
0,106
Meio ambiente
Grupo I
33
61,27
19,75
25,00
93,75
Grupo II
29
58,30
21,33
15,63
100,00
Total
62
59,88
20,39
15,63
100,00
0,571
Teste t de Student. * Mínimo: 0;
** Máximo: 100. Média e Desvio Padrão (DP) dos domínios do WHOQOL-BREF de acordo com o Grupo (I e II) e no geral, na amostra de
indivíduos expostos ao césio-137. Goiânia, Goiás, Brasil, 2011. Esses resultados são consistentes com os
apresentados por Miranda et al. 19 em um estudo
constituído por um follow up da população de
radioacidentados de Goiânia em dois períodos (3
e 15 anos após o evento), cujo objetivo foi anali-
sar a percepção das pessoas diretamente atingi-
das sobre os efeitos do acidente. Os resultados
apontaram que a percepção de problemas psico-
lógicos e de saúde geral permaneceu elevada nos
dois períodos estudados, tanto para os indivídu-
os do Grupo I como para os do Grupo II. domínio relações sociais 46,67/55,56; domínio
meio ambiente 59,79/51,88). A Tabela 4 mostra a correlação entre os domí-
nios do WHOQOL-BREF. Verifica-se que todos os
domínios foram significativos e apresentaram re-
lação moderada. Os maiores coeficientes foram
encontrados nos domínios psicológico e físico. O
domínio das relações sociais apresentou os coe-
ficientes de correlação mais baixos. Cad. Saúde Pública, Rio de Janeiro, 29(7):1301-1310, jul, 2013 Discussão Os resultados encontrados no presente tra-
balho são semelhantes aos verificados na po-
pulação envolvida no acidente de Chernobyl,
Ucrânia. Havenaar et al. 9, em um levantamen-
to epidemiológico realizado após o desastre de
Chernobyl, revelaram que os envolvidos tive-
ram substancialmente maiores taxas de queixas
de saúde e sofrimento psíquico do que os habi-
tantes de uma região não afetada. Além disso,
Rahu 20 ressaltou que o desastre de Chernobyl
teve um impacto de longo prazo sobre o bem-
estar físico e psicológico, doenças e na qualida-
de de vida das pessoas envolvidas. Os resultados apresentados pelos indivíduos ex-
postos ao césio-137 no questionário WHOQOL-
BREF, considerando uma escala de 0 a 100, apre-
sentaram, de forma crescente, para os domínios
psicológico, relações sociais, físico e meio am-
biente valores entre 53,02 e 59,88. Sendo que,
as pessoas com mais de 41 anos, mulheres e os
sujeitos do Grupo I, indicaram menores escores,
principalmente quanto à avaliação geral da pró-
pria saúde e nos domínios psicológico e relações
sociais. Fuini SC et al. 1306 Tabela 3 Tabela 3 Comparação dos domínios do WHOQOL-BREF em relação ao sexo e faixa etária na amostra de indivíduos expostos ao
césio-137. Goiânia, Goiás, Brasil, 2011. Domínios WHOQOL-BREF
n
Média
Valor de p
Sexo
Físico
Masculino
32
56,03
Feminino
30
53,33
Total
62
54,72
0,528
Psicológico
Masculino
32
54,17
Feminino
30
51,81
Total
62
53,02
0,610
Relações sociais
Masculino
32
55,99
Feminino
30
51,11
Total
62
53,63
0,345
Meio ambiente
Masculino
32
63,67
Feminino
30
55,84
Total
62
59,88
0,131
Idade (anos)
Físico
18-40
32
59,68
41 ou mais
30
49,77
Total
62
54,72
0,018
Psicológico
18-40
32
59,81
41 ou mais
30
46,24
Total
62
53,02
0,002
Relações sociais
18-40
32
61,56
41 ou mais
30
45,70
Total
62
53,63
0,001
Meio ambiente
18-40
32
63,81
41 ou mais
30
55,95
Total
62
59,88
0,130
Teste t Student. Comparação dos domínios do WHOQOL-BREF em relação ao sexo e faixa etária na amostra de indivíduos expostos ao
césio-137. Goiânia, Goiás, Brasil, 2011. das pessoas, assim como ao estigma e à discrimi-
nação. Nesse contexto, o acidente ocorrido em
Goiânia constituiu-se em um evento bastante
negativo, gerando, enquanto evento social, as-
pectos relacionados à segregação, desorgani-
zação e migração. Ao mesmo tempo, enquanto
evento psicológico, o choque, o estresse, o medo
e o trauma 13,15,19. Cad. Saúde Pública, Rio de Janeiro, 29(7):1301-1310, jul, 2013 Discussão Especificamente quanto aos menores esco-
res apresentados neste estudo referentes aos do-
mínios psicológicos e relações sociais, sabe-se
que nos últimos anos os acidentes com subs-
tâncias radioativas trouxeram com eles graves
efeitos psicossociais, que em combinação com
outros fatores produziram sérios impactos nas
populações atingidas. Essas consequências são
bastante complexas pelo fato de não estarem re-
lacionadas somente com o acidente em si, mas
aos efeitos de deslocamento e remanejamento Com relação ao aspecto físico, estudos ante-
riores a respeito da população de radioaciden-
tados pelo césio-137 em Goiânia demonstraram QUALIDADE DE VIDA DOS INDIVÍDUOS EXPOSTOS AO CÉSIO-137
1307 Tabela 4 Correlação entre os domínios do WHOQOL-BREF na amostra de indivíduos expostos ao césio-137. Goiânia, Goiás, Brasil,
2011. Domínios WHOQOL-BREF
Psicológico
Relações Sociais
Meio Ambiente
Físico
r
0,738
0,712
0,736
p
< 0,001
< 0,001
< 0,001
Psicológico
r
-
0,737
0,638
p
-
< 0,001
< 0,001
Relações Sociais
r
-
-
0,617
p
-
-
< 0,001
Teste Pearson. p: diferem signifi cativamente para p < 0,001; r: relação. Correlação entre os domínios do WHOQOL-BREF na amostra de indivíduos expostos ao césio-137. Goiânia, Goiás, Brasil, Correlação entre os domínios do WHOQOL-BREF na amostra de indivíduos expostos ao césio-137. Goiânia, Goiás, Brasil,
2011. formas mais intrapessoais, como a reflexão e o
isolamento social 21,22. que o impacto na saúde física desta população
não foi significativo 3,5. É provável que os impac-
tos negativos da exposição à radiação foram evi-
denciados na percepção dos indivíduos quan-
to à sua qualidade de vida e permaneceram ao
longo do tempo. Segundo Miranda et al. 19, os
problemas de saúde física relatados pelos radio-
acidentados de Goiânia não se referem direta-
mente aos efeitos esperados em acidentes dessa
natureza, mas atuam de forma indireta entre o
aspecto orgânico e o mental, por meio de sinto-
mas somáticos. No entanto, apesar do instrumento
WHOQOL-BREF requerer uma autoavaliação
considerando as duas últimas semanas da da-
ta de preenchimento do questionário, deve-se
considerar o fato de que na época do acidente ra-
dioativo de Goiânia alguns eram crianças e ado-
lescentes. Nesse aspecto, Compas et al. Cad. Saúde Pública, Rio de Janeiro, 29(7):1301-1310, jul, 2013 Discussão 1308 bastante complexas pelo fato de não estarem re-
lacionadas somente com o acidente em si, mas
por preocupações com doenças físicas em longo
prazo e pelo impacto decorrente de deslocamen-
tos, segregação, estigma e discriminação 8,19,29. que são mães e por isto preocupam-se com os fi-
lhos 8,25. A vertente de gênero tem sido identifica-
da como uma questão bastante relacionada com
a avaliação da qualidade de vida; especialmente
nos casos de eventos traumáticos pode-se veri-
ficar que as mulheres geralmente desenvolvem
doenças e apresentam prejuízo na saúde mental
mais do que os homens 9,25. Nessa perspectiva, uma das principais carac-
terísticas do acidente com o césio-137 é que ele
não fazia parte da experiência habitual e carre-
gava uma enorme capacidade para gerar pressão
física, econômica, social e psicológica sobre os
envolvidos. Sendo assim, os resultados demons-
traram que as questões preocupantes no início
do acidente ainda perduram no presente e no
futuro das pessoas afetadas. Essa constatação
corrobora com os resultados de outros estudos,
confirmando que pelo fato de se tratar de um
acidente radioativo, a condição de vítimas per-
manece, tornando-se crônica 19,20,29. Em outro estudo de avaliação da qualidade
de vida com pessoas expostas a doses baixas e
prolongadas pela radiação ionizante por Cobalto
60 em uma região da cidade de Taiwan, Repúbli-
ca da China, onde foi utilizado o WHOQOL-BREF,
observou-se que os indivíduos mais jovens, as
mulheres e as pessoas com maior tempo de ex-
posição apresentaram os menores escores em to-
dos os domínios, com exceção do domínio meio
ambiente 26. Esses resultados são semelhantes
aos do presente estudo, excetuando-se a variável
idade em que as pessoas mais velhas tiveram os
escores mais baixos. Talvez isso se deva ao fato
de que o acidente de Goiânia foi ocasionado por
características bem diferentes em comparação
ao de Taiwan, pois aconteceu de forma mais trau-
mática, repentina e impactante e, por sua vez, foi
mais presenciado e experimentado pelas pessoas
que hoje estão com mais de 41 anos. Dentre as limitações deste estudo pode-se
destacar a preferência de alguns radioaciden-
tados em se manter na condição de “vítimas”, o
que pode ter contribuído para uma tendência em
uma pior avaliação da qualidade de vida. Discussão 23 apon-
tam a necessidade de alterações para aplicar as
noções de estresse e coping para as crianças, já
que precisam ser consideradas a dependência
da criança em relação ao adulto e as caracterís-
ticas básicas do seu desenvolvimento cognitivo
e social, pois a idade e as pessoas envolvidas na
situação estressante são fatores determinantes
na escolha da estratégia a ser utilizada. Geral-
mente, as estratégias utilizadas por crianças e
adolescentes estão mais relacionadas às respos-
tas correspondentes às demandas situacionais,
procurando uma adaptação ao ambiente social,
indicando que coping é um processo mais si-
tuacional do que disposicional, apesar de que
não existem respostas adaptativas universais,
adequadas para todos os indivíduos, em todas
as situações e em todo o tempo 24. Ainda no presente estudo, observou-se que
os maiores escores concentraram-se no domínio
meio ambiente, talvez porque os aspectos que
influenciam uma maior satisfação podem estar
relacionados com a salubridade no ambiente fí-
sico (clima, ruído, poluição), o que coincide com
a maioria das respostas dos sujeitos. Sabe-se que as estratégias de coping (estilo
de enfrentamento) são definidas como um con-
junto de esforços, cognitivos e comportamentais,
utilizado pelos indivíduos com o objetivo de lidar
com demandas específicas, internas ou exter-
nas, que surgem em situações de estresse e são
avaliadas como sobrecarregando ou excedendo
seus recursos pessoais. Nesta pesquisa a variável
idade apresentou um considerável índice de sig-
nificância, mostrando que as pessoas com mais
de 41 anos autoavaliaram sua qualidade de vi-
da com escores menores do que as mais jovens. Alguns estudos têm sugerido que os padrões de
coping estão associados à idade, ou seja, pessoas
mais jovens usam formas de coping mais intera-
tivas, tais como a expressão de emoções e a busca
de informações, enquanto as mais velhas usam Considerando os aspectos relacionados ao
gênero, foi observado neste estudo que a percep-
ção da qualidade de vida para as mulheres foi
inferior à dos homens em todas as faixas etárias,
nos dois grupos e em todos os domínios, exce-
tuando-se o domínio físico, porém não signifi-
cativas. Pesquisas têm revelado que as mulheres
tendem ao sofrimento psíquico mais do que os
homens em situações relacionadas com a saúde
e questões sociais, com destaque para aquelas Fuini SC et al. Discussão Isso se
justifica, tendo em consideração que os radio-
acidentados pelo césio-137 de Goiânia sofrem
as consequências da violência provocada pelo
imprevisível e inesperado, bem como os efeitos
prolongados resultantes de acidentes com subs-
tâncias radioativas. Geralmente esse efeito se ca-
racteriza pela ameaça crônica e exacerbada por
acidentes dessa natureza, gerando necessidades
de suporte em áreas de subsistência de suas vi-
das, tais como: econômica, social, saúde física
e mental 17. Sendo assim, manter a condição de
“vítima” pode significar a garantia da sociedade
e do governo em gerar respostas a essas neces-
sidades para que as dificuldades sentidas sejam
superadas e/ou minimizadas. Como foi demonstrado, a avaliação dos es-
cores médios dos domínios de qualidade de vi-
da mensurados nesta pesquisa estiveram entre
49,75 e 61,27. Apesar de não terem sido encon-
trados estudos em populações semelhantes re-
alizados no Brasil, torna-se interessante compa-
rar com trabalhos que utilizaram o instrumento
WHOQOL-BREF em populações diversas. Entre
eles destaca-se um estudo sobre a Qualidade de
Vida e Trauma Psíquico em Vítimas da Violência
por Arma de Fogo, cujos resultados demonstra-
ram médias baixas que variaram entre 44,71 e
52,84, em que o escore médio do domínio meio
ambiente foi menor que o dos domínios físico,
relações sociais e psicológico 27. Outra limitação foi a dificuldade de conven-
cimento dos radioacidentados, pois, a maioria
manifestou revolta com os serviços públicos
oferecidos e que não mais serviria de “cobaias”
de pesquisa, se negando de forma agressiva a
participar do presente estudo. Nesse caso, foi ne-
cessário uma articulação com a Associação das
Vítimas do Césio-137 (AVCESIO) para ajudar na
adesão dos mesmos, até alcançar o quantitativo
mínimo do cálculo amostral. Outro trabalho que avaliou a Percepção de
Qualidade de Vida de Pessoas com Lesão Medu-
lar Traumática, apresentou escores entre 49,61
e 55,81, sendo o menor escore representado no
domínio meio ambiente e o maior no domínio
relações sociais 28. Percebe-se nos estudos citados que os me-
nores escores concentraram no domínio meio
ambiente, contrariando os resultados da presen-
te pesquisa, cujo escore mais alto foi nesse domí-
nio. Cad. Saúde Pública, Rio de Janeiro, 29(7):1301-1310, jul, 2013 Discussão Talvez isso se deva aos efeitos comumente
chamados “indiretos”, os quais podem ser cau-
sados por desastres sem ter uma relação direta
com o evento, como é o caso dos impactos psi-
cológicos e sociais, sobretudo em eventos com
substâncias radioativas, cujas consequências são Concluindo, os resultados deste estudo mos-
traram que os indivíduos expostos à radiação pe-
lo césio-137 sofrem considerável impacto na sua
qualidade de vida. Observou-se a persistência de
problemas psicossociais, na medida em que, os
menores escores foram associados ao domínio
psicológico, seguido de relações sociais, sobretu-
do para os indivíduos do Grupo I, as mulheres e
pessoas com mais de 41 anos. Ou seja, enquanto
evento psicossocial, esse acidente pode ter pro- QUALIDADE DE VIDA DOS INDIVÍDUOS EXPOSTOS AO CÉSIO-137
1309 duzido trauma, medo, estresse, choque, entre
outras possíveis reações, até os dias de hoje. Os
resultados apontados mostraram que não houve
associação ente as variáveis sociodemográficas
(religião e escolaridade) com os domínios do
WHOQOL-BREF. condizentes e influenciar as decisões e condutas
terapêuticas, voltadas a esse segmento da po-
pulação, incluindo o fortalecimento da atenção
psicossocial no intuito de reduzir o sofrimento
das mesmas. Faz-se necessário, também, a realização de
novas pesquisas, principalmente porque são
muito escassos os estudos que tratam das conse-
quências indiretas em populações de vítimas de
acidentes com substâncias radioativas. Enfim, há
muito o que se fazer para responder efetivamen-
te às demandas das pessoas que sofrem com os
efeitos da radiação ou, pelo menos, minimizar
a potencialidade do desenvolvimento de efeitos
futuros. Contudo, mesmo com a manutenção regular
do monitoramento dos indivíduos dos Grupos I
e II em Goiânia, verifica-se ainda a existência de
incertezas quanto aos efeitos do acidente, pois
essas dúvidas desempenham um papel negativo
na saúde e no bem-estar dessas pessoas. Espera-
se com este trabalho que a compreensão sobre
a qualidade de vida dos radioacidentados pos-
sa subsidiar a elaboração de políticas públicas Resumen Estudio transversal observacional realizado en Goiâ-
nia, Goiás, Brasil, con un 56% de 111 individuos para
evaluar la calidad de vida de las personas expuestas
al cesio-137 y su asociación con factores sociodemo-
gráficos. Se utilizó el WHOQOL-BREF y el Sistema de
Monitoreo de Radio accidentados. Los participantes
fueron divididos: Grupo I - Dosimetría radiodermitis
y/o citogenética mayor de 20 rads (n = 33) y Grupo II -
dosimetría citogenética ≤ 20 rad (n = 29), de un total de
62 sujetos. Entre los dominios del WHOQOL-BREF, el
medio ambiente tenía los puntos más altos de prome-
dio (59,88; SD = 20,39) y el promedio más bajo fue el
psicológico (53,02; SD = 17,98). Las asociaciones entre
las relaciones físicas, psicológicas y sociales fueron sig-
nificativas para la variable edad. No hubo diferencias
entre los grupos y entre los factores sociodemográficos
y de calidad de vida. Los radioaccidentados sufren un
impacto considerable en la calidad de vida, con la per-
sistencia de los problemas psicosociales, especialmente
para aquellos con más de 41 años. S. C. Fuini contribuiu na concepção e design, aquisição
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https://openalex.org/W2028130894
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https://curis.ku.dk/ws/files/127547653/Control_region_sequences_indicate_that_multiple_externae_represent_multiple_infections_by_Sacculina_carcini.pdf
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English
| null |
Control region sequences indicate that multiple externae represent multiple infections by Sacculina carcini (Cirripedia: Rhizocephala)
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Ecology and evolution
| 2,014
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cc-by
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university of copenhagen university of copenhagen
Control region sequences indicate that multiple externae represent multiple infections
by Sacculina carcini (Cirripedia: Rhizocephala) university of copenhagen Control region sequences indicate that multiple externae represent multiple infections
by Sacculina carcini (Cirripedia: Rhizocephala) Citation for published version (APA):
Rees, D., & Glenner, H. (2014). Control region sequences indicate that multiple externae represent multiple
infections by Sacculina carcini (Cirripedia: Rhizocephala). Ecology and Evolution, 4(16), 3290-3297.
https://doi.org/10.1002/ece3.1177 Download date: 24. Oct. 2024 ª 2014 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use,
distribution and reproduction in any medium, provided the original work is properly cited. Control region sequences indicate that multiple externae represent multiple infections
by Sacculina carcini (Cirripedia: Rhizocephala) Rees, David; Glenner, Henrik Document version
Publisher's PDF, also known as Version of record Citation for published version (APA):
Rees, D., & Glenner, H. (2014). Control region sequences indicate that multiple externae represent multiple
infections by Sacculina carcini (Cirripedia: Rhizocephala). Ecology and Evolution, 4(16), 3290-3297. https://doi.org/10.1002/ece3.1177 Download date: 24. Oct. 2024 Funding Information Funding Information
The study was finaced by Carlsberg
Foundation (grant no. 2008-01-0491). Received: 17 January 2014; Revised: 4 July
2014; Accepted: 5 July 2014 Ecology and Evolution 2014; 4(16): 3290–
3297 Ecology and Evolution 2014; 4(16): 3290–
3297 doi: 10.1002/ece3.1177 2001; for further details of the development of Sacculina
carcini and its morphological and behavioral impacts on
Carcinus maenas see, e.g., Kristensen et al. 2012; Høeg
1995; Høeg and Lutzen 1995). Control region sequences indicate that multiple externae
represent multiple infections by Sacculina carcini
(Cirripedia: Rhizocephala) 1Department of Biology, Marine Biodiversity, University of Bergen, Thormøhlensgate 53a, Box 7800, Bergen N-5020, Norway
2Center for Macroecology, Evolution and Climate, University of Copenhagen, Universitetsparken 15, 2100 Copenhagen, Denmark Correspondence
Henrik Glenner Correspondence
Henrik Glenner
Department of Biology, Marine Biodiversity,
University of Bergen, Thormøhlensgate 53a,
Box 7800, Bergen N-5020, Norway. Tel: +47 45 86 46 47; Fax: +47 55 58 44 00;
E-mail: henrik.glenner@bio.uib.no Funding Information
The study was finaced by Carlsberg
Foundation (grant no. 2008-01-0491). Keywords y
Carcinus maenas, control region,
mitochondrial DNA, parasitism, population
genetics, rhizocephala. Carcinus maenas, control region,
mitochondrial DNA, parasitism, population
genetics, rhizocephala. The rhizocephalan barnacle, Sacculina carcini, is a common parasite of the
European shore crab, Carcinus maenas, in which it causes significant detrimen-
tal physical and behavioral modifications. In the vast majority of cases, the
external portion of the parasite is present in the form of a single sac-like exter-
na; in rare cases, double or even triple externae may occur on the same individ-
ual host. Here, we use a highly variable DNA marker, the mitochondrial
control region (CR), to investigate whether multiple externae in S. carcini rep-
resent infection by multiple parasites or asexual cloning developed by a single
parasite individual. Sequences for multiple externae from C. maenas hosts from
the Danish inlet, Limfjorden, and from the mud flates at Roscoff, France, were
compared. In almost all cases, double or triple externae from an individual host
yielded different haplotypes. In the few cases where identical haplotypes were
identified from externae on a multiple-infected host, this always represented the
most commonly found haplotype in the population. This indicates that in Sac-
culina carcini, the presence of multiple externae on a single host reflects infec-
tion by different individual parasites. A haplotype network of CR sequences
also suggests a degree of geographical partitioning, with no shared haplotypes
between the Limfjorden and Roscoff. Our data represent the first complete CR
sequences for a rhizocephalan, and a unique gene order was also revealed. Although the utility of CR sequences for population-level work must be investi-
gated further, the CR has proved a simple to use and highly variable marker
for studies of S. carcini and can easily be applied to a variety of studies in this
important parasite. Introduction The rhizocephalan barnacle, Sacculina carcini, is a para-
sitic castrator of the European shore crab, Carcinus mae-
nas. The adult parasite consists of an external sac-like
structure located at the ventral side of the host abdomen
at a position where adult female crabs carry their eggs
(see Fig. 1A, B). This sac, the externa, contains the repro-
ductive organs of the parasite and communicates with an
internal root-like structure, the interna, via a stalk that
penetrates the abdominal cuticle of the host. The interna
is an extensive structure that infiltrates most of the larger
blood sinuses of the crab and serves as a trophic organ
extracting nutrients from the crab hemolymph (Glenner Occasionally, more than one externa occurs on the
same crab (Rainbow et al. 1979). This can be interpreted
as either a double infection, where two individual parasite
specimens are competing for the same food resource
entailing that each externa is feeding from two indepen-
dent internas (see Fig. 1C). Alternatively, the phenome-
non can be regarded as asexual budding of two externae
connected to, and feeding from, the same interna. Which
of the two alternatives is in play is of crucial importance
in order to understand the biology of the parasite and the 3290 Multiple Infections of C. maenas by S. carcini D. Rees & H. Glenner (A)
(B)
(C)
Figure 1. Comparison of a gravid female crab carrying its egg m
under the abdomen and mimicking rhizocephalan parasites situa
at the same position. (A) Gravid female Carcinus maenas; (B) fem
C. maenas
with
a
single
mature
S. carcini
parasite;
(C)
m
C. maenas with three recently emerged Sacculina carcini extern
The abdomen has been considerable broadened (feminized) as ef
of the presence of the parasites. (A) collection of an unprecedented number of crabs with mul-
tiple externae. This allowed a comprehensive examination
of whether the observed multiple externae were caused by a
single infection, multiple infections, or a combination of
both alternatives. Multiple externae are documented as
being developed by clonal, or asexual reproduction, in one
sacculinid genus, Polyascus, where multiple externae on the
same host is the rule (Glenner et al. 2003). Introduction In other sacculi-
nid genera, the presence of multiple externae on the same
host is rare, but notable exceptions do exist, as in Heteros-
accus dolfusi, which parasitizes the swimming crab, Charyb-
dis longicollis, a Lessepsian invader of the Mediterranean
sea (Galil and Lutzen 1995). In this parasite–host system,
parasitized crabs predominantly have more than one exter-
na and 3–5 per host is common. With an extraordinary
prevalence of 60–90%, the chances of a cypris larva finding
a host already occupied by another parasites are much
higher than finding an un-parasitized host, and it is there-
fore believed that the intensity observed in most cases is
due to multiple infections – not asexual reproduction
(Glenner and Hebsgaard 2006). However, since morpho-
logical inspections of the internal parasite is unable to dis-
tinguish the presence of more than one externa, the
question of asexual budding or multiple infection can only
be addressed by developing, and employing, high-resolu-
tion molecular markers. (A) (B) (B) (C) (C) (C) Mitochondrial (mt) DNA is a popular marker for phylo-
genetic, phylogeographic, and population genetic studies at
a wide range of taxonomic and geographical scales. Notable
benefits of mtDNA markers include relative ease of amplifi-
cation, presence of variable regions flanked by conserved
stretches suitable for primer design, and predominantly
nonrecombinant inheritance. The mtDNA control region
(CR) is a noncoding portion of the mt genome, responsible
for replication and transcription, and is usually the fastest
evolving mtDNA region in invertebrates (Avise 2000; Bil-
lington 2003). The CR exhibits an evolutionary rate three-
fold to fivefold higher than other regions of the mt genome
(Brown et al. 1993), making it a popular marker for genetic
studies involving a wide range of taxa. This marker has
been widely utilized in vertebrates and insects but also been
applied to studies of genetic variability and population
structure in a number of commercially important marine
crustaceans, for example, the swimming crab Portunus tri-
tuberculatus (Guo et al. 2012), the scalloped lobster Panuli-
rus homarus (Farhadi et al. 2013), and the mantis shrimp
Oratosquilla oratoria (Lui et al. 2010). Figure 1. Comparison of a gravid female crab carrying its egg mass
under the abdomen and mimicking rhizocephalan parasites situated
at the same position. (A) Gravid female Carcinus maenas; (B) female
C. maenas
with
a
single
mature
S. carcini
parasite;
(C)
male
C. maenas with three recently emerged Sacculina carcini externae. ª 2014 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. Introduction The
three most common haplotypes are also indicated, as in Fig. 2, by a,
b, and c. 2007; Tsang et al. 2007; Dawson et al. 2010). No studies
involving Rhizocephala, and no complete control region
sequences for this group, have been published to date. The
high variability associated with the CR made this an ideal
candidate for our work involving Sacculina carcini, both for
assessing population – and individual-level variation. An
initial test of control region sequence data for Sacculina
carcini, aimed at assessing suitability for population studies,
indicated surprisingly high levels of variability. This pre-
sented the opportunity to investigate another aspect of
S. carcini biology: whether (rare) double and triple infec-
tions (indicated by multiple externae) are the result of
emergence by multiple individuals of S. carcini, or whether
multiple externae can emerge from a single parasite. In this
study, we present new primers and a simple method for tar-
geting the CR in Sacculina carcini and examine DNA
sequence variation in multiple externa from two disjunct
populations. Externa
Host No. Introduction Location
Haplotype
GenBank accession
SA01
1
Roscoff
R01
KF649275
SA02
2
Roscoff
R02
KF649276
SA03
3
Roscoff
R03
KF649277
SA04
4
Limfjorden
L08 b
KF649263
SA05
5
Limfjorden
L17 c
KF649272
SA06
6
Limfjorden
L18
KF649273
TC01A
7
Limfjorden
L17 c
TC01B
Limfjorden
L09
KF649264
TC02A
8
Limfjorden
L16
KF649271
TC02B
Limfjorden
L11 a
KF649266
TC03A
9
Limfjorden
L11 a
TC03B
Limfjorden
L13
KF649268
TC04A
10
Limfjorden
L06
KF649261
TC04B
Limfjorden
L01
KF649256
TC05A
11
Limfjorden
L19
KF649274
TC05B
Limfjorden
L08 b
TC06A
12
Limfjorden
L11 a
TC06B
Limfjorden
L08 b
502A
13
Roscoff
R04
KF649278
502B
Roscoff
R05
KF649279
503A
14
Roscoff
R06
KF649280
503B
Roscoff
R07
KF649281
504A
15
Roscoff
R08
KF649282
504B
Roscoff
R09
KF649283
512A
16
Roscoff
R10
KF649284
512B
Roscoff
R11
KF649285
513A
17
Roscoff
R12
KF649286
513B
Roscoff
R13
KF649287
SAC11A
18
Limfjorden
L05
KF649260
SAC12B
Limfjorden
L17 c
SAC13C
Limfjorden
L11 a
SAC14A
19
Limfjorden
L14
KF649269
SAC15B
Limfjorden
L11 a
SAC16C
Limfjorden
L03
KF649258
SAC17A
20
Limfjorden
L17 c
SAC18B
Limfjorden
L04
KF649259
SAC19C
Limfjorden
L15
KF649270
SAC20A
21
Limfjorden
L01
SAC21B
Limfjorden
L08 b
SAC22A
22
Limfjorden
L17 c
SAC23B
Limfjorden
L08 b
SAC24A
23
Limfjorden
L02
KF649257
SAC25B
Limfjorden
L08 b
SAC26A
24
Limfjorden
L10
KF649265
SAC27B
Limfjorden
L08 b
SAC28A
25
Limfjorden
L17 c
SAC29B
Limfjorden
L17 c
SAC30A
26
Limfjorden
L07
KF649262
SAC31B
Limfjorden
L09
SAC32A
27
Limfjorden
L17 c
SAC33B
Limfjorden
L08 b Sample collection and DNA extraction Sacculina carcini externa were collected from the shore
crab/green crab Carcinus maenas from Limfjorden, Den-
mark, and from near Roscoff, north-west France, and pre-
served in 96% ethanol prior to DNA extraction. Genomic
DNA extraction was performed for a total of 57 externa
comprising six single infections, 21 double, and three triple
infections (double and triple infections being characterized
by two or three externa from a single host specimen. Five
double and three single externae were collected from
C. maenas near Roscoff and the remainder (three triple, 16
double, and three single externae) came from Limfjorden
(see the data accessibility section, Table 1 for sample
details). Approximately 1 mm3 of mantle tissue was care-
fully excised from individual Sacculina carcini externae for
DNA extraction. Alternatively, eggs or nauplii larvae from
the mantle cavity of the female externae were DNA
extracted. Tissue from the receptacle region were carefully
omitted to avoid DNA contamination from a dwarf male. All extractions were carried out using the Qiagen DNeasy
Blood and Tissue kit (QIAGEN Inc., Valencia, CA, USA) or
a GeneMole extraction robot, following the manufacturers’
standard protocols. Multiple parallel extractions were also
performed for individual externa, along with PCR amplifi-
cation and sequencing, to confirm reproducibility and
specificity of DNA sequence data. Introduction The abdomen has been considerable broadened (feminized) as effect
of the presence of the parasites. Figure 1. Comparison of a gravid female crab carrying its egg mass
under the abdomen and mimicking rhizocephalan parasites situated
at the same position. (A) Gravid female Carcinus maenas; (B) female
C. maenas
with
a
single
mature
S. carcini
parasite;
(C)
male
C. maenas with three recently emerged Sacculina carcini externae. The abdomen has been considerable broadened (feminized) as effect
of the presence of the parasites. host–parasite interaction. Due to the delicacy of the
interna, which consists of a dense network of extremely fine
root extensions, it is impossible to distinguish morphologi-
cally whether two externae are connected to a common
interna (asexual externa budding), or to separate internas
(independent specimens). As part of a 4-years, large-scale
study on the population dynamics and biology of the Euro-
pean shore crab, Carcinus maenas, in the Danish inlet Limf-
jorden (see www.Carcinus.com), specimens with the rare
occurrence of multiple externae were collected. The large
quantity of crabs examined during the project allowed host–parasite interaction. Due to the delicacy of the
interna, which consists of a dense network of extremely fine
root extensions, it is impossible to distinguish morphologi-
cally whether two externae are connected to a common
interna (asexual externa budding), or to separate internas
(independent specimens). As part of a 4-years, large-scale
study on the population dynamics and biology of the Euro-
pean shore crab, Carcinus maenas, in the Danish inlet Limf-
jorden (see www.Carcinus.com), specimens with the rare
occurrence of multiple externae were collected. The large
quantity of crabs examined during the project allowed Use of CR sequences in published studies of Cirripedia is
few, and its use seems to be limited mainly to population
and taxonomic studies involving a small number of Tho-
racican barnacles; Chthamalus stellatus (Sasson et al. 2012),
Semibalanus balanoides (Flight et al. 2012), Catomerus poly-
merus (York et al. 2008), and Tetraclita spp. (Chan et al. 3291 ª 2014 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. Multiple Infections of C. maenas by S. carcini D. Rees & H. Glenner D. Rees & H. Glenner Table 1. Sacculina carcini host, location, and haplotype information. Individual hosts are numbered and double or triple externa denoted
by A/B/C. Associated haplotypes indicated for each externa and Gen-
Bank accession numbers are listed for each unique haplotype. ª 2014 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. Sequence characteristics The final aligned dataset, comprising DNA sequence data
for 57 individual externa, was 812 base-pair (bp) long. The
presence of insertions and deletions (indels) resulted in
unaligned individual sequence lengths ranging from 795 to
803 bp. BLAST searches confirmed partial matches to
S. carcini 12S and COI genes (http://blast.ncbi.nlm.nih. gov/Blast.cgi) and subsequent alignment of the sequenced
region with the complete mitochondrial genome sequence
for S. carcini (unpublished data) confirmed the relative
positions of the flanking regions and the presence of the
putative control region. Annotation of the sequenced
region using the MITOS package indicated that the
sequenced region comprised approximately 175 bp of 12S
and 35 bp of COI, flanking a 600-bp segment of noncoding
A+T-rich sequence (84% A+T). Analyses also indicated that
the noncoding region between the 12S and COI genes was
split by one 56-bp tRNA (tRNA Cysteine; gca), resulting in
a 145 bp of noncoding sequence adjacent to 12S and
400 bp adjacent to COI. The overall length of the amplified
fragment was significantly shorter than expected (see
below) but with primers located in conserved, coding
flanking regions, we minimized potential problems with
amplification (or nondetection) of nuclear mitochondrial
copies (Numts) or CR paralogs (see, e.g., Walther et al. 2011). Comparison of our coding sequence data indicates
complete congruence with those held in GenBank, as well
as complete agreement for our total sequence reads with an
independently generated full mitochondrial S. carcini gen-
ome sequence (unpublished data). Analysis of this full
mtDNA genome sequence identified all 13 protein coding
genes, two rRNA genes, and 22 tRNA genes. sequences; the forward primer was designed from the single
12S rRNA sequence available in GenBank (AY520690), and
the reverse primer was designed from an alignment of
multiple GenBank cytochrome c oxidase 1 gene (COI)
sequences. Primer design was performed using the Pri-
mer3Plus web interface (Untergasser et al. (2007); http://
www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus. cgi) and the following primers were selected: 12SF_Sacc
(50-TGAATTCAGATTAGGTGCAAAGA-30)
and
COIR_
Sacc (50-CCCCCACTAAACCTGATCATA-30). PCR ampli-
fications were carried out in 25 lL volumes containing 19
PCR buffer, 1.2-lL 2-mmol/L dNTPs, 0.4 lmol/L of each
primer, 0.75 units of Takara polymerase, and 1 lL of tem-
plate (and ddH2O up to 25 lL). Primer design and PCR Specific polymerase chain reaction (PCR) primers were
designed from flanking regions using published S. carcini ª 2014 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. 3292 Multiple Infections of C. maenas by S. carcini D. Rees & H. Glenner Table 1. Continued. Externa
Host No. Location
Haplotype
GenBank accession
SAC34A
28
Limfjorden
L11 a
SAC35B
Limfjorden
L17 c
SAC36A
29
Limfjorden
L12
KF649267
SAC37B
Limfjorden
L17 c
SAC38A
30
Limfjorden
L17 c
SAC39B
Limfjorden
L17 c Table 1. Continued. genome for S. carcini (unpubl. data Podsiadlowski , L.,
Hecht, J., Rees D., Noever, D., Glenner, H.) using the MI-
TOS web server (Bernt et al. 2012; http://mitos.bioinf. uni-leipzig.de/). Sequence characteristics PCRs were performed on
a Bio-Rad C1000 Thermal with the following cycling pro-
file: initial denaturation at 94°C for 5 min, then 35 cycles
of 94°C for 30 sec, annealing at 54°C for 30 sec, and exten-
sion at 72°C for 2 min, followed by a final 72°C extension
for 7 min. Amplification products were visualized on 1.5%
agarose gels to confirm fragment size and quality. PCR
purification was carried out by the addition of 1 unit each
of exonuclease I and shrimp alkaline phosphatase (plus
0.9 lL ddH2O) to 8 lL of each reaction, with reactions
subsequently heated to 37°C for 30 min and then 85°C for
15 min. In a small number of cases, PCR products were
gel-purified using the Qiagen MinElute gel extraction kit,
with the target band excised directly from the gel prior to
sequencing. ª 2014 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. Sequencing and data analysis With our primers located in the 12S and CO1 genes,
we had expected to amplify the NADH dehydrogenase
subunit 2 (ND2) gene along with the control region, since
the gene order for all Cirripedia for which data is avail-
able (with one exception, see below), as well as for the
majority of crustaceans, is 12S, CR, ND2, and CO1. This
also applies to ancestral pancrustaceans and arthropods
(Lavrov et al. 2004). However, our sequence data indi-
cated that for Sacculina carcini, ND2 was not present in
this position (and the fragment was therefore ~1 kb
shorter than expected). Both strands of all PCR products were sequenced on an
ABI 3730 capillary sequencer using the BigDye v3.1 cycle
sequencing kit (Applied Biosystems, Inc., Norwalk, CT,
USA) and the same primers as in the initial PCR. Forward
and reverse sequences were aligned and edited in Sequen-
cher v.5.0.1 (Gene Codes) and a contig of all sequences
exported to eBioX v.1.5.1 (www.ebioinformatics.org) for
final alignment using MUSCLE (Edgar 2004). A haplotype
network was subsequently estimated using the TCS pro-
gram (v.1.21; Clement et al. 2000) using the default 95%
connection limit. In addition, annotation of the sequenced
region was accomplished by analyses of the full mitochondrial A survey of full mitochondrial genome sequences held in
GenBank for crustaceans (18 August 2013) indicated that 3293 ª 2014 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. Multiple Infections of C. maenas by S. carcini D. Rees & H. Glenner the majority of taxa present the expected order for genes
flanking the control region (12S, CR, ND2, COI). This gene
order exists in 49 of 78 noncirriped crustaceans in which
the position of the control region has been annotated (an
additional 23 full crustacean mitochondrial genomes have
no control region specified). Different flanking genes are
reported for 27 taxa (including ten species in a single
amphipod genus), but all Cirripedia for which data are
available conform to the majority pattern, with the excep-
tion of the single rhizocephalan barnacle for which a near-
complete mitochondrial genome has been published: Pol-
yascus gregaria (Yan et al. 2012). Although Yan et al. were
unable to present full sequence for the control region in
P. Sequencing and data analysis gregaria due to problematic repetitive elements, the gene
order presented by the authors is consistent with other cir-
ripeds except for the additional presence of ND1 between
the control region and ND2. appears variable in some taxa and if a few cases, two
control regions have been annotated. Position and percent-
age A+T content support the assumption that the region
sequenced for this study is likely to be the control region. However, there is a possibility that Sacculina carcini also
possesses a second control region, since annotation of the
full mitochondrial sequence for S. carcini (unpublished
data) indicates an A+T-rich (85%) 655-bp noncoding
region between the cytochrome b (cob) and 12S genes. Haplotype diversity The initial group of S. carcini externa examined included
three single externa from two locations; Roscoff, north-west
France, and Limfjorden in Denmark. These six individual
externa yielded six haplotypes, with 0.4–0.6% pairwise
divergence among the Roscoff samples (mean = 0.5%) and
0.1–1.1% among those from Limfjorden (mean = 0.8%). Together with sequences from double or triple externa, the
final dataset of 57 externa yielded 32 haplotypes, shown as a
TCS haplotype network in Fig. 2. Representatives of all
haplotypes shown in the TCS network have been deposited As such, the flanking gene order for Sacculina carcini
is, at present, unique among Cirripedia and also Crusta-
cea. Although a predominant flanking gene order was evi-
dent from the survey of crustacean full mitochondrial
genome sequences, the position of the control region L04
L05
L18
L02
L06
L10
L08
L14
L13
L09
L12
L01
L15
L07
L03
L11
L16
L19
L17
R12
R13
R07
R08
R02
R10
R04
R01
R11
R03
R09
R05
R06
α
γ
β
n = 2
n = 2
n = 6
n = 12
n = 8
Figure 2. Statistical parsimony network inferred from mtDNA sequence data, with haplotypes coded “R” for Roscoff or “L” for Limfjorden. Ovals
represent sampled haplotypes, branches connecting haplotypes represent nucleotide substitution steps, and small circles represent missing
haplotypes. The square represents the haplotype (L17/C) inferred by TCS to be ancestral. Oval sizes are relative to haplotype frequency; details of
individuals represented, along with single, double, or triple externa status and GenBank accession numbers, are presented in Table 1. The bold
bar indicates the division between parts of the network corresponding to Limfjorden and Roscoff samples. Figure 2. Statistical parsimony network inferred from mtDNA sequence data, with haplotypes coded “R” for Roscoff or “L” for Limfjorden. Ovals
represent sampled haplotypes, branches connecting haplotypes represent nucleotide substitution steps, and small circles represent missing
haplotypes. The square represents the haplotype (L17/C) inferred by TCS to be ancestral. Oval sizes are relative to haplotype frequency; details of
individuals represented, along with single, double, or triple externa status and GenBank accession numbers, are presented in Table 1. The bold
bar indicates the division between parts of the network corresponding to Limfjorden and Roscoff samples. Figure 2. Statistical parsimony network inferred from mtDNA sequence data, with haplotypes coded “R” for Roscoff or “L” for Limfjorden. ª 2014 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. Life history conclusions extracted from the
study Observations of multiple externae are extraordinarily rare
in crab populations infested by Sacculina carcini. This is
true even in crab populations with high prevalence of the
parasite, as in this study. The dataset is based on the
examination of 24878 crabs collected between May 2011
and April 2012 in the Danish inlet, Limfjorden. On average,
9% of these crabs were visibly infested by Sacculina carcini
due to the presence of externae. Of these 2239 (9%), 40
(1.79%) were double infected, and of those two (5%) were
triple infected (female crabs were slightly but significantly
more parasitized than male crabs). The small number of
multiple-infected crabs is dramatically lower than would be
expected if cypris infection on parasitized and un-parasit-
ized crabs were indiscriminate and random. This is in
accordance with the study of (Rainbow et al. 1979). The absence of shared haplotypes among the initial set
of single externa samples from Roscoff and Limfjorden
populations was also consistent in the full dataset. No
haplotypes were shared by individuals from these two
locations and on examination of the haplotype network
(Fig. 2) suggested that a degree of geographic structuring
was present in the data. All samples (single and double
infections) from Roscoff form one part of the network,
distinct from the Limfjorden samples (Fig. 2). The most
similar haplotypes from the two populations are still sep-
arated by five substitutions and the mean pairwise dis-
tance between externa from Limfjorden and Roscoff is
0.9%. Although preliminary, this is in contrast to the
findings of Sasson et al. (2012) who reported no phyloge-
ographic pattern among CR sequences from populations
of the thoracican barnacle Chthamalus stellatus. Sasson
et al. (2012) also reported a higher proportion of single-
ton haplotypes (68%) among sequenced individuals than
we observed in S. carcini (47%). Considering the rarity of multiple S. carcini externae on
infected hosts, it is worth speculating as to possible mecha-
nisms that might be involved in the observed pattern, that
is, why multiple infections are so rare. The relative rarity
of multiple externa may be linked to a possible preference
of infecting cypris larvae for uninfected crabs, with chemi-
cal cues involved in signaling infection status. In other
barnacles, attraction of conspecifics via chemical and other
cues has been documented (see, e.g., Clare et al. 1994;
Dreanno et al. 2007) but signaling of infection status by
S. carcini remains uninvestigated. Haplotype diversity Ovals
represent sampled haplotypes, branches connecting haplotypes represent nucleotide substitution steps, and small circles represent missing
haplotypes. The square represents the haplotype (L17/C) inferred by TCS to be ancestral. Oval sizes are relative to haplotype frequency; details of
individuals represented, along with single, double, or triple externa status and GenBank accession numbers, are presented in Table 1. The bold
bar indicates the division between parts of the network corresponding to Limfjorden and Roscoff samples. ª 2014 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. ª 2014 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. 3294 Multiple Infections of C. maenas by S. carcini D. Rees & H. Glenner in GenBank under the accession numbers KF649256–
KF649287. All 13 externa from Roscoff and one-third of
those from Limfjorden gave unique haplotypes. Within the
Limfjorden sample, three haplotypes were found to be more
common; one haplotype was shared by six individual exter-
na, one by eight and one by 12 samples (denoted by in the
TCS network (Fig. 2) by A, B, and C, respectively). The
most
common
Limfjorden
haplotype
(C)
contained
sequences from two pairs of externa (i.e., two double infec-
tions with identical haplotypes). Although these could be
viewed as possibly resulting from asexual budding in single
S. carcini individuals, the fact that distinct haplotypes were
found in all other double and triple externa suggest that
these are more likely to be the result of a shared, relatively
common haplotype in the Limfjorden population. Pairwise
divergences among the 57 externa ranged from 0% (the two
pairs of externa sharing the common Limfjorden “C” hap-
lotype) to 1.5%, and overall pairwise divergence was 0.7%. in studies of genetic differentiation at finer scales, as in the
case of multiple externa. Combined with specific primers
and ease of amplification, the control region offers a great
deal of promise as a marker that bridges the gap between
phylogenetics and population genetics and is a welcome
additional tool to studies of S. carcini. ª 2014 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. Conflict of Interest None declared. Flight, P. A., M. A. O’Brien, P. S. Schmidt, and D. M. Rand. 2012. Genetic structure and the North American postglacial
expansion of the barnacle, Semibalanus balanoides. J. Hered. 103:153–165. Life history conclusions extracted from the
study Active avoidance of
infected hosts in favor of searching for an uninfected crab
may be unlikely due to the small size (250 lm) and limited
energy resources (5 days; Glenner et al. 1989; Glenner and
Werner 1998) of cypris larvae. However, if two (or in even
rarer cases, three) cypris larvae successfully infect a new
host at the same time, then this could occasionally lead to
simultaneous establishment and later emergence of multi-
ple externae. Unfortunately,
detailed
geographical
information
regarding specific sites of individual samples was not
available for the material used in this study, but this will
be addressed in forthcoming work. Large-scale sampling
of S. carcini, involving multiple localities including Ro-
scoff and Limfjorden, is underway and material will be
analyzed both with microsatellites and sequencing of the
control region. This will allow us to assess congruence
between the two datasets and to properly determine what
degree of geographic resolution can be achieved from
analyses of control region sequences. Alternatively, rarity of multiple externae might be
explained by the fact that once a virgin externa has emerged
through the abdominal cuticle of the host, host molting is
arrested and competing parasites are unable to emerge
(Høeg 1995; Høeg and Lutzen 1995). The result of this
“first past the post” scenario would be that although multi-
ple cypris larvae might initially infect a host, external emer-
gence by one parasite would prevent others from utilizing
the hosts’ resources for developing their reproductive Our work with the mitochondrial control region in Sac-
culina carcini has shown this A+T-rich region, flanked by
the 12S rRNA and COI genes, to be highly variable. The
level of variability observed makes this marker suitable for
population-level studies and has also demonstrated utility 3295 Multiple Infections of C. maenas by S. carcini D. Rees & H. Glenner apparatus. Again, in rare cases, two or more parasites might
simultaneously emerge, giving rise to multiple externae. single, double (A/B), or triple (A/B/C) externa present on
a single C. maenas host. Haplotype codes are the same as
those used in Fig. 2; GenBank accession numbers are
indicated for each of the 32 unique haplotypes. References The most widely used methods of genotyping compromises
restriction-fragment-length polymorphism (RFLP), ran-
dom-amplified polymorphism detection (RAPD), ampli-
fied-fragment-length polymorphism (AFLP), microsatellite
genotyping, and single-nucleotide polymorphisms (SNP). For nonmodel species, with no previously existing proce-
dure protocols, the time and money spent for developing
and optimizing the methods to a new species are often dis-
proportionally high compared to the outcome. Our study
demonstrates that DNA sequence data from the highly vari-
able mitochondrial control region is an inexpensive, easy,
and robust alternative method to genetically differentiate
specimens of the parasitic barnacles, Sacculina carcini. This
marker also possesses broad utility; as well as testing for the
presence/absence of asexual reproduction in adult parasites,
we have successfully used Sacculina-specific CR amplifica-
tion to screen for endoparasitic internas in potentially
infected hosts without visible externae, and we will be fur-
ther testing the resolution of CR sequences for small-scale
population studies. Ease of use, along with the level of vari-
ation observed in S. carcini, makes the application of this
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antennular secretion of the cyprid of Balanus amphitrite,
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computer program to estimate gene genealogies. Mol. Ecol. 9:1657–1660. The authors wish to thank the Carlsberg Foundation for
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barnacle, Catomerus polymerus (Darwin). Mol. Ecol. 17:1948–1961. Kristensen, T., Nielsen, A. I., Jørgensen , A. I., Mouritsen, K. N., Glenner, H., Christensen, J. T., et al. 2012. The selective
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https://www.frontiersin.org/articles/10.3389/fgene.2023.1137017/pdf
|
English
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Regulatory mechanisms of microRNAs in endocrine disorders and their therapeutic potential
|
Frontiers in genetics
| 2,023
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cc-by
| 12,865
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Abbreviations: miRNA, microRNA; DM, Diabetes mellitus; T1D, Type 1 diabetes; T2D, Type 2 diabetes;
Treg Regulatory T cells AITDs, Autoimmune thyroid disorders; GD, Graves’ disease; HT, Hashimoto’s
thyroiditis; BMD, Bone mineral density; MSC, Mesenchymal stem cells; APA, Aggressive pituitary
adenoma; CAB, Cabergoline. KEYWORDS miRNAs, endocrine disorders, diabetes mellitus, thyroid diseases, osteoporosis, pituitary
tumors OPEN ACCESS OPEN ACCESS
EDITED BY
Lingling Wang,
Hainan Normal University, China
REVIEWED BY
Zhongxin Lu,
Huazhong University of Science and
Technology, China
Kinga Fulopne Nemeth,
University of Texas MD Anderson Cancer
Center, United States
*CORRESPONDENCE
Luis M. Ruiz-Manriquez,
a01701195@tec.mx
Asim K. Duttaroy,
a.k.duttaroy@medisin.uio.no
Sujay Paul,
spaul@tec.mx
†These authors have contributed equally
to this work
SPECIALTY SECTION
This article was submitted to RNA,
a section of the journal
Frontiers in Genetics
RECEIVED 03 January 2023
ACCEPTED 09 February 2023
PUBLISHED 21 February 2023
CITATION
Ledesma-Pacheco SJ, Uriostegui-Pena AG,
Rodriguez-Jacinto E, Gomez-Hernandez E,
Estrada-Meza C, Banerjee A, Pathak S,
Ruiz-Manriquez LM, Duttaroy AK and
Paul S (2023), Regulatory mechanisms of
microRNAs in endocrine disorders and
their therapeutic potential. Front. Genet. 14:1137017. doi: 10.3389/fgene.2023.1137017 S. Janin Ledesma-Pacheco1†, Andrea G. Uriostegui-Pena 1†,
Estefania Rodriguez-Jacinto1, Elizabeth Gomez-Hernandez1,
Carolina Estrada-Meza 1, Antara Banerjee2, Surajit Pathak2,
Luis M. Ruiz-Manriquez1,3*, Asim K. Duttaroy4* and Sujay Paul1* 1Tecnologico de Monterrey, School of Engineering and Sciences, Queretaro, Mexico, 2Chettinad
Academy of Research and Education (CARE), Chettinad Hospital and Research Institute (CHRI),
Department of Medical Biotechnology, Faculty of Allied Health Sciences, Chennai, India, 3Tecnologico de
Monterrey, Escuela de Medicina, Monterrey, Mexico, 4Department of Nutrition, Institute of Basic Medical
Sciences, Faculty of Medicine, University of Oslo, Oslo, Norway MicroRNAs (miRNAs) are small endogenous non-coding RNA molecules capable
of regulating gene expression at the post-transcriptional level either by
translational
inhibition
or
mRNA
degradation
and
have
recently
been
importantly related to the diagnosis and prognosis of the most relevant
endocrine
disorders. The
endocrine
system
comprises
various
highly
vascularized ductless organs regulating metabolism, growth and development,
and sexual function. Endocrine disorders constitute the fifth principal cause of
death worldwide, and they are considered a significant public health problem due
to their long-term effects and negative impact on the patient’s quality of life. Over
the last few years, miRNAs have been discovered to regulate various biological
processes associated with endocrine disorders, which could be advantageous in
developing new diagnostic and therapeutic tools. The present review aims to
provide an overview of the most recent and significant information regarding the
regulatory mechanism of miRNAs during the development of the most relevant
endocrine disorders, including diabetes mellitus, thyroid diseases, osteoporosis,
pituitary
tumors,
Cushing’s
syndrome,
adrenal
insufficiency
and
multiple
endocrine neoplasia, and their potential implications as disease biomarkers. OPEN ACCESS CITATION
Ledesma-Pacheco SJ, Uriostegui-Pena AG,
Rodriguez-Jacinto E, Gomez-Hernandez E,
Estrada-Meza C, Banerjee A, Pathak S,
Ruiz-Manriquez LM, Duttaroy AK and
Paul S (2023), Regulatory mechanisms of
microRNAs in endocrine disorders and
their therapeutic potential. Front. Genet. 14:1137017. doi: 10.3389/fgene.2023.1137017 © 2023 Ledesma-Pacheco, Uriostegui-
Pena, Rodriguez-Jacinto, Gomez-
Hernandez, Estrada-Meza, Banerjee,
Pathak, Ruiz-Manriquez, Duttaroy and
Paul. 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 Review
PUBLISHED 21 February 2023
DOI 10.3389/fgene.2023.1137017 TYPE Review
PUBLISHED 21 February 2023
DOI 10.3389/fgene.2023.1137017 TYPE Review
PUBLISHED 21 February 2023
DOI 10.3389/fgene.2023.1137017 1 Introduction (
,
)
Recently, miRNAs have been demonstrated to regulate various
processes associated with endocrine disorders, such as high-glucose-
induced apoptosis, insulin secretion, and proliferation (Wang et al.,
2020), as well as glycolipid metabolism (Zhang et al., 2020) in
diabetes mellitus; abnormal ciliogenesis (Martínez-Hernández
et al., 2019) in thyroid diseases;
osteoclast
and osteoblast
differentiation (Li et al., 2018; Wang et al., 2021) in osteoporosis;
proliferation and apoptosis (He et al., 2018; He et al., 2020) in
pituitary tumors; proliferation (Vetrivel et al., 2022) in Cushing’s
syndrome;
steroidogenesis
(Bitetto
et
al.,
2022)
in
adrenal
insufficiency; and adrenocortical proliferation (Li et al., 2021) in
multiple endocrine neoplasia; among others. The association of
these small RNA molecules in endocrine disorders could be
advantageous in developing novel diagnostic and therapeutic tools. The present review aims to illuminate the most recent and
relevant information about the potential role of microRNAs in
endocrine disorders such as diabetes mellitus, thyroid diseases,
osteoporosis,
pituitary
tumors,
Cushing’s
syndrome,
adrenal
insufficiency, multiple endocrine neoplasia, as well as their
diagnostic and therapeutic potential. Recently, miRNAs have been demonstrated to regulate various
processes associated with endocrine disorders, such as high-glucose-
induced apoptosis, insulin secretion, and proliferation (Wang et al.,
2020), as well as glycolipid metabolism (Zhang et al., 2020) in
diabetes mellitus; abnormal ciliogenesis (Martínez-Hernández
et al., 2019) in thyroid diseases;
osteoclast
and osteoblast
differentiation (Li et al., 2018; Wang et al., 2021) in osteoporosis;
proliferation and apoptosis (He et al., 2018; He et al., 2020) in
pituitary tumors; proliferation (Vetrivel et al., 2022) in Cushing’s
syndrome;
steroidogenesis
(Bitetto
et
al.,
2022)
in
adrenal
insufficiency; and adrenocortical proliferation (Li et al., 2021) in
multiple endocrine neoplasia; among others. The association of
these small RNA molecules in endocrine disorders could be
advantageous in developing novel diagnostic and therapeutic tools. Alternatively, numerous non-canonical miRNA biogenesis
pathways have been identified and thoroughly revised (Treiber
et al., 2018; Stavast and Erkeland, 2019). These pathways employ
various combinations of the canonical pathway proteins, primarily
Drosha, Dicer, exportin 5, and AGO2 (Kim et al., 2016; O’Brien
et al., 2018). The non-canonical miRNA biogenesis can be divided
into
Dicer-independent
and
Drosha/DGCR8-independent
pathways (Annese et al., 2020). Pre-miRNAs produced by the
Drosha/DGCR8-independent pathway resemble Dicer substrates. Mirtrons, which are created during splicing from the introns of
mRNA,
is
an
illustration
of
such
pre-miRNAs. The
7-
methylguanosine (m7G)-capped pre-miRNA serves as another
illustration. 1 Introduction The main function of the endocrine system’s highly vascularized ductless organs is to
produce hormones that can control metabolism, growth, development, and sexual function
(Thi et al., 2017; Perle, 2021). The major glands that comprise the endocrine system are the
adrenal, hypothalamus, pituitary, parathyroids, pineal body, thyroid, and ovaries or testes Frontiers in Genetics 01 frontiersin.org 10.3389/fgene.2023.1137017 Ledesma-Pacheco et al. 10.3389/fgene.2023.1137017 human
genome
(Zhang
and
Wang,
2017),
and
currently,
1,917 precursors (hairpin portion of a miRNA transcript) and
2,654 mature human miRNA sequences are deposited to the
microRNA database/miRbase (miRBase, 2022; Kozomara et al.,
2019; Kozomara and Griffiths-Jones, 2014). human
genome
(Zhang
and
Wang,
2017),
and
currently,
1,917 precursors (hairpin portion of a miRNA transcript) and
2,654 mature human miRNA sequences are deposited to the
microRNA database/miRbase (miRBase, 2022; Kozomara et al.,
2019; Kozomara and Griffiths-Jones, 2014). (Malespin & Nassri, 2019). The epithelial cells of the organs secrete
their specific hormone product into the bloodstream, which binds to
specific receptors in the target organs, affecting cellular functions
(Perle, 2021). Endocrine disorders occur with the dysregulation of
hormone levels or if the body does not respond to hormones
appropriately
(Lause
et
al.,
2017)
and
constitute
the
fifth
principal cause of death worldwide. Moreover, they are a
significant
public
health
issue
since
they
can
substantially
decrease patients’ quality of life and cause long-term disabilities
(Crafa et al., 2021). (Malespin & Nassri, 2019). The epithelial cells of the organs secrete
their specific hormone product into the bloodstream, which binds to
specific receptors in the target organs, affecting cellular functions
(Perle, 2021). Endocrine disorders occur with the dysregulation of
hormone levels or if the body does not respond to hormones
appropriately
(Lause
et
al.,
2017)
and
constitute
the
fifth
principal cause of death worldwide. Moreover, they are a
significant
public
health
issue
since
they
can
substantially
decrease patients’ quality of life and cause long-term disabilities
(Crafa et al., 2021). Over the past decades, it has been demonstrated that miRNAs
can
regulate
hormone
production,
activity,
and
target
cell
responsiveness (Peng and Wang, 2018). 1 Introduction Without Drosha cleavage, these developing RNAs are
exported directly to the cytoplasm by exportin 1. Conversely, Dicer-
independent miRNAs are processed by Drosha from endogenous
short hairpin RNA (shRNA) transcripts. These pre-miRNAs
demand AGO2 terminate their maturation within the cytoplasm
since they are of insufficient length to be Dicer-substrates. The present review aims to illuminate the most recent and
relevant information about the potential role of microRNAs in
endocrine disorders such as diabetes mellitus, thyroid diseases,
osteoporosis,
pituitary
tumors,
Cushing’s
syndrome,
adrenal
insufficiency, multiple endocrine neoplasia, as well as their
diagnostic and therapeutic potential. Frontiers in Genetics frontiersin.org 1 Introduction For instance, miRNAs
can
directly
target
genes
encoding
hormones
or
enzymes
involved
in
hormone
production
or
metabolism,
affecting
hormone concentrations; and proteins that modulate hormone
actions, such as antagonists, can also be regulated by miRNAs;
also, many studies have demonstrated that hormones regulate
miRNAs,
and
such
regulation
can
occur
at
the
level
of
transcription or processing resulting in a complex regulatory
network that ultimately impacts cell homeostasis (Peng and Li,
2022). Moreover, miRNAs can target hormone receptors and
intracellular signaling molecules to alter target cell responses
(Derghal et al., 2016; Bayraktar et al., 2017). Recently, evidence
postulated that miRNAs could be secreted to the extracellular milieu
and act as endocrine factors, performing endocrine and paracrine
crosstalk between cells and tissues (Wu et al., 2022). MiRNAs are small (21–24 nucleotides long) endogenous non-
coding RNA molecules that post-transcriptionally regulate gene
expression either by degradation of mRNA or translational
inhibition (Xiang et al., 2017; de Sousa et al., 2019). Biogenesis
and processing of the miRNA molecules start in the cell’s nucleus,
where RNA polymerase II transcribes the genes that codify the
miRNA into the hairpin-structured primary miRNA, which is
converted into a shorter stem-loop precursor miRNA by the
microprocessor complex that consists of the enzymes Drosha and
DGCR8. Afterward, the precursor miRNA is exported to the
cytoplasm due to an interaction with Exportin 5, to be further
processed into a mature miRNA/miRNA* duplex by the action of
RNase III Dicer and trans-activation responsive RNA-binding
protein. Next, a helicase separates the duplex so that the guide
strand can be integrated into the RNA-induced silencing complex,
also known as RISC, which is guided by the AGO2 protein. Finally,
the RISC-miRNA complex identifies specific mRNA targets by base
complementarity leading to translational inhibition or mRNA
degradation (Paul et al., 2021; Ruiz-Manriquez et al., 2022). 1.1 Diabetes mellitus Diabetes mellitus (DM) is a chronic metabolic disorder
characterized by persistently elevated blood glucose levels or
hyperglycemia (Wang et al., 2020). Hyperglycemia can be caused
by inefficiency in insulin action (also known as insulin resistance),
secretion (also known as insulin deficiency), or both, as well as
perturbations in the metabolism of carbohydrates, fats, or proteins
(Cannata et al., 2020). The progression and development of DM are
known to be a consequence of malfunctioned pancreatic β-cells,
given that these cells are involved in insulin production (Wang et al.,
2020). Type 1 (T1D) and type 2 diabetes (T2D) are the most
common types of diabetes (Wang et al., 2020). T1D is caused by
the autoimmune destruction of β-cells, leading to complete insulin According to most studies, miRNAs induce translational
repression, mRNA deadenylation, and decapping by binding to a
specific sequence at the 3′UTR of their target mRNAs (Dexheimer
and Cochella, 2020). Nevertheless, miRNA binding sites have also
been found in the 5′UTR, coding sequence, promoter regions, and
other mRNA regions (Treiber et al., 2018). While the interaction of
miRNAs with the promoter region has been reported to induce
transcription, miRNA binding to the 5′UTR and coding regions has
been shown to silence the expression of specific genes (O’Brien et al.,
2018; Liu et al., 2021; Xiang et al., 2022). Interestingly, these
molecules are found to regulate about 60% of genes of the 02 frontiersin.org Biological mechanism
Source
References
Oxidative stress, high-glucose-induced β-cell apoptosis,
insulin secretion, proliferation, β-cell protection
Pancreatic and lung mice tissue
Wang et al. (2018)
Proliferation in acute pancreatitis and apoptosis
Pancreatic rat tissue
Ling et al. (2020)
Proliferation, insulin secretion, β-cell apoptosis
Blood from diabetic patients
Li et al. (2016)
Treg cell function
Peripheral blood and lymph nodes of
T1D patients
Sebastiani et al. (2017)
Treg cell induction and stability, autoimmune
activation, and progression
Human and mice CD4+ cells during
the onset of islet autoimmunity
and T1D
Scherm et al. (2019)
Glycogen synthesis, hepatic gluconeogenesis and
lipogenesis
T2D mice and rat liver tissue
Xu et al. (2018)
Hepatocytes
Insulinitis, insulin resistance and secretion, pancreatic
inflammation
β cells of βTG mice
Sun et al. (2021)
R1, EXT1, MTPN,
B2, RHOQ, SOX5,
Insulin sensitivity, preservation of β-cell function
Serum exosomes of obese mice and
humans
Xu et al. (2020)
Immune response
Thyroid tissue from AITD patients
Martínez-Hernández et al. (2018)
Ciliogenesis
Thyroid tissue from AITD patients
Martínez-Hernández et al. 1.1 Diabetes mellitus (2019)
(Continued on following page) Biological mechanism
Source
References
Immune response
Peripheral blood mononuclear cells
from HT and GD patients
Kagawa et al. (2016)
Hormone regulation, tumor proliferation migration and
agiogenesis
Plasmatic cells from GD patients
Yao et al. (2019)
Osteoclastogensis
Serum samples and lumbar spine
BMD of post-menopausal osteoporotic
patients
Li et al. (2018)
Osteoclast differentiation and apoptosis
Blood and bone tissue from patients
with osteoporosis
Li et al. (2020)
Cell viability, apoptosis and osteoblast differentiation
Human osteoblasts
Wang et al. (2021)
Formation of adipocytes, osteogenic and macrophage
differentiation, bone development
Mice bone marrow MSCs and stromal
ST2 cell lines during adipogenesis
Tian et al. (2017)
Osteogenic differentiation
Osteoporosis patients
Chen et al. (2019)
Osteoblast differentiation
Primary rat MSC cells
Li et al. (2019)
Osteogenesis, osteoblastogenesis. Osteoporosis dignosis
Serum from post-menopausal
osteoporotic patients
Du et al. (2015); Kerschan-Schindl et al. (2021); Lei et al. (2019); Sun et al. (2017)
(Continued on following page) Biological mechanism
Source
References
Obsteoblast proliferation, osteoclast, and osteoblast
differentiation, oxidative stress
Mice PBMC
Lu et al. (2021)
Metastasis
Human pituitary tumor tissue
He et al. (2020)
Proliferation, migration, apoptosis
HP75 and MMQ tumor cell lines
Metastasis, glycolysis
Human pituitary tumor tissue
He et al. (2018)
Proliferation, apoptosis
HP75 and MMQ tumor cell lines
APA metastasis, tumor size, temozolomide resistance
Human APA tissues
Lou et al. (2021)
(Continued on following page) Biological mechanism
Source
References
Metastasis, apoptosis, proliferation, migration, invasion,
temozolomide resistance, cancer cell differentiation,
survival and movement
GH3 tumor cell line
Cabergoline resistance
DA-resistant human prolactinoma
tissues
Wu et al. (2019)
Apoptosis, autophagy
MMQ and GH3 tumor cell lines
DNA synthesis, proliferation, cell cycle progression
GH-producing human pituitary tumor
cells and GH3 cell line
He et al. (2019)
Tumor cell’s invasiveness
Patients with non-functional pituitary
neuroendocrine tumors
Wang et al. (2020)
Cell cycle transition, viability, proliferation
aT3-1 cell line
Tumor progression
Plasma from pituitary adenomas
Németh et al. (2019)
Proliferation, metastasis
Peripheral blood samples from
untreated NFPA patients
Lin et al. (2018); Zhang et al. (2021)
Proliferation, metastasis, prognosis
You et al. (2019); Zhang et al. (2021)
Proliferation, migration
Park et al. (2018); Zhang et al. (2021)
Tumor growth
Han et al. (2018); Zhang et al. (2021)
Autoimmune response modulation
Serum from CS patients
Vetrivel et al. (2021)
Androstenedione production, proliferation
Adrenal tissue from CS patients
Vetrivel et al. (2022)
ENTPD1,
RY2,
HRB,
Immune response
Peripheral blood samples from
Adisson’s disease patients
Fichna et al. Frontiers in Genetics 1.1 Diabetes mellitus (2021)
Steroidogenesis
Adrenal gland tissue from patients
with adrenal insufficiency
Bitetto et al. (2022)
Adrenocortical proliferation, adrenal tumorogenesis
Men1+/−mice adrenal glands
Li et al. (2021)
Growth induction, growth arrest
Serum from MEN1 patients
Kooblall et al. (2022) 10.3389/fgene.2023.1137017 Ledesma-Pacheco et al. FIGURE 1
miRNAs expression profiles observed in diabetes mellitus type I, type II, and unspecified, and the biological mechanisms involved. FIGURE 1
miRNAs expression profiles observed in diabetes mellitus type I, type II, and unspecified, and the biological mechanisms involved. GURE 1
miRNAs expression profiles observed in diabetes mellitus type I, type II, and unspecified, and the biological mechanisms involved. FIGURE 2
Dysregulation of miRNA expression profiles in Hashimoto’s thyroiditis and Grave’s disease, and the biological mechanism involved. FIGURE 2
Dysregulation of miRNA expression profiles in Hashimoto’s thyroiditis and Grave’s disease, and the biological mechanism involved 2019). According to the World Health Organization (2021),
1.5 million deaths were estimated to be caused directly by
diabetes, placing this disease as the ninth leading cause of death
worldwide. Given the high occurrence of DM and its complication, deficiency. In contrast, T2D is attributed to a continuous loss of β-
cell insulin secretion, often in the context of insulin resistance
(Association,
2019). Diabetes
prevalence
increases
yearly,
affecting around 500 million people worldwide (Saeedi et al., 07 frontiersin.org 10.3389/fgene.2023.1137017 Ledesma-Pacheco et al. FIGURE 3
miRNAs expressions profile involved in osteoblast, osteoclast, and osteogenic differentiation, and apoptosis that disrupts bone homeostasis,
ultimately leading to osteoporosis. FIGURE 3
miRNAs expressions profile involved in osteoblast, osteoclast, and osteogenic differentiation, and apoptosis that disrupts bone homeostasis,
ultimately leading to osteoporosis. xpressions profile involved in osteoblast, osteoclast, and osteogenic differentiation, and apoptosis that disrupts bone homeostasis,
leading to osteoporosis. SOCS3 axis dysregulation in T2D development. Furthermore, they
noticed that the expression of miR-19-3p was notably downregulated
in diabetic patients’ blood and that there was an inverse correlation
between blood glucose concentration and plasma miR-19a-3p level (Li
et al., 2016). Further, the authors demonstrated that miR-19a-3p
promotes cell proliferation and insulin secretion while inhibiting
pancreatic β-cell apoptosis by directly targeting SOCS3 (Li et al.,
2016). several investigations have been carried out to establish the role of
miRNAs during the development of this disease to propose novel
therapeutic approaches (Table 1) (Figure 1). Frontiers in Genetics frontiersin.org 1.1 Diabetes mellitus According to the American
Thyroid Association (2016), about 20 million Americans suffer from
thyroid problems, and up to 60% of them are unaware of their
condition. MiRNAs have been discovered to regulate numerous
biological processes, including immune activation, making them
promising candidates for the management of thyroid problems
(Table 1) (Figure 2) (Martínez-Hernández et al., 2019). miR-142-3p was upregulated in both human and murine models
with islet autoimmunity and T1D. Furthermore, miR-142-3p was
identified to affect FOXP3 CNS2-induced DNA methylation and
directly target TET2, a modulator of DNA methylation, and is
suggested to be crucial to T cell activation and Treg induction. Overall,
it
has
been
concluded
that
the
miR142-3p/TET2/
FOXP3 axis hampers efficient Treg cell induction, compromising
Treg stability and contributing to autoimmune activation and
progression (Scherm et al., 2019). Dysregulated glycolipid metabolism is one of the crucial causes
of T2D development (Zhang et al., 2020). In this sense, Xu et al. (2018) showed that the enhancement of miR-125a-5p expression
appears advantageous for glycolipid metabolism in T2D mice. Putatively, the authors evidenced a downregulation of miR-125a-
5p in mouse and rat livers with T2D. Also, they demonstrated that
miR-125a-5p targets the STAT3 in hepatocytes. Due to the
involvement of STAT3 in glycolipid metabolism, it was revealed
that
the
inhibition
of
miR-125a-5p
increased
hepatic
gluconeogenesis and lipogenesis, decreased glycogen synthesis,
and aggravated hyperglycemia and hyperlipidemia. Conversely,
the
elevation
of
miR-125a-5p
mitigated
glucose
and
lipid
metabolic
disorders. This
information
suggests
a
critical
association between miR-125a-5p and glycolipid metabolism (Xu
et al., 2018). Martínez-Hernández et al. (2018) found that individuals with
AITDs have severely dysregulated levels of a variety of miRNAs
connected to critical immunological mechanisms that might be
involved in thyroid disorders. Eight miRNAs were found to be
differentially expressed (miR-21-5p, miR-142-3p, miR-146a-5p,
miR-146b-5p, miR-155-5p, miR-338-5p, miR-342-5p, and miR-
766-3p)
in
patients
with
AITD. Furthermore,
when
the
expression
of
these
miRNAs
was
compared
to
additional
miRNAs previously linked to AITD in peripheral blood, the
expression of four of them (miR-Let7d-5p, miR-21-5p, miR-96-
5p, miR-142-3p, and miR-301a-3p) was found to be notably higher
in AITDs and, in patients with GD. Only miR-Let-7d was
downregulated. Besides, the expression level of those miRNAs
was associated with higher disease severity, including active
ophthalmopathy, higher antibody titers, goiter, and/or higher
recurrence rates. Interestingly, prior research has linked miR-
Let7d-5p, miR-126-3p, miR-142-5p, miR-223-3p, and miR-301a-
3p to autoimmune illnesses, including in AITD. 1.1 Diabetes mellitus Furthermore, the
authors found a link between miR-Let7d-5p, miR-21-5p, miR-96-
5p, miR-142-3p, and miR-301-3p and the levels of all three thyroid
autoantibodies, suggesting that these miRNAs could be used to
predict the likelihood of getting AITD (Martínez-Hernández et al.,
2018). Pancreatic inflammatory diseases, including acute or chronic
pancreatitis, could result in diabetes (Gál et al., 2021). Pancreatic
inflammation has been found to be promoted by the overexpression
of miR-29 in β cells of βTG mice (Sun et al., 2021). Explicitly, miR-29
overexpression causes the suppression of the TRAF3, whose axis
regulates
the
response
to
metabolic
stress
associated
with
inflammation. Additionally, the inhibition of miR-29 improves
insulitis, insulin resistance, and dysfunction of glucose-stimulates
insulin secretion, causing the attenuation of inflammation and
diabetes (Sun et al., 2021). Primary cilia are a sensory organelle that reacts to mechanical
and chemical stimuli in the environment and transmits that
information to the inside of the cell. Martínez-Hernández et al. (2018) examined the distribution and length of primary cilia, which
act as sensory regulators in the thyroid to modulate hormone
secretion in thyroid tissues from AITD patients, finding that both
the number and length were significantly lower in AITDs than in
healthy tissue. It was noticed that certain miRNAs, including miR-
21-5p,
miR-146b-3p,
miR-5571-3p,
and
miR-6503-3p,
were
upregulated
in
the
cilia,
therefore,
associated
with
these
conditions. Therefore, the study suggests the potential use of
miRNAs to detect AITD (Martínez-Hernández et al., 2019). Given the importance of insulin secretion and sensitivity in
maintaining normal glucose levels, their disruption results in DM
(Bełtowski et al., 2018). In this regard, miR-26a has been shown to
improve peripheral insulin sensitivity and preservation of β cell
function (Xu et al., 2020). Putatively, miR-26a expression was
downregulated in the serum exosomes of obese mice and humans. Interestingly, by triggering miR-26a expression, glucose dysregulation
(hyperinsulinemia) could be averted since target genes of miR-26a
such as CACNA1C, CTGF, CREBRF, ESR1, EXT1, MTPN, ONECUT2,
PJA2, PLCB1, PFKFB2, RHOQ, and SOX5, are the regulators involved
in insulin secretion and β cell proliferation/survival (Xu et al., 2020). Additionally, other target genes such as INSR, PDX1, AKT1, and
IGF1R (β cell hyperplasia activators) were downregulated in the islet
of RIP TG mice (Xu et al., 2020). IL-10 is a cytokine directly involved in reducing inflammation
induced by macrophages and T cells. 1.1 Diabetes mellitus One of the essential functions
of IL-10 is inhibiting Th1 cells, a type of T cell that synthesizes
inflammatory cells (Ouyang and O’Garra, 2019). Interestingly, Let-
7e has been found to target the interleukin IL-10 genes (Kagawa
et al., 2016). Precisely, Kagawa et al. (2016) observed that IL-10
production was inhibited in peripheral blood mononuclear cells of
patients with HT and that serum levels of Th1 cytokines were
elevated. Importantly, let-7e expression was considerably higher
in HT patients compared to GD patients or control subjects. However,
there
were
no
significant
variations
in
Let-7e
expression levels between GD patients and control subjects. Moreover, Let-7e expression levels in severe HT patients were Altogether, the above information proves that the dysregulation
of a great number of miRNAs contributes to the development and
progression of diabetes. Therefore, these miRNAs could be potential
biomarkers and therapeutic tools for treating diabetic patients. Frontiers in Genetics 1.1 Diabetes mellitus For example, it has been shown that myeloid KLF2 overexpression
exerts a protective effect in mice against insulin resistance; therefore, it
is considered a crucial regulator of obesity and its sequelae, such as
diabetes (Sweet et al., 2020). Interestingly, Wang et al., 2018 suggested
that miRNA-92a can negatively regulate the expression of KLF2, which
is thought to function via modulating the Notch signaling pathway. The authors also revealed that miRNA-92a was predominantly
expressed in the pancreas and lungs in mice models, which was an
indicator of the involvement of miRNA-92a in pancreatic function. Moreover, they validated that miRNA-92a might modulate oxidative
stress, inhibit high-glucose-induced apoptosis of β-cells, increase
insulin secretion and proliferation, and protect pancreatic β-cell
function (Wang et al., 2018). Consistent with these findings,
another analogous study highlighted that the downregulation of
miRNA-92a-3p enhances KLF2 expression and inhibits apoptosis,
thus promoting proliferation in acute pancreatitis in pancreatic rat
tissue (Ling et al., 2020). It is well known that Regulatory T cells (Treg) are important
regulators of peripheral immune tolerance and that the development
of autoimmunity disorders, including T1D, can be due to their
insufficiency (Visperas and Vignali, 2016). In this context, an
upregulation of miR-125a-5p in Treg cells obtained from T1D
patients’
peripheral
blood
and
lymph
nodes
was
noted,
suggesting
that
this
overexpression
could
impede
Treg-cell
function (Sebastiani et al., 2017). Moreover, miR-125a-5p targets
TNFR2 and CCR2. Remarkably, CCR2 has been implicated in T1D
pathogenesis, and its expression is thought to modulate Treg cell
function. This data suggests that the upregulation of miR-125a-5p in
Treg cells of T1D patients leads to a decrease in CCR2 expression,
which might hinder the migration of critical immune cells to the
pancreas required for the maintenance of peripheral tolerance
(Sebastiani
et
al.,
2017). Similarly,
Scherm
et
al.,
2019
demonstrated that by inhibiting miR-142-3p, Treg induction and
stability could be enhanced. More precisely, the authors showed that Over the past years, SOCS3 has been associated with glucose
metabolism and diabetes progression, given that it is a crucial negative
regulator of insulin signaling (Iqbal et al., 2020). Notably, Li et al. (2016) found an inverse correlation between SOCS3 plasma levels and
miR-19a-3p, which led them to suggest the role of the miR-19a-3p/ 08 frontiersin.org 10.3389/fgene.2023.1137017 Ledesma-Pacheco et al. 10.3389/fgene.2023.1137017 metabolic
processes
(Kyritsi
and
Kanaka-Gantenbein,
2020). Thyroid diseases include hypothyroidism, hyperthyroidism, and
autoimmune thyroid disorders (AITDs), such as Graves’ disease
(GD) and Hashimoto’s thyroiditis (HT). frontiersin.org 1.2 Thyroid diseases Thyroid diseases are conditions in which the thyroid gland is
affected. This gland plays an essential role in regulating various 09 frontiersin.org frontiersin.org 10.3389/fgene.2023.1137017 Ledesma-Pacheco et al. FIGURE 4
Oncogenic and tumor suppressor miRNAs expression profile involved in pituitary tumors, ultimately affecting cabergoline resistance, proliferation,
metastasis, apoptosis, and migration. FIGURE 4
Oncogenic and tumor suppressor miRNAs expression profile involved in pituitary tumors, ultimately affecting cabergoline resistance, proliferation,
metastasis, apoptosis, and migration. have proven to be a valuable tool for the early detection of thyroid
diseases such as HT and GD; however, further research is required to
use them as potential disease biomarkers. Moreover, investigating
the roles of miRNAs in hyper and hypothyroidism might open a new
arena of thyroid disease management. found to be considerably lower than in mild HT patients (Kagawa
et al., 2016). These findings lead to the understanding of Let-7e in
the regulation of the immune response of people with HT, which
might eventually help with HT diagnosis and treatment. Graves’ disease is an autoimmune condition known as the most
common cause of hypothyroidism (Yao et al., 2019). It is
characterized by an activated immune system, which results in
the
production
of
thyroid-stimulating
antibodies
(TSAs);
consequently, it makes the thyroid gland overactive, producing
more hormones than usual (Razmara et al., 2021). GD is a
complex condition resulting from environmental and genetic
factors (including mutations in the TSHR and CD40 genes). Yao
et al. (2019) demonstrated that various plasmatic miRNAs are
associated
with
GD
and
might
serve
as
biomarkers
for
diagnosing this condition. They found that five miRNAs were
substantially differentially expressed in GD. Among them, miR-
16-1-3p, miR-122-5p, miR-221-3p, and miR-762 were upregulated,
while miR-144-3p was downregulated. Intriguingly, miR-762 was
positively associated with FT3, a thyroid hormone, and TRab (Yao
et al., 2019), which is considered the gold standard diagnostic test for
the autoimmunity of GD (Bell et al., 2018). Moreover, miR-144-3p
has been shown to be downregulated in many kinds of carcinoma
and is associated
with tumor proliferation,
migration, and
angiogenesis (He et al., 2020). Frontiers in Genetics frontiersin.org 1.3 Osteoporosis (2019a) consistently studied miR-449b-5p expression in
primary rat MSC cells, and miR-449b-5p was reported to be
underexpressed
in
osteogenic
differentiation;
moreover,
the
differentiation could be inhibited by the miRNA overexpression,
thus revealing the mechanism of miR-449b-5p in suppressing bone
formation. In addition, miR-449b-5p was proved to target the
SATB2 gene, which enhances osteoblast differentiation, regulates
skeletal development, and promotes bone formation. Therefore,
miR-449b-5p is thought to repress the osteogenic differentiation
of MSCs via SATB2, therefore promoting the progression of
osteoporosis (Li et al., 2019). Similarly, miR-483-5p has also been suggested to promote
osteoclast differentiation in osteoporosis. Putatively, in samples of
blood and bone tissue from osteoporotic patients, miR-483-5p was
found to be significantly upregulated, whereas its predicted target
IGF2 was underexpressed. Interestingly, the upregulation of miR-
483-5p promoted the differentiation of the M-CSF, a relevant
cytokine for osteoclast differentiation in mammals, and RANKL-
induced CD14+ peripheral blood mononuclear cells into osteoclasts. Moreover, overexpression of IGF2 was discovered to revert the
miRNA effects on osteoclast differentiation and thus enhance
osteoclast apoptosis. Therefore, a dysregulation in miR-483-5p
expression
might
contribute
to
osteoclast
differentiation
by
targeting IGF2, causing dysregulation in the balance between
bone formation and resorption, ultimately causing osteoporosis
(Li et al., 2020). Kerschan-Schindl et al. (2021) used a signature panel of nineteen
circulating miRNAs to serve as biomarkers to diagnose osteoporosis
in post-menopausal patients based on the WHO criteria and with
the ability to identify patients that had previously suffered fractures. The miRNAs in the panel represent different independent factors
related to bone disease and the risk of fractures. The panel includes
miR-127-3p, miR-133b, miR-203a, miR-214-3p, miR-31-5p, and
miR-375 (all of which the effects of hemolysis did not bias
measurement); miR-143-3p, miR-188-5p, and miR-582-5p (these
miRNAs along with miR-375 strongly contributed with the WHO
criteria); miR-550a-3p and miR-152-3p (which contributed to the
major osteoporotic fractures); and miR-141-3p, miR-144-5p, miR-
17-5p, miR-19b-3p, miR-29-3p, miR-320a, miR-335-5p, and let-7b-
5p. Out of these, the upregulation of serum miR-375 indicated the
presence of osteoporosis according to the WHO criteria and has
been associated with reducing the bone-formation effect of
teriparatide, a drug to treat severe osteoporosis (Lei et al., 2019). MiR-375 has been found to negatively modulate osteogenesis The bone formation also relies on osteoblast differentiation
(Wang et al., 2021); therefore, an alteration in this process may
lead to skeletal disorders such as osteoporosis. 1.3 Osteoporosis MiRNAs have an important role in
the regulation of bone formation and resorption (Li et al., 2018;
Chen et al., 2019) that allows a balance to maintain the healthy state
of the bones, and their dysregulation is associated with osteoporosis;
however,
the
precise
underlying
mechanism
is
still
poorly
understood. Hence, there is a current need to elucidate the
regulatory pathways of miRNAs in osteoporosis to prevent and
treat this global disease (Table 1) (Figure 3). It is estimated that one in three 50 + aged women will suffer a
fracture in their remaining lifetime (International Osteoporosis
Foundation, 2022), strongly influenced by estrogen deficiency
and aging (Li et al., 2018). One of the hallmarks of post-
menopausal osteoporosis is a low bone mineral density (BMD)
derived from the altered activity of osteoblasts and osteoclasts. Notably, miR-133a has been significantly upregulated in serum
samples and showed a negative correlation with lumbar spine
BMD
of
post-menopausal
patients
with
osteoporosis. Interestingly,
this
miRNA
was
upregulated
during
osteoclastogenesis,
thus
promoting
the
differentiation
of
RAW264.7 and THP-1 cells into osteoclasts, which would be
induced
by
RANKL,
a
crucial
cytokinin
for
osteoclast
development (Li et al., 2018). Consistently, an in vivo analysis in
OVX rats demonstrated that a knockdown of miR-133a augmented
lumbar spine BMD, dysregulated the serum levels of factors related
to osteoclastogenesis, and modified the histomorphology of the bone
mainly by perforation, connectivity loss, and thinning of the
trabecular bone. Altogether, it was demonstrated that miR-133a
promotes osteoclast differentiation, thus contributing to the
regulation of osteoporosis in post-menopausal women (Li et al.,
2018). Although bone mass recovery strategies are limited, miRNA-
mediated osteogenic differentiation has become a target of interest
for osteoporosis therapeutics. In that matter, Chen et al. (2019)
discovered that serum levels of miR-19a-3p were underexpressed in
patients with osteoporosis. Furthermore, the levels of miR-19a-3p,
RUNX2, and OCN (these last being osteogenesis-related genes)
would be gradually upregulated as the osteogenic differentiation
of MSCs prolongated. miR-19a-3p was found to target the HDAC4
gene, a critical factor in bone formation, bone metabolism, and
osteoblast differentiation. Thus, the analyzed miRNA was suggested
to reduce osteoporosis progression by accelerating the osteogenic
differentiation of MSCs by inhibiting HDAC4 (Chen et al., 2019). Li
et al. 1.3 Osteoporosis Osteoporosis is a multifactorial condition considered as the
most prevailing metabolic bone disorder in humans (Scimeca
et al., 2017). It is caused by complex interactions between genetic
and environmental factors (Li et al., 2018) and is characterized by an
imbalance between the bone formation mediated by osteoblasts and
the bone resorption mediated by osteoclasts. Bone homeostasis is
commonly altered by dysregulation in osteoblasts and osteoclasts’
proliferation, differentiation, or apoptosis (Wang et al., 2021), which
leads to bone mass and strength decrease, degenerative change of
bone microarchitecture, and non- or low-trauma fractures (Cosman
et al., 2014; Scimeca et al., 2017). Osteoporosis usually has no
symptoms prior to a first fracture, which is associated with a risk
increment
of
a
subsequent
fracture
by
86%
(International
Osteoporosis Foundation, 2022). Osteoporosis has become a
serious health concern, affecting around 50% of post-menopausal
females and 20% of males over 50 (Gennari et al., 2016). Furthermore, it causes over 8.9 million fractures annually (Hu
et al., 2018), many of which lead to mortality (International Since thyroid diseases affect a large number of people, most of
whom are unaware of it, it is crucial to find new ways to detect
thyroid diseases to implement an adequate early treatment. MiRNAs 10 frontiersin.org 10.3389/fgene.2023.1137017 10.3389/fgene.2023.1137017 Ledesma-Pacheco et al. MSCs generates a bone mass decrease associated with osteoporosis
(Hu et al., 2018; Li et al., 2019), the regulation mechanism of
miRNAs in these processes has been extensively studied in order
to develop potential target treatments for this skeletal disorder. In
that context, miR-148a-3p was overexpressed in mice bone marrow
MSCs and stromal ST2 cell lines during adipogenesis (Tian et al.,
2017). miR-148a-3p potentiated the formation of adipocytes in the
adipogenic medium while inhibiting osteoblast differentiation. Further study revealed KDM6b as the direct target of miR-148a-
3p. In the investigation, KDM6b was found to influence the
differentiation into mature adipocytes; however, it has also been
related to the modulation of osteogenic commitment of MSCs,
osteogenic
and
macrophage
differentiation,
and
bone
development. Altogether it was concluded that miR-148a-3p has
a positive adipogenic and negative osteogenic function by directly
targeting and negatively regulating KDM6B, affecting the MSCs fate
decision (Tian et al., 2017). Osteoporosis Foundation, 2022). Frontiers in Genetics frontiersin.org Ledesma-Pacheco et al. 1.3 Osteoporosis A study in human
osteoblasts
revealed
the
downregulation
of
miR-133b
and
subsequent overexpression of its target GNB4 in osteoporotic
patients (Wang et al., 2021). Intriguingly, the induction of miR-
133b led to higher cell viability, diminished apoptosis, and
promotion of osteoblast differentiation (Wang et al., 2021);
hence, the underlying mechanism of the miR-133b/GNB4 might
be of use for osteoporosis treatment. Correspondingly, miRNAs can
also influence bone marrow mesenchymal stem cells (MSCs), known
to be osteoblast and adipocyte progenitor cells (Hu et al., 2018). Since an imbalance of osteoblast and adipogenic differentiation of 11 frontiersin.org 10.3389/fgene.2023.1137017 Ledesma-Pacheco et al. (Kerschan-Schindl et al., 2021), possibly by targeting RUNX2 (Du
et al., 2015; Lei et al., 2019), and suppressing WNT pathways by
targeting LRP5 and ß-catenin (Sun et al., 2017). Moreover, the
upregulation of miR-203a-3p was a potential marker for fragility
fractures
and
has
been
identified
to
negatively
regulate
osteoblastogenesis via BMP-2 and DLX5, which activate the
transcription factors for osteoblast differentiation: RUNX2 and
Osterix. Altogether, the analyzed panel could serve as a novel
and non-invasive tool for osteoporosis diagnosis and identifying
patients with fracture history (Kerschan-Schindl et al., 2021). decreased levels of vimentin were also noticed in HP75 and
MMQ cells overexpressing miR-448 (He et al., 2020). Caspase-3
and PARP are pro-apoptotic proteins (Hsu et al., 2018), while
E-cadherin and vimentin are biomarkers that, in this case,
suggest the inhibition of epithelial to mesenchymal transition of
pituitary tumor cells (Liao et al., 2019; He et al., 2020). Hence, the
elucidated miR-448–BCL2 pathway might be of use as a therapeutic
strategy to regulate the malignant behavior of pituitary tumor cells. (Kerschan-Schindl et al., 2021), possibly by targeting RUNX2 (Du
et al., 2015; Lei et al., 2019), and suppressing WNT pathways by
targeting LRP5 and ß-catenin (Sun et al., 2017). Moreover, the
upregulation of miR-203a-3p was a potential marker for fragility
fractures
and
has
been
identified
to
negatively
regulate
osteoblastogenesis via BMP-2 and DLX5, which activate the
transcription factors for osteoblast differentiation: RUNX2 and
Osterix. Altogether, the analyzed panel could serve as a novel
and non-invasive tool for osteoporosis diagnosis and identifying
patients with fracture history (Kerschan-Schindl et al., 2021). He et al. (2018) also observed the downregulation of miR-1 in
human pituitary tumor tissues. Moreover, a much lower abundance
of miR-1 was found in the presence of lymphatic metastasis,
associated with a worse prognosis in patients. 1.3 Osteoporosis Other assessments
showed that the overexpression of miR-1 in HP75 and MMQ cell
lines suppresses the proliferation and increases the apoptosis rate of
the cells. Also, it was demonstrated that miR-1 negatively regulates
the protein expression of its target gene G6PD, limiting its function
of
generating
ribose-5-phosphate
and
NADPH,
which
are
promoters of glycolysis and nucleotide biosynthesis in tumor
cells; therefore, pituitary tumor tissues under expressing miR-1
showed
an
augmentation
of
G6PD
(C. He
et
al.,
2018),
suggesting miR-1 as a target to diagnose and treat pituitary tumors. Currently, microRNA biomarkers for osteoporosis are found to
be
very
promising
since
conventional
protein
osteoporosis
biomarkers are not very efficient and often produce false positive
results. In this matter, Lu et al. (2021) found thirteen significantly
consistent overexpressed miRNAs (miR-1271-5p, miR-132-3p,
miR-153a-3p, miR-153b-3pA, miR-153-3p, miR-15a-5p, miR-
15b-5p,
miR-182-5p,
miR-223-3p,
miR-27a-3p,
miR-370-3p,
miR-486-5p, and miR-9-5p) in mice PBMC that were positively
correlated with osteoporosis progression, therefore elucidating their
potential role as biomarkers for this disease. The mentioned
miRNAs were FOXO1 regulators, an important molecule in bone
metabolism
that
activates
osteoblasts’
proliferation
and
differentiation, suppresses the differentiation and viability of
osteoclasts, and diminishes the negative effects of oxidative stress
to favor bone cell function (Lu et al., 2021). gg
g
g
g
p
y
Pituitary tumors are referred to as aggressive pituitary adenomas
(APAs) when they exhibit aggressive behaviors such as solid
invasiveness and resistance to conventional therapies (Lou et al.,
2021). The role of miRNA-146b-5p in APA was studied by Lou et al. (2021). MiRNA-146b-5p was found to be downregulated in human
APA tissues and pituitary tumor cell lines (AtT-20, HP75, GH3,
GT1-1) compared to non-tumor tissues and normal human
astrocytes,
respectively. Furthermore,
the
downregulation
of
miRNA-146b-5p in APA patients was associated with poor
prognosis, poor survival rate, and features indicative of advanced
tumor stage related to APA metastasis, such as temozolomide
resistance, larger tumor size, poorer Hardy grade, and poorer
Knosp grade (Lou et al., 2021); these last grades refer to
classification systems indicating local invasion and cavernous
sinus
invasion
(Araujo-Castro
et
al.,
2021). Subsequent
assessments demonstrated that the induced overexpression of
miRNA-146b-5p in GH3 cells suppressed metastasis by triggering
cell apoptosis and negatively regulating cell proliferation, migration,
and invasion. The direct target of miRNA-146b-5p, EPHA7 was also
found to be overexpressed in APA tissues as well as in the pituitary
tumor
cell
lines
previously
mentioned
(Lou
et
al.,
2021). 1.3 Osteoporosis EPHA7 mediates the inflammatory response to cell injury, and
its low expression has been found in different carcinomas (Gajdzis
et al., 2020). The anti-metastatic properties of miRNA-146b-5p in
GH3 cells were reversed by the induced overexpression of EPHA7
(Lou et al., 2021). Interestingly, compared to chemosensitive tissues,
chemoresistant tissues showed overexpression of EPHA7 and
underexpression of miRNA-146b-5p. The same results were
found in a temozolomide-resistant cell line of GH3 compared to
the parental cell line. It was speculated that miRNA-146b-5p might
regulate autophagy since treatment with this miRNA decreased the
autophagic activity of temozolomide-resistant cells, while Lv-
EPHA7 treatment enhanced it. Thus, it was suggested that
abnormal expressions of miRNA-146b-5p and EPHA7 might be
involved
in
the
temozolomide
resistance
in
APA. Finally,
IRAK4
and
TRAF6
protein
expression
and
NF-κB
phosphorylation were suppressed in GH3 cells overexpressing Although osteoporosis is a common concern in the elderly and
post-menopausal
population,
bone
restoration
strategies
are
currently limited. Recently, several investigations have elucidated
the role of miRNAs in osteoporosis which could be promising in
discovering
novel
miRNA-based
diagnostic
and
therapeutic
strategies that could increase the patient’s quality of life. Frontiers in Genetics frontiersin.org 1.4 Pituitary tumors Pituitary tumors, also called pituitary neuroendocrine tumors,
are adenomas that arise from the anterior pituitary gland and
constitute around 10 to 15 percent of intracranial tumors (Asa
et al., 2017; Lou et al., 2021). Pituitary tumors arise sporadically from
any
of
the
five
hormone-producing
cell
types
of
the
adenohypophysis, but despite being monoclonal, they contain
plenty of multi-responsive and multi-hormonal cells whose role
is not yet fully understood (Fiordelisio et al., 2021). Many
neoplasms, including pituitary tumors, have exhibited altered
miRNA expression, thus being a significant field of research for
understanding, diagnosing, and treating pituitary tumors (Table 1)
(Figure 4). Although pituitary tumors are mostly benign, they have the
potential to develop into cancer. He et al. (2020) noticed a reduced
expression of miR-448 in human pituitary tumor tissues and an even
lower expression in tissues from patients with metastasis. The
induced overexpression of miR-448 was evaluated in HP75 and
MMQ cells (pituitary adenoma cell lines), showing inhibition of
proliferation and migration of both cell lines and an increase in the
apoptosis rate. It was suggested that miR-448 negatively regulates its
target gene, BCL2, an integral outer mitochondrial membrane
protein that inhibits apoptosis in HP75 and MMQ cells. In
addition, increased levels of PARP, E-cadherin, caspase-3, and 12 frontiersin.org Ledesma-Pacheco et al. 10.3389/fgene.2023.1137017 10.3389/fgene.2023.1137017 miRNA-146b-5p; however, restoration of EPHA7 expression
reversed
these
inhibitory
effects. The
IRAK4/TRAF6/NF-κB
signaling
pathway
is
essential
in
regulating
cancer
cell
differentiation, survival, and movement, and EPHA7 is required. Hence, miRNA-146b-5p may inhibit invasion and metastasis by
directly targeting EPHA7 and suppressing the IRAK4/TRAF6/NF-
κB signaling pathway (Lou et al., 2021). These findings suggest that
miRNA-146b-5p could be used as a molecular marker to evaluate
and predict pituitary tumor aggressiveness and chemotherapeutic
drug resistance. since it modulates the cell cycle via SKP2/p27Kip1, thus revealing
a novel mechanism that might have significant therapeutic
implications. miRNA-146b-5p; however, restoration of EPHA7 expression
reversed
these
inhibitory
effects. The
IRAK4/TRAF6/NF-κB
signaling
pathway
is
essential
in
regulating
cancer
cell
differentiation, survival, and movement, and EPHA7 is required. Hence, miRNA-146b-5p may inhibit invasion and metastasis by
directly targeting EPHA7 and suppressing the IRAK4/TRAF6/NF-
κB signaling pathway (Lou et al., 2021). These findings suggest that
miRNA-146b-5p could be used as a molecular marker to evaluate
and predict pituitary tumor aggressiveness and chemotherapeutic
drug resistance. 1.4 Pituitary tumors Afterward, the miRNA-93-5p inhibitor
was used to transfect MMQ cells which were then injected
subcutaneously
into
nude
mice;
the
results
showed
an
enhancement
of
the
therapeutic
effectiveness
of
CAB,
a
diminution in the prolactin expression in mice, an upregulation
of LC3-II protein, and the overproduction of autophagosomes. Therefore,
the
investigation
suggests
that
miRNA-93-5p
inhibition enhances CAB efficiency in vivo by upregulating
autophagy. With the objective of identifying circulating miRNAs that could
serve as biomarkers for pituitary adenomas, Nemeth et al. (2019)
investigated the dysregulation of several miRNAs in plasma and
extracellular vesicles from patients with pituitary adenomas at a
preoperative, as well as early and late postoperative stages. A total of
29 miRNAs were found promising to distinguish preoperative
plasma samples from normal controls. Interestingly, the study
demonstrated that miR-143-3p was substantially differentially
expressed (downregulated) in late postoperative samples of FSH/
LH + adenomas compared to preoperative samples, indicating a
successful surgery. Nevertheless, no significant alteration of this
miRNA has been noticed when comparing early postoperative
samples
with
preoperative
ones. Although
the
results
are
promising, further investigation is needed to determine the
potential role of this miRNA as a marker for tumor recurrence
(Németh et al., 2019). Growth hormone (GH) pituitary tumors are another subtype
of functioning pituitary tumors characterized by excessive
production of GH by the pituitary gland leading to acromegaly
and gigantism (Ji et al., 2022). He et al. (2019) reported a reduced
expression of miR-186 in GH-producing human pituitary tumors. The miR-186 regulates the expression of its direct target, the
SKP2, an oncogene that targets p27Kip1 to inhibit its expression
(He et al., 2019). The p27Kip1 is a tumor suppressor that inhibits
the transition of the cell cycle from the G1 to the S phase (Li et al.,
2021). The role of miR-186 was tested in human GH-secreting
pituitary tumor cells and GH3 cells, and its inhibition resulted in
the overexpression of SKP2 and underexpression of p27Kip1,
causing
an
induction
in
the
DNA
synthesis
and
cell
proliferation. Consistently, the overexpression of miR-186 had
the opposite effect on the cell lines and induced G0/G1 cell cycle
arrest. In addition, miR-186 and p27Kip1 were downregulated in
GH-producing human pituitary tumor tissues compared to
normal human pituitary tissues, while SKP2 was upregulated
(He et al., 2019). 1.4 Pituitary tumors Non-functional pituitary neuroendocrine tumors remain a
challenge since the only first-line treatment choices are surgery
and radiotherapy, and early detection of invasiveness is scarce due to
a lack of diagnostic biomarkers (Bao et al., 2021). Wang et al. (2020)
analyzed specimens of patients with non-functional pituitary
neuroendocrine tumors and reported that miR-134 might inhibit
the tumor cell’s invasiveness and expression of Ki-67, a marker of
cellular proliferation. The verified target of miR-134 was the VEGFA
(Wang
et
al.,
2020),
the
most
functional
isoform
of
the
proangiogenic factors VEGF, which contribute to angiogenesis,
successively helping the generation and development of cancer
(Zhang et al., 2019). Additionally, they found that by influencing
the expression of miR-134, SDF-1α could promote the development
of tumors. SDF-1α and VEGFA expression levels were relatively
high in the analyzed invasive specimens, defining invasiveness as
resistance to conventional treatment and numerous recurrences. It
was confirmed in mouse pituitary aT3-1 cells that due to miR-134
upregulation, VEGFA expression and transition of the cell cycle
from the G1 to the S phase are inhibited. Interestingly, SDF-1α
treatment
reduces
miR-134
expression
and
boosts
VEGFA
expression, which promotes cell viability and proliferation by
allowing cell cycle transition (Wang et al., 2020). For that reason,
the SDF-1α/miR-134/VEGFA axis might be a potential target for
detecting and treating non-functional pituitary neuroendocrine
tumors. Pituitary tumors can be functioning (which involves the
overproduction of certain hormones) or non-functioning (low
hormone production) (Butt and Srinivasan, 2022). The most
common functioning pituitary tumors are prolactin-secreting
tumors called prolactinomas (Kim et al., 2022). In most cases,
cabergoline (CAB) or other dopamine agonists are used for the
treatment, but there are dopamine agonist-resistant prolactinomas
whose management remains a challenge (Wu et al., 2019). Wu et al. (2019) proposed that miRNA-93-5p, a miRNA overexpressed in
dopamine agonists-resistant prolactinoma tissues, targets ATG7 and
intervenes in CAB resistance. Autophagy is a crucial pathway for cell
survival under stress that degrades large structures such as
organelles and proteins (Li et al., 2019), and ATG7 is an essential
protein downregulated in dopamine agonists-resistant human
prolactinoma tissues (Wu et al., 2019). Assays performed with
MMQ and GH3 rat prolactinoma cell lines indicated that the
upregulation of miRNA-93-5p may decrease the CAB-induced
autophagic cell death by inhibiting the expression level of cleaved
caspase-8 and LC3-II, proteins related with CAB-induced apoptosis
and autophagy, respectively. Frontiers in Genetics frontiersin.org 1.5.3 Multiple endocrine neoplasia Multiple endocrine neoplasia type 1 (MEN1) is an autosomal
dominant disorder characterized by various endocrine and non-
endocrine tumors. It is a rare disorder, given that it affects between
3–20 in every 100,000 people (Brandi et al., 2021). Recently, miR-
486-3p was found to be notably downregulated in Men1+/−mice’s
adrenal glands (Li et al., 2021). miR-486-3p was proven to target
fatty acid synthase (FASN), which is involved with adrenocortical
proliferation. Thus, it is theorized that miR-486-3p downregulation
might play a crucial role in adrenal tumorigenesis (Li et al., 2021). Moreover, a significant downregulation of miR-3156-5p was
observed in serum samples of MEN1 patients compared to
controls. This downregulation may be the result of a reduction in
MEN1 expression (Kooblall et al., 2022). It was also found that miR-
3156-5p directly targets MORF4L2, given that miR-3156-5p
upregulation causes a decrease of MORF4L2 expression, which
plays an important role in the activation of oncogene and proto-
oncogene-mediated
growth
induction,
as
well
as
in
tumor
suppressor-mediated growth arrest. Additionally, MORF4L2 is
part of the NETest, a useful tool for neuroendocrine tumor
(NET) subtype management and diagnosis, therefore, it might be
of utility for MEN1 diagnosis (Kooblall et al., 2022). 1.4 Pituitary tumors Similarly, miRNA-455 and miRNA-125a were significantly
downregulated in the adrenal gland of Allgrove syndrome patients
with adrenal insufficiency (Bitetto et al., 2022). These miRNAs are
known to be involved in the regulation of Scavenger receptor class B-1
(SCARB1), an important factor in steroidogenesis found to be
downregulated in the samples. Due to the downregulation of miR-
455 and miR-125a, there is an induction of SCARB1 on the cell surface
(Bitetto et al., 2022). proliferation, metastasis, and prognosis of colon cancer cells (You et al.,
2019); miR-206 has been reported to increase the migration and
proliferation of colorectal cancer cells (Park et al., 2018); and miR-
let-7e has been reported to diminish tumor growth (Han et al., 2018). Altogether this information could imply using these four miRNAs as
novel targets for the clinical management of NFPA (Zhang et al., 2021). Because of their essential roles in regulating the development of
pituitary tumors, miRNAs have significant clinical value. More
specifically, miRNAs could be used to diagnose the presence and
progression of a tumor and classify it more accurately, predict
invasiveness or aggressiveness, treat or regulate the behavior of
pituitary tumors, and evaluate the success of an operation. 1.5.1 Cushing’s disease Cushing’s syndrome (CS) is a rare disease caused by an
overproduction
of
cortisol
in
most
cases
due
to
adrenocorticotropic
hormone
(ACTH)-producing
pituitary
adenomas (Belaya et al., 2020). In a study where circulating
miRNA expression profiles were compared in serum samples of
patients with and without CS, miR-182-5p was identified as a
potential biomarker due to its persistent considerable differential
expression between the samples (Vetrivel et al., 2021). Along with
miR-182-5p, other members of the miR-183 cluster, including miR-
96-5p
and
miR-183-5p,
were
found
to
be
substantially
downregulated in the samples. This cluster is associated with
auto-immune, neurological, and psychiatric disorders, as well as
with cancer (Vetrivel et al., 2021). In a recent study, upregulation of
miR-1247-5p and miR-379-5p was observed in the adrenal tissues of
patients with different forms of CS in comparison to controls
(Vetrivel et al., 2022). One target of miR-1247-5p and miR-379-
5p was found to be Cytochrome b5 (CYB5A), which was notably
downregulated in all forms of CS and is known to intervene in the
regulation of androstenedione production. However, other target
genes were identified as involved in the WNT signaling pathway,
whose dysregulation promotes proliferation in ACTH-secreting
pituitary adenomas (Vetrivel et al., 2022). 2 Discussion Endocrine disorders are a significant health issue that decreases
the quality of patients’ life and sometimes could be fatal. To date,
none of the conventional diagnoses or therapies for endocrine
disorders are efficient or precise, which has outstood the urge to
develop novel and more functional techniques. Recently, miRNAs
have been shown to regulate various vital processes associated with
endocrine disorders such as diabetes mellitus, thyroid diseases,
osteoporosis,
pituitary
tumors,
Cushing’s
syndrome,
adrenal
insufficiency, and multiple endocrine neoplasia. Although the
precise roles of several miRNAs in various signaling pathways
during the development of endocrine disorders have yet not been
fully elucidated, these small molecules have drawn the attention of
global researchers to be used for novel therapeutic purposes. Furthermore, miRNAs have shown promising results as disease
biomarkers to detect the disease at an early stage, distinguish among
different conditions of the patients, as well as to determine the
disease’s severity. Altogether, miRNAs have revealed their potential
for developing novel tools and techniques that allow the precise
detection and management of endocrine disorders, resulting in a
general improvement in the patient’s life quality. 1.4 Pituitary tumors The above-mentioned information indicates
that miR-186 has a tumor-suppressive role in pituitary tumors Dysregulation of miRNAs has also been associated with tumor
incidence and progression, although the regulatory mechanism of
these tiny molecules behind non-functional pituitary adenomas
(NFPA) has not yet been clarified. In this context, Zhang et al. (2021) explored the correlation between miRNAs expression levels
and NFPA by comparing miRNA expression levels in peripheral
blood samples from untreated NFPA patients and healthy controls. Four specific miRNAs: miR-26b, miR-138, miR-206, and miR-let-7e
were significantly downregulated in NFPA patients than healthy
controls, suggesting the involvement of these molecules in the
occurrence and progression of NFPAs. Relevantly, miR-26b has
been previously associated with the inhibition of proliferation and
metastasis in hepatocellular carcinoma (Lin et al., 2018); miR-138
has
been
reported
as
a
tumor-suppressor
gene
related
to 13 frontiersin.org 10.3389/fgene.2023.1137017 10.3389/fgene.2023.1137017 Ledesma-Pacheco et al. SEMA4C, SIRPA, SLC11A, THRB, and VGLL3. These genes are
thought to be involved in immune response, primary cellular
component regulation, and metabolic processes (Fichna et al.,
2021). Similarly, miRNA-455 and miRNA-125a were significantly
downregulated in the adrenal gland of Allgrove syndrome patients
with adrenal insufficiency (Bitetto et al., 2022). These miRNAs are
known to be involved in the regulation of Scavenger receptor class B-1
(SCARB1), an important factor in steroidogenesis found to be
downregulated in the samples. Due to the downregulation of miR-
455 and miR-125a, there is an induction of SCARB1 on the cell surface
(Bitetto et al., 2022). proliferation, metastasis, and prognosis of colon cancer cells (You et al.,
2019); miR-206 has been reported to increase the migration and
proliferation of colorectal cancer cells (Park et al., 2018); and miR-
let-7e has been reported to diminish tumor growth (Han et al., 2018). Altogether this information could imply using these four miRNAs as
novel targets for the clinical management of NFPA (Zhang et al., 2021). Because of their essential roles in regulating the development of
pituitary tumors, miRNAs have significant clinical value. More
specifically, miRNAs could be used to diagnose the presence and
progression of a tumor and classify it more accurately, predict
invasiveness or aggressiveness, treat or regulate the behavior of
pituitary tumors, and evaluate the success of an operation. SEMA4C, SIRPA, SLC11A, THRB, and VGLL3. These genes are
thought to be involved in immune response, primary cellular
component regulation, and metabolic processes (Fichna et al.,
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Addison’s disease, is the consequence of the malfunction of the
adrenal
cortex
that
causes
chronic
glucocorticoid
or
mineralocorticoid deficiency (Barthel et al., 2019). PAI affects 1 in
every 5,000–7,000 humans worldwide (Saverino and Falorni, 2020). Recent studies have proven the involvement of miRNAs in this
condition. A significant upregulation of miR-7977 was found in
CD4+ T cells obtained from the peripheral blood of patients with
autoimmune Addison’s disease compared to healthy controls (Fichna
et al., 2021). Additionally, a positive correlation was found between
the amount of co-occurring autoimmune disorders and the
abundance of miR-7977, as well as with the expression of miR-
7977 and circulating autoantibodies against thyroid peroxidase
(aTPO). Although the particular target of miR-7977 is still
unknown, 18 potential targets involved in autoimmunity were
identified, and they are ARRB1, CD80, CEBPG, CMKLR1, CRP,
ENTPD1, HIPK2, HRH4, IL16, JAG1, MAVS, P2RY2, SEMA3G, 14 Frontiers in Genetics frontiersin.org Ledesma-Pacheco et al. 10.3389/fgene.2023.1137017 Conflict of interest The Supplementary Material for this article can be found online
at: https://www.frontiersin.org/articles/10.3389/fgene.2023.1137017/
full#supplementary-material 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. Publisher’s note SJL-P and AGU-P conceived, performed the literature search
and wrote the manuscript. ER-J, EG-H, CE-M, and LR-M performed
the literature search and contributed to writing the manuscript. AB,
SRP, AKD, and SJP critically revised the manuscript. All authors
have reviewed and approved the final manuscript. 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
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Selective synthesis and crystal structures of manganese(I) complexes with a bi- or tridentate terpyridine ligand
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Acta crystallographica. Section E, Crystallographic communications
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research communications Selective synthesis and crystal structures of
manganese(I) complexes with a bi- or tridentate
terpyridine ligand Selective synthesis and crystal structures of
manganese(I) complexes with a bi- or tridentate
terpyridine ligand ISSN 2056-9890 Kosei Wadayama,a Tsugiko Takaseb and Dai Oyamab* aGraduate School of Science and Engineering, Fukushima University, 1 Kanayagawa, Fukushima 960-1296, Japan, and
bDepartment of Natural Sciences and Informatics, Fukushima University, 1, Kanayagawa, Fukushima 960-1296, Japan. *Correspondence e-mail: daio@sss.fukushima-u.ac.jp Received 3 June 2020
Accepted 19 June 2020
Edited by M. Weil, Vienna University of
Technology, Austria
Keywords: crystal structure; manganese(I)
complex; terpyridyl ligand; distinct coordination
mode; disorder. CCDC references: 2010792; 2010791
Supporting information: this article has
supporting information at journals.iucr.org/e Received 3 June 2020
Accepted 19 June 2020 The crystal structures of two manganese(I) complexes with a different coord-
ination mode of the supporting ligand are reported: fac-bromidotricarbonyl(40-
phenyl-2,20:60,200-terpyridine-2N,N0)manganese(I), [MnBr(C21H15N3)(CO)3], I,
and cis-bromidodicarbonyl(40-phenyl-2,20:60,200-terpyridine-3N,N0,N00)mangan-
ese(I), [MnBr(C21H15N3)(CO)2], II. In both complexes, the manganese(I) atom
is coordinated by terminal carbonyl ligands, a bromide ion, and a 40-phenyl-
2,20:60,200-terpyridine ligand within a distorted octahedral environment. In I, the
metal ion is facially coordinated by three carbonyl ligands and the terpyridine
ligand binds in a bidentate fashion. The non-coordinating nitrogen atom in the
terpyridine ligand is positioned on the side opposite to the bromido ligand. In II,
the metal ion is coordinated by two carbonyl ligands in a cis configuration and
the terpyridine ligand binds in a tridentate fashion; notably, one carbonyl and
the trans bromido ligand are mutually disordered over two positions. In I, the
complex molecules are linked by C—H Br hydrogen bonds. In II, aromatic
– contacts are present, as well as pairs of C—H Br and C—H O hydrogen
bonds. Edited by M. Weil, Vienna University of
Technology, Austria Keywords: crystal structure; manganese(I)
complex; terpyridyl ligand; distinct coordination
mode; disorder. 1. Chemical context 1. Chemical context 1. Chemical context Carbonylmanganese(I) complexes with polypyridyl ligands
are of particular interest as novel active molecules that are
able
to
release
CO
in
response
to
photoirradiation
(Carrington et al., 2013; Chakraborty et al., 2014; Jimenez et al.,
2015) or as electrocatalysts of CO2 reduction (Grills et al.,
2018; Stanbury et al., 2017). Among these compounds, studies
have
concentrated
mainly
on
tricarbonyl
complexes
comprising bidentate polypyridyl supporting ligands; by
contrast, only few reports exist on dicarbonyl complexes
bearing tridentate ligands (Compain et al., 2015; Machan &
Kubiak, 2016). In fact, even though the typically tridentate
ligands 2,20:60,200-terpyridine and derivatives thereof coordin-
ate to an MnI ion, the majority of them bind the metal ion in a
bidentate manner (Compain et al., 2014; Moya et al., 2001). As indicated by the results of studies focusing on the
comparison
between
carbonylmanganese
complexes
containing bidentate and tridentate terpyridines (Compain et
al., 2015; Machan & Kubiak, 2016), investigating the rela-
tionship between reactivity and molecular structure is a key
research objective. However, comparing these two systems
experimentally is difficult, particularly considering that avail-
able structural data on complexes comprising tridentate
terpyridine ligands are quite scarce. https://doi.org/10.1107/S2056989020008178
1139 1139 Acta Cryst. (2020). E76, 1139–1142 Acta Cryst. (2020). E76, 1139–1142 research communications pyridyl rings remains outside the coordination sphere. The
corresponding non-coordinating N atom, N3, is positioned on
the side opposite to the Br atom. As a result, the torsion angle
between the coordinating and non-coordinating pyridyl rings
in I (N2—C13—C14—N3) is much smaller [47.9 (3)] than
those reported for related MnI complexes with bidentate
terpyridine derivatives (Compain et al., 2014, 2015). The non-
coordinating N atom is positioned in proximity of the equa-
torial carbonyl ligand (C2
O2), with a short value for the
interatomic distance between C2 and N3 [2.900 (4) A˚ ]. Since
this distance is considerably shorter than the sum of the two
atoms’ van der Waals radii (3.25 A˚ ; Bondi, 1964), evidence
suggests that an interaction exists between the free pyridine
and the adjacent CO ligand. This interaction may explain the
observation that the Mn1—C2 distance [1.840 (3) A˚ ] is longer
than the other two corresponding distances in I [Mn1—C1 =
1.805 (3) and Mn1—C3 = 1.796 (3) A˚ ]. Herein,
we
report
the
structural characterization of
complex fac(CO)-[Mn(tpyPh-2N,N0)(CO)3Br] (I; tpyPh = 40-
phenyl-2,20:60,200-terpyridine) comprising a bidentate terpyr-
idine-based ligand, which has been synthesized by Moya et al. 1. Chemical context (2001), and the synthesis and characterization of the corre-
sponding complex cis(CO)-[Mn(tpyPh-3N,N0,N00)(CO)2Br]
(II), whereby the same terpyridine-based ligand is tridentate. The crystal structures of MnI dicarbonyl complexes with
tridentate terpyridines have very rarely been reported
(Compain et al., 2015), because of the instability in solution of
compounds of this type. In II, the carbonyl ligands are in cis
configuration, again in accordance with IR data. Differently
from I, in II the MnI ion is coordinated by a tridentate
terpyridyl ligand, as well as two CO ligands and a Br ion. Only the central Mn—N2 bond is slightly shortened (by
0.05 A˚ ) as a result of geometric constraints. In contrast to I,
where no disorder is observed, in II one of the CO ligands
(C2
O2) and the Br ligand are mutually disordered over
two positions. The dihedral angle between the phenyl pendant
and the central pyridyl ring in II is slightly larger than the
corresponding angle in I. Specifically, the C10—C11—C19—
C20 torsion angle has a value of 19.3 (5) in II and 9.9 (4)
in I, but both values indicate an essential quasi-coplanarity. Notably, the extended conjugation made possible by the
mentioned quasi-planarity may contribute to an increased
stability of these compounds. 5. Synthesis and crystallization In the crystal structure of II, weak C—H Br and C—
H O hydrogen bonding interactions (Table 2) exist between
the terpyridyl ligand and the disordered CO/Br ligands. Additional – interactions [Cg3 Cg2iv = 4.000 (2) and
Cg1 Cg1i = 4.128 (3) A˚ ; Cg1, Cg2 and Cg3 are the centroids
of the N1/C4–C8, N2/C9–C13 and N3/C14–C18 rings, respec-
tively; symmetry codes: (i) 1 x, y, 2 z; (iv) x, y + 1
2, z 1
2]
consolidate the crystal packing. These interactions lead to the
formation of a three-dimensional network structure (Fig. 4). All the manganese(I) complexes were handled and stored in
the dark to minimize exposure to light. Compound I was
synthesized as described by Moya et al. (2001). The compound
thus obtained proved to be analytically and spectroscopically
pure (as determined by microanalysis, IR, UV–vis, and
1H NMR data). Crystals suitable for use in X-ray diffraction
experiments were grown by vapor diffusion of diethyl ether
into an acetone solution of I. For the synthesis of compound II, bromidopentacarbonyl-
manganese(I) (30 mg, 0.11 mmol) and 40-phenyl-2,20:60,200-
terpyridine (31 mg, 0.10 mmol) were dissolved in an acetone–
water mixture (20/30 ml). The solution thus obtained was
refluxed for 24 h; the solvent was then evaporated under
reduced pressure, and the resulting solid was placed in diethyl
ether (50 ml); the resulting mixture was stirred for 30 min to
remove the starting materials and subsequently filtered; the
isolated residue was washed with diethyl ether to obtain a
yield for the desired complex of 43 mg (86%). Single crystals
suitable for X-ray diffraction experiments were grown by slow
vapor diffusion of n-hexane into an acetone solution of II. FTIR CO (KBr pellet): 1916 (s), 1838 (s) cm1. research communications Table 1
Hydrogen-bond geometry (A˚ , ) for I. D—H A
D—H
H A
D A
D—H A
C7—H4 Br1i
0.95
2.83
3.754 (3)
165
C16—H8 Br1ii
0.95
2.88
3.612 (4)
135
C20—H11 Br1i
0.95
2.92
3.844 (2)
163
Symmetry codes: (i) x þ 1; y 1
2; z þ 3
2; (ii) x þ 1; y þ 1
2; z þ 3
2. Table 2
Hydrogen-bond geometry (A˚ , ) for II. D—H A
D—H
H A
D A
D—H A
C5—H2 Br1i
0.95
2.84
3.528 (4)
130
C7—H4 Br1ii
0.95
2.86
3.771 (4)
162
C12—H6 Br2iii
0.95
2.75
3.688 (7)
171
C12—H6 O2iii
0.95
2.55
3.491 (7)
173
C15—H7 Br2iii
0.95
2.81
3.759 (7)
175
C15—H7 O2iii
0.95
2.50
3.447 (7)
172
C16—H8 Br2iv
0.95
2.52
3.286 (7)
138
C16—H8 O2iv
0.95
2.57
3.363 (7)
141
C20—H11 Br1ii
0.95
2.81
3.743 (4)
168
C20—H11 O3ii
0.95
2.55
3.446 (18)
158
C24—H15 Br2iii
0.95
2.84
3.611 (7)
139
Symmetry codes: (i) x þ 1; y; z þ 2; (ii) x; y þ 1
2; z þ 1
2; (iii) x; y þ 1
2; z þ 3
2; (iv)
x; y þ 1
2; z 1
2. Table 2
Hydrogen-bond geometry (A˚ , ) for II. 3. Supramolecular features In the crystal structure of I, complex molecules display three
kinds of C—H Br hydrogen bonds (i.e., between the Br
ligand and the C—H groups in the coordinating pyridyl ring,
the free pyridyl ring, and the phenyl pendant), forming a
three-dimensional supramolecular structure (Table 1 and
Fig. 3). 2. Structural commentary The molecular structures of compounds I and II are displayed
in Figs. 1 and 2, respectively. Although I was prepared by
Moya et al. (2001), its structure has not previously been
determined. In I and II, the manganese(I) atoms exhibit
distorted octahedral coordination environments, similar to
those reported for other structurally related complexes
(Compain et al., 2014, 2015). In I, the fac configuration of the
three CO ligands around the central manganese(I) atom is in
agreement with the IR data of the complex and similar to
those
previously
reported
for
complexes
of
this
type
(Compain et al., 2014, 2015). As can be evinced from Fig. 1, the
terpyridine ligand exhibits a bidentate coordination with
respect to the central MnI atom, so that one of the outer Figure 1
The molecular structure of compound I, with atom labeling and
displacement ellipsoids drawn at the 50% probability level. Figure 2
The molecular structure of compound II, with atom labeling and
displacement ellipsoids drawn at the 50% probability level. Only the
major components (Br1/C2
O2) of the disordered groups are shown. Figure 1
The molecular structure of compound I, with atom labeling and
displacement ellipsoids drawn at the 50% probability level. Figure 2
The molecular structure of compound II, with atom labeling and
displacement ellipsoids drawn at the 50% probability level. Only the
major components (Br1/C2
O2) of the disordered groups are shown. i Figure 2 Figure 1
The molecular structure of compound I, with atom labeling and
displacement ellipsoids drawn at the 50% probability level. Figure 2
The molecular structure of compound II, with atom labeling and
displacement ellipsoids drawn at the 50% probability level. Only the
major components (Br1/C2
O2) of the disordered groups are shown. g
The molecular structure of compound II, with atom labeling and
displacement ellipsoids drawn at the 50% probability level. Only the
major components (Br1/C2
O2) of the disordered groups are shown. 1140
Wadayama et al. [MnBr(C21H15N3)(CO)3] and [MnBr(C21H15N3)(CO)2] 1140
Wadayama et al. [MnBr(C21H15N3)(CO)3] and [MnBr(C21H15N3)(CO)2] Acta Cryst. (2020). E76, 1139–1142 4. Database survey With respect to manganese(I) complexes with a tridentate
terpyridine
derivative
ligand
of
the
form
cis(CO)-
[Mn(tpyR)(CO)2Br], only a single structure, whereby R = p-
tolyl, has been reported (Compain et al., 2015). In contrast,
some structures of bidentate terpyridine derivative-coordi-
nated manganese(I) complexes have been reported by
Compain et al. (2014, 2015). Figure 3
The crystal packing of compound I with C—H Br hydrogen bonds
shown as dashed lines. Figure 3
The crystal packing of compound I with C—H Br hydrogen bonds FTIR CO (KBr pellet): 1916 (s), 1838 (s) cm
. Figure 3
The crystal packing of compound I with C—H Br hydrogen bonds
shown as dashed lines. Figure 4
The crystal packing of compound II with C—H Br and C—H O
hydrogen bonds (blue) and – contacts (green) shown as dashed lines;
ring centroids are shown as red spheres. Figure 4
The crystal packing of compound II with C—H Br and C—H O
hydrogen bonds (blue) and – contacts (green) shown as dashed lines;
ring centroids are shown as red spheres. Figure 3
The crystal packing of compound I with C—H Br hydrogen bonds
shown as dashed lines. Figure 4
The crystal packing of compound II with C—H Br and C—H O
hydrogen bonds (blue) and – contacts (green) shown as dashed lines;
ring centroids are shown as red spheres. Figure 3
The crystal packing of compound I with C—H Br hydrogen bonds
shown as dashed lines. g
The crystal packing of compound II with C—H Br and C—H O
hydrogen bonds (blue) and – contacts (green) shown as dashed lines;
ring centroids are shown as red spheres. Wadayama et al. [MnBr(C21H15N3)(CO)3] and [MnBr(C21H15N3)(CO)2]
1141 Wadayama et al. [MnBr(C21H15N3)(CO)3] and [MnBr(C21H15N3)(CO)2]
1141 1141 Acta Cryst. (2020). E76, 1139–1142 II Crystal data
Chemical formula
[MnBr(C21H15N3)(CO)3]
[MnBr(C21H15N3)(CO)2]
Mr
528.24
500.23
Crystal system, space group
Monoclinic, P21/c
Monoclinic, P21/c
Temperature (K)
93
93
a, b, c (A˚ )
11.6630 (3), 11.6691 (3), 15.8892 (4)
10.497 (3), 14.123 (5), 13.504 (4)
()
103.0774 (7)
96.767 (3)
V (A˚ 3)
2106.39 (10)
1988.0 (11)
Z
4
4
Radiation type
Mo K
Mo K
(mm1)
2.56
2.71
Crystal size (mm)
0.15 0.08 0.03
0.20 0.08 0.05
Data collection
Diffractometer
Rigaku Saturn70
Rigaku Saturn70
Absorption correction
Multi-scan (REQAB, Rigaku, 1998)
Multi-scan (REQAB, Rigaku, 1998)
Tmin, Tmax
0.774, 0.926
0.795, 0.873
No. of measured, independent and
observed [F 2 > 2.0(F 2)] reflections
21455, 4813, 4253
19872, 4518, 4016
Rint
0.030
0.028
(sin /
)max (A˚ 1)
0.649
0.649
Refinement
R[F 2 > 2(F 2)], wR(F 2), S
0.035, 0.092, 1.06
0.046, 0.096, 1.27
No. of reflections
4813
4518
No. of parameters
289
289
No. of restraints
0
3
H-atom treatment
H-atom parameters constrained
H-atom parameters constrained
max, min (e A˚ 3)
0.96, 0.32
0.83, 0.80
Computer programs: PROCESS-AUTO (Rigaku, 1998), CrystalClear (Rigaku, 2008), SIR97 (Altomare et al., 1999), SHELXL2018/3 (Sheldrick, 2015), Mercury (Macrae et al., 2020),
ORTEP-3 for Windows (Farrugia, 2012), CrystalStructure (Rigaku, 2019), PLATON (Spek, 2020) and publCIF (Westrip, 2010). Crystal data
Chemical formula
Mr
Crystal system, space group
Temperature (K)
a, b, c (A˚ )
()
V (A˚ 3)
Z
Radiation type
(mm1)
Crystal size (mm)
Data collection
Diffractometer
Absorption correction
Tmin, Tmax
No. of measured, independent and
observed [F 2 > 2.0(F 2)] reflections
Rint
(sin /
)max (A˚ 1) Computer programs: PROCESS-AUTO (Rigaku, 1998), CrystalClear (Rigaku, 2008), SIR97 (Altomare et al., 1999), SHELXL2018/3 (Sheldrick, 2015), Mercury (Macrae et al., 2020),
ORTEP-3 for Windows (Farrugia, 2012), CrystalStructure (Rigaku, 2019), PLATON (Spek, 2020) and publCIF (Westrip, 2010). Computer programs: PROCESS-AUTO (Rigaku, 1998), CrystalClear (Rigaku, 2008), SIR97 (Altomare et al., 1999), SHELXL2018/3 (Sheldrick, 2015), Mercury (Macrae et al., 2020),
ORTEP-3 for Windows (Farrugia, 2012), CrystalStructure (Rigaku, 2019), PLATON (Spek, 2020) and publCIF (Westrip, 2010). Funding information Wood, P. A. (2020). J. Appl. Cryst. 53, 226–235. Funding for this research was provided by: Japan Society for
the Promotion of Science (grant No. JP17K05799). Moya, S. A., Pastene, R., Le Bozec, H., Baricelli, P. J., Pardey, A. J. & Moya, S. A., Pastene, R., Le Bozec, H., Baricelli, P. J., Pardey, A. J. &
Gimeno, J. (2001). Inorg. Chim. Acta, 312, 7–14. Moya, S. A., Pastene, R., Le Bozec, H., Baricelli, P. Gimeno, J. (2001). Inorg. Chim. Acta, 312, 7–14. Rigaku (1998). REQAB and PROCESS-AUTO. Rigaku Corpora-
tion, Tokyo, Japan. 6. Refinement Chakraborty, I., Carrington, S. J. & Mascharak, P. K. (2014). ChemMedChem, 9, 1266–1274. Crystal data, data collection, and structure refinement details
are summarized in Table 3. All hydrogen atoms were placed at
calculated positions (C—H = 0.95 A˚ ) and refined using a
riding model with Uiso(H) = 1.2Ueq(C). In compound II, the
CO group and the Br atom trans to it were refined as being
disordered over two sets of sites, (Br1/C2
O2) and (Br2/
C3
O3), respectively, with an occupancy ratio of 0.807 (2):
0.193 (2). Compain, J.-D., Bourrez, M., Haukka, M., Deronzier, A. & Chardon-
Noblat, S. (2014). Chem. Commun. 50, 2539–2542. Compain, J.-D., Bourrez, M., Haukka, M., Deronzier, Compain, J.-D., Stanbury, M., Trejo, M. & Chardon-Noblat, S. (2015). Eur. J. Inorg. Chem. pp. 5757–5766. Farrugia, L. J. (2012). J. Appl. Cryst. 45, 849–854. Grills, D. C., Ertem, M. Z., McKinnon, M., Ngo, K. T. & Rochford, J. (2018). Coord. Chem. Rev. 374, 173–217. Jimenez, J., Chakraborty, I. & Mascharak, P. K. (2015). Eur. J. Inorg. Chem. pp. 5021–5026. Machan, C. W. & Kubiak, C. P. (2016). Dalton Trans. 45, 17179–17186. Macrae, C. F., Sovago, I., Cottrell, S. J., Galek, P. T. A., McCabe, P.,
Pidcock, E., Platings, M., Shields, G. P., Stevens, J. S., Towler, M. &
Wood, P. A. (2020). J. Appl. Cryst. 53, 226–235. research communications Table 3
Experimental details. Table 3
Experimental details. Altomare, A., Burla, M. C., Camalli, M., Cascarano, G. L.,
Giacovazzo, C., Guagliardi, A., Moliterni, A. G. G., Polidori, G.
& Spagna, R. (1999). J. Appl. Cryst. 32, 115–119.
Bondi, A. (1964). J. Phys. Chem. 68, 441–451.
Carrington, S. J., Chakraborty, I. & Mascharak, P. K. (2013). Chem.
Commun. 49, 11254–11256. References y
p
Rigaku (2008). CrystalClear. Rigaku Corporation, Tokyo, Japan. Altomare, A., Burla, M. C., Camalli, M., Cascarano, G. L.,
Giacovazzo, C., Guagliardi, A., Moliterni, A. G. G., Polidori, G. & Spagna, R. (1999). J. Appl. Cryst. 32, 115–119. Bondi, A. (1964). J. Phys. Chem. 68, 441–451. Carrington, S. J., Chakraborty, I. & Mascharak, P. K. (2013). Chem. Commun. 49, 11254–11256. Rigaku (2019). CrystalStructure. Rigaku Corporation, Tokyo, Japan. Altomare, A., Burla, M. C., Camalli, M., Cascarano, G. L.,
Giacovazzo, C., Guagliardi, A., Moliterni, A. G. G., Polidori, G. & Spagna, R. (1999). J. Appl. Cryst. 32, 115–119. Bondi, A. (1964). J. Phys. Chem. 68, 441–451. Carrington, S. J., Chakraborty, I. & Mascharak, P. K. (2013). Chem. Commun. 49, 11254–11256. Sheldrick, G. M. (2015). Acta Cryst. C71, 3–8. Spek, A. L. (2020). Acta Cryst. E76, 1–11. (
)
Stanbury, M., Compain, J.-D., Trejo, M., Smith, P., Goure´, E. &
Chardon-Noblat, S. (2017). Electrochim. Acta, 240, 288–299. Stanbury, M., Compain, J.-D., Trejo, M., Smith, P., Goure´, E. & Stanbury, M., Compain, J.-D., Trejo, M., Smith, P., Goure, E. &
Chardon-Noblat, S. (2017). Electrochim. Acta, 240, 288–299. Westrip S P (2010) J Appl Cryst 43 920 925 Chardon-Noblat, S. (2017). Electrochim. Acta, 240, 288–299. Westrip, S. P. (2010). J. Appl. Cryst. 43, 920–925. Chardon Noblat, S. (2017). Electrochim. Acta, 2
Westrip, S. P. (2010). J. Appl. Cryst. 43, 920–925. 1142
Wadayama et al. [MnBr(C21H15N3)(CO)3] and [MnBr(C21H15N3)(CO)2] Acta Cryst. (2020). E76, 1139–1142 supporting information supporting information a Cryst. (2020). E76, 1139-1142 [https://doi.org/10.1107/S2056989020008178] Selective synthesis and crystal structures of manganese(I) complexes with a bi-
or tridentate terpyridine ligand Kosei Wadayama, Tsugiko Takase and Dai Oyama Kosei Wadayama, Tsugiko Takase and Dai Oyama Computing details Data collection: PROCESS-AUTO (Rigaku, 1998) for (I); CrystalClear (Rigaku, 2008) for (II). Cell refinement:
PROCESS-AUTO (Rigaku, 1998) for (I); CrystalClear (Rigaku, 2008) for (II). Data reduction: PROCESS-AUTO
(Rigaku, 1998) for (I); CrystalClear (Rigaku, 2008) for (II). For both structures, program(s) used to solve structure:
SIR97 (Altomare et al., 1999); program(s) used to refine structure: SHELXL2018/3 (Sheldrick, 2015); molecular
graphics: Mercury (Macrae et al., 2020), ORTEP-3 for Windows (Farrugia, 2012); software used to prepare material for
publication: CrystalStructure (Rigaku, 2019), PLATON (Spek, 2020), publCIF (Westrip, 2010). fac-Bromidotricarbonyl(4′-phenyl-2,2′:6′,2′′-terpyridine-κ2N,N′)manganese(I) (I) sup-1
Acta Cryst. (2020). supporting information E76, 1139-1142
Crystal data
[MnBr(C21H15N3)(CO)3]
Mr = 528.24
Monoclinic, P21/c
a = 11.6630 (3) Å
b = 11.6691 (3) Å
c = 15.8892 (4) Å
β = 103.0774 (7)°
V = 2106.39 (10) Å3
Z = 4
F(000) = 1056.00
Dx = 1.666 Mg m−3
Mo Kα radiation, λ = 0.71075 Å
Cell parameters from 18973 reflections
θ = 3.0–27.5°
µ = 2.56 mm−1
T = 93 K
Platelet, orange
0.15 × 0.08 × 0.03 mm
Data collection
Rigaku Saturn70
diffractometer
Detector resolution: 7.143 pixels mm-1
ω scans
Absorption correction: multi-scan
(REQAB, Rigaku, 1998)
Tmin = 0.774, Tmax = 0.926
21455 measured reflections
4813 independent reflections
4253 reflections with F2 > 2.0σ(F2)
Rint = 0.030
θmax = 27.5°, θmin = 3.0°
h = −15→15
k = −15→15
l = −20→19
Refinement
Refinement on F2
R[F2 > 2σ(F2)] = 0.035
wR(F2) = 0.092
S = 1.06
4813 reflections
289 parameters
0 restraints
Primary atom site location: structure-invariant
direct methods
Secondary atom site location: difference Fourier
map
Hydrogen site location: inferred from
neighbouring sites
H-atom parameters constrained Crystal data
[MnBr(C21H15N3)(CO)3]
Mr = 528.24
Monoclinic, P21/c
a = 11.6630 (3) Å
b = 11.6691 (3) Å
c = 15.8892 (4) Å
β = 103.0774 (7)°
V = 2106.39 (10) Å3
Z = 4
F(000) = 1056.00
Dx = 1.666 Mg m−3
Mo Kα radiation, λ = 0.71075 Å
Cell parameters from 18973 reflections
θ = 3.0–27.5°
µ = 2.56 mm−1
T = 93 K
Platelet, orange
0.15 × 0.08 × 0.03 mm 4813 independent reflections
4253 reflections with F2 > 2.0σ(F2)
Rint = 0.030
θmax = 27.5°, θmin = 3.0°
h = −15→15
k = −15→15
l = −20→19 supporting information w = 1/[σ2(Fo2) + (0.0416P)2 + 2.6282P]
where P = (Fo2 + 2Fc2)/3
(Δ/σ)max = 0.001
Δρmax = 0.96 e Å−3
Δρmin = −0.32 e Å−3
Special details
Refinement. Refinement was performed using all reflections. The weighted R-factor (wR) and goodness of fit (S) are
based on F2. R-factor (gt) are based on F. The threshold expression of F2 > 2.0 sigma(F2) is used only for calculating R-
factor (gt). w = 1/[σ2(Fo2) + (0.0416P)2 + 2.6282P]
where P = (Fo2 + 2Fc2)/3
(Δ/σ)max = 0.001
Δρmax = 0.96 e Å−3
Δρmin = −0.32 e Å−3
S
i l d t il Δρmax = 0.96 e Å−3
Δρmin = −0.32 e Å−3 p
Refinement. Refinement was performed using all reflections. The weighted R-factor (wR) and goodness of fit (S) are
based on F2. R-factor (gt) are based on F. The threshold expression of F2 > 2.0 sigma(F2) is used only for calculating R-
factor (gt). Refinement. Refinement was performed using all reflections. The weighted R-factor (wR) and goodness of fit (S) are
based on F2. R-factor (gt) are based on F. The threshold expression of F2 > 2.0 sigma(F2) is used only for calculating R-
factor (gt). Refinement Primary atom site location: structure-invariant
direct methods
Secondary atom site location: difference Fourier
map
Hydrogen site location: inferred from
neighbouring sites
H-atom parameters constrained sup-1 Acta Cryst. (2020). E76, 1139-1142 supporting information supporting information Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2)
x
y
z
Uiso*/Ueq
Br1
0.61476 (2)
0.17999 (2)
0.88742 (2)
0.02812 (9)
Mn1
0.78248 (3)
0.17015 (3)
0.81054 (2)
0.02513 (10)
O1
0.99149 (17)
0.17222 (19)
0.73649 (14)
0.0400 (5)
O2
0.7925 (2)
0.42515 (18)
0.81993 (14)
0.0444 (5)
O3
0.95375 (19)
0.1798 (2)
0.97692 (14)
0.0457 (5)
N1
0.76212 (17)
−0.00325 (19)
0.81015 (13)
0.0252 (4)
N2
0.65411 (17)
0.14582 (19)
0.69692 (13)
0.0238 (4)
N3
0.7794 (2)
0.3305 (2)
0.62983 (17)
0.0359 (5)
C1
0.9075 (2)
0.1690 (2)
0.76117 (18)
0.0320 (6)
C2
0.7848 (3)
0.3278 (3)
0.81319 (19)
0.0354 (6)
C3
0.8852 (2)
0.1758 (2)
0.91343 (18)
0.0327 (6)
C4
0.8291 (2)
−0.0776 (2)
0.86473 (17)
0.0313 (6)
H1
0.893046
−0.048384
0.907298
0.038*
C5
0.8095 (2)
−0.1941 (3)
0.86198 (19)
0.0358 (6)
H2
0.860275
−0.243958
0.900658
0.043*
C6
0.7144 (2)
−0.2373 (3)
0.80188 (19)
0.0350 (6)
H3
0.698453
−0.317169
0.798723
0.042*
C7
0.6431 (2)
−0.1615 (2)
0.74643 (18)
0.0313 (6)
H4
0.576614
−0.188952
0.705236
0.038*
C8
0.6693 (2)
−0.0454 (2)
0.75134 (15)
0.0247 (5)
C9
0.6038 (2)
0.0406 (2)
0.69157 (15)
0.0232 (5)
C10
0.4996 (2)
0.0143 (2)
0.63271 (15)
0.0232 (5)
H5
0.466483
−0.060220
0.632112
0.028*
C11
0.44359 (19)
0.0971 (2)
0.57461 (15)
0.0214 (5)
C12
0.5003 (2)
0.2023 (2)
0.57681 (16)
0.0248 (5)
H6
0.467288
0.260028
0.536409
0.030*
C13
0.6042 (2)
0.2244 (2)
0.63698 (16)
0.0254 (5)
C14
0.6644 (2)
0.3362 (2)
0.63063 (17)
0.0280 (5)
C15
0.6033 (2)
0.4378 (2)
0.62349 (19)
0.0326 (6)
H7
0.522062
0.438538
0.624227
0.039*
C16
0.6619 (3)
0.5393 (3)
0.6152 (2)
0.0418 (7)
H8
0.621545
0.610530
0.610537
0.050*
C17
0.7798 (3)
0.5349 (3)
0.6138 (2)
0.0446 (7)
H9
0.822144
0.602872
0.607979
0.054*
C18
0.8345 (3)
0.4297 (3)
0.6212 (2)
0.0403 (7)
H10
0.915543
0.426919
0.620071
0.048*
C19
0.3301 (2)
0.0749 (2)
0.51219 (15)
0.0215 (5) Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) l atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) Acta Cryst. (2020). supporting information E76, 1139-1142 sup-2 supporting information
C20
0.2826 (2)
−0.0347 (2)
0.49944 (17)
0.0316 (6)
H11
0.324220
−0.097276
0.530401
0.038*
C21
0.1756 (3)
−0.0542 (3)
0.44231 (19)
0.0389 (7)
H12
0.144475
−0.129640
0.434142
0.047*
C22
0.1145 (2)
0.0361 (3)
0.39740 (19)
0.0373 (6)
H13
0.039539
0.023966
0.359983
0.045*
C23
0.1626 (3)
0.1429 (3)
0.4072 (2)
0.0453 (8)
H14
0.122516
0.204709
0.374184
0.054*
C24
0.2688 (3)
0.1626 (2)
0.4644 (2)
0.0402 (7)
H15
0.300073
0.238042
0.470790
0.048*
Atomic displacement parameters (Å2)
U11
U22
U33
U12
U13
U23
Br1
0.02474 (13)
0.03228 (14)
0.02610 (14)
0.00130 (9)
0.00315 (10)
−0.00370 (10)
Mn1
0.01744 (18)
0.0327 (2)
0.0221 (2)
−0.00087 (14)
−0.00207 (14)
−0.00159 (15)
O1
0.0209 (9)
0.0603 (14)
0.0372 (11)
−0.0046 (9)
0.0033 (8)
−0.0054 (10)
O2
0.0517 (13)
0.0352 (11)
0.0424 (12)
−0.0087 (10)
0.0023 (10)
−0.0043 (9)
O3
0.0325 (11)
0.0571 (14)
0.0377 (12)
−0.0050 (9)
−0.0125 (9)
−0.0002 (10)
N1
0.0163 (9)
0.0359 (11)
0.0211 (10)
0.0032 (8)
−0.0007 (8)
−0.0008 (8)
N2
0.0168 (9)
0.0321 (11)
0.0209 (10)
0.0015 (8)
0.0005 (8)
−0.0036 (8)
N3
0.0263 (11)
0.0384 (13)
0.0427 (14)
−0.0042 (9)
0.0072 (10)
−0.0009 (10)
C1
0.0241 (13)
0.0399 (15)
0.0271 (13)
−0.0049 (10)
−0.0045 (10)
−0.0031 (11)
C2
0.0300 (14)
0.0417 (16)
0.0323 (15)
−0.0015 (11)
0.0028 (11)
−0.0017 (12)
C3
0.0303 (14)
0.0343 (14)
0.0312 (14)
−0.0039 (11)
0.0019 (11)
−0.0009 (11)
C4
0.0212 (12)
0.0407 (15)
0.0271 (13)
0.0043 (10)
−0.0049 (10)
−0.0010 (11)
C5
0.0266 (13)
0.0432 (16)
0.0323 (15)
0.0071 (11)
−0.0041 (11)
0.0091 (12)
C6
0.0300 (13)
0.0368 (15)
0.0345 (15)
−0.0005 (11)
−0.0005 (11)
0.0060 (12)
C7
0.0252 (12)
0.0385 (15)
0.0266 (13)
−0.0036 (10)
−0.0017 (10)
0.0053 (11)
C8
0.0178 (11)
0.0354 (13)
0.0198 (11)
0.0014 (9)
0.0019 (9)
0.0013 (10)
C9
0.0178 (10)
0.0328 (13)
0.0186 (11)
0.0010 (9)
0.0031 (9)
0.0008 (9)
C10
0.0187 (10)
0.0280 (12)
0.0212 (11)
−0.0022 (9)
0.0009 (9)
0.0020 (9)
C11
0.0165 (10)
0.0282 (12)
0.0184 (11)
0.0000 (9)
0.0016 (8)
−0.0003 (9)
C12
0.0185 (11)
0.0276 (12)
0.0263 (12)
0.0017 (9)
0.0007 (9)
0.0011 (10)
C13
0.0196 (11)
0.0287 (12)
0.0267 (13)
0.0008 (9)
0.0027 (10)
−0.0034 (10)
C14
0.0246 (12)
0.0308 (13)
0.0276 (13)
−0.0025 (10)
0.0038 (10)
−0.0005 (10)
C15
0.0259 (12)
0.0313 (13)
0.0412 (15)
−0.0036 (10)
0.0089 (11)
−0.0030 (11)
C16
0.0374 (15)
0.0309 (14)
0.0568 (19)
−0.0007 (12)
0.0103 (14)
0.0005 (13)
C17
0.0380 (16)
0.0354 (15)
0.060 (2)
−0.0105 (13)
0.0105 (14)
0.0009 (14)
C18
0.0279 (14)
0.0428 (16)
0.0506 (18)
−0.0074 (12)
0.0100 (13)
−0.0054 (14)
C19
0.0179 (10)
0.0283 (12)
0.0172 (11)
0.0023 (9)
0.0018 (9)
−0.0002 (9)
C20
0.0300 (13)
0.0320 (13)
0.0278 (13)
−0.0007 (10)
−0.0039 (11)
0.0056 (11)
C21
0.0344 (15)
0.0347 (15)
0.0399 (16)
−0.0088 (12)
−0.0078 (12)
0.0015 (12)
C22
0.0246 (13)
0.0417 (16)
0.0368 (15)
0.0031 (11)
−0.0115 (11)
−0.0062 (12)
C23
0.0420 (17)
0.0332 (15)
0.0465 (18)
0.0079 (13)
−0.0194 (14)
0.0018 (13)
C24
0.0377 (16)
0.0266 (13)
0.0432 (17)
−0.0041 (11)
−0.0182 (13)
0.0049 (12) supporting information supporting information Atomic displacement parameters (Å2) sup-3 Acta Cryst. (2020). E76, 1139-1142 sup-3 supporting information supporting information supporting information C9—N2—Mn1
113.14 (16)
C15—C16—H8
120.6
C13—N2—Mn1
128.74 (17)
C18—C17—C16
118.4 (3)
C18—N3—C14
117.3 (2)
C18—C17—H9
120.8
O1—C1—Mn1
174.2 (2)
C16—C17—H9
120.8
O2—C2—Mn1
175.2 (3)
N3—C18—C17
123.6 (3)
O3—C3—Mn1
177.3 (3)
N3—C18—H10
118.2
N1—C4—C5
123.3 (2)
C17—C18—H10
118.2
N1—C4—H1
118.4
C24—C19—C20
117.6 (2)
C5—C4—H1
118.4
C24—C19—C11
120.9 (2)
C4—C5—C6
118.8 (3)
C20—C19—C11
121.5 (2)
C4—C5—H2
120.6
C21—C20—C19
121.1 (2)
C6—C5—H2
120.6
C21—C20—H11
119.5
C7—C6—C5
118.6 (3)
C19—C20—H11
119.5
C7—C6—H3
120.7
C22—C21—C20
119.8 (3)
C5—C6—H3
120.7
C22—C21—H12
120.1
C6—C7—C8
119.7 (2)
C20—C21—H12
120.1
C6—C7—H4
120.2
C23—C22—C21
119.3 (2)
C8—C7—H4
120.2
C23—C22—H13
120.3
N1—C8—C7
121.7 (2)
C21—C22—H13
120.3
N1—C8—C9
114.5 (2)
C22—C23—C24
120.9 (3)
C7—C8—C9
123.7 (2)
C22—C23—H14
119.6
N2—C9—C10
122.9 (2)
C24—C23—H14
119.6
N2—C9—C8
115.1 (2)
C19—C24—C23
121.2 (3)
C10—C9—C8
122.0 (2)
C19—C24—H15
119.4
C9—C10—C11
120.0 (2)
C23—C24—H15
119.4
C8—N1—C4—C5
1.8 (4)
Mn1—N2—C13—C14
20.3 (3)
Mn1—N1—C4—C5
178.9 (2)
C11—C12—C13—N2
−1.1 (4)
N1—C4—C5—C6
−1.9 (5)
C11—C12—C13—C14
175.4 (2)
C4—C5—C6—C7
0.4 (4)
C18—N3—C14—C15
−0.3 (4)
C5—C6—C7—C8
1.0 (4)
C18—N3—C14—C13
177.8 (3)
C4—N1—C8—C7
−0.3 (4)
N2—C13—C14—N3
47.9 (3)
Mn1—N1—C8—C7
−177.7 (2)
C12—C13—C14—N3
−128.7 (3)
C4—N1—C8—C9
−177.3 (2)
N2—C13—C14—C15
−134.0 (3)
Mn1—N1—C8—C9
5.3 (3)
C12—C13—C14—C15
49.5 (4)
C6—C7—C8—N1
−1.1 (4)
N3—C14—C15—C16
−0.1 (4)
C6—C7—C8—C9
175.7 (2)
C13—C14—C15—C16
−178.1 (3)
C13—N2—C9—C10
−5.6 (3)
C14—C15—C16—C17
0.4 (5)
Mn1—N2—C9—C10
164.82 (19)
C15—C16—C17—C18
−0.3 (5)
C13—N2—C9—C8
173.0 (2)
C14—N3—C18—C17
0.5 (5)
Mn1—N2—C9—C8
−16.7 (3)
C16—C17—C18—N3
−0.2 (5)
N1—C8—C9—N2
7.9 (3)
C12—C11—C19—C24
−10.3 (4)
C7—C8—C9—N2
−169.1 (2)
C10—C11—C19—C24
170.7 (3)
N1—C8—C9—C10
−173.6 (2)
C12—C11—C19—C20
169.1 (2)
C7—C8—C9—C10
9.4 (4)
C10—C11—C19—C20
−9.9 (4)
N2—C9—C10—C11
1.4 (4)
C24—C19—C20—C21
−1.8 (4)
C8—C9—C10—C11
−177.0 (2)
C11—C19—C20—C21
178.7 (3)
C9—C10—C11—C12
2.9 (3)
C19—C20—C21—C22
−0.2 (5) 113.14 (16)
C15—C16—H8
120.6
128.74 (17)
C18—C17—C16
118.4 (3)
117.3 (2)
C18—C17—H9
120.8
174.2 (2)
C16—C17—H9
120.8
175.2 (3)
N3—C18—C17
123.6 (3)
177.3 (3)
N3—C18—H10
118.2
123.3 (2)
C17—C18—H10
118.2
118.4
C24—C19—C20
117.6 (2)
118.4
C24—C19—C11
120.9 (2)
118.8 (3)
C20—C19—C11
121.5 (2)
120.6
C21—C20—C19
121.1 (2)
120.6
C21—C20—H11
119.5
118.6 (3)
C19—C20—H11
119.5
120.7
C22—C21—C20
119.8 (3)
120.7
C22—C21—H12
120.1
119.7 (2)
C20—C21—H12
120.1
120.2
C23—C22—C21
119.3 (2)
120.2
C23—C22—H13
120.3
121.7 (2)
C21—C22—H13
120.3
114.5 (2)
C22—C23—C24
120.9 (3)
123.7 (2)
C22—C23—H14
119.6
122.9 (2)
C24—C23—H14
119.6
115.1 (2)
C19—C24—C23
121.2 (3)
122.0 (2)
C19—C24—H15
119.4
120.0 (2)
C23—C24—H15
119.4
1.8 (4)
Mn1—N2—C13—C14
20.3 (3)
178.9 (2)
C11—C12—C13—N2
−1.1 (4)
−1.9 (5)
C11—C12—C13—C14
175.4 (2)
0.4 (4)
C18—N3—C14—C15
−0.3 (4)
1.0 (4)
C18—N3—C14—C13
177.8 (3)
−0.3 (4)
N2—C13—C14—N3
47.9 (3)
−177.7 (2)
C12—C13—C14—N3
−128.7 (3)
−177.3 (2)
N2—C13—C14—C15
−134.0 (3)
5.3 (3)
C12—C13—C14—C15
49.5 (4)
−1.1 (4)
N3—C14—C15—C16
−0.1 (4)
175.7 (2)
C13—C14—C15—C16
−178.1 (3)
−5.6 (3)
C14—C15—C16—C17
0.4 (5)
164.82 (19)
C15—C16—C17—C18
−0.3 (5)
173.0 (2)
C14—N3—C18—C17
0.5 (5)
−16.7 (3)
C16—C17—C18—N3
−0.2 (5)
7.9 (3)
C12—C11—C19—C24
−10.3 (4)
−169.1 (2)
C10—C11—C19—C24
170.7 (3)
−173.6 (2)
C12—C11—C19—C20
169.1 (2)
9.4 (4)
C10—C11—C19—C20
−9.9 (4)
1.4 (4)
C24—C19—C20—C21
−1.8 (4)
−177.0 (2)
C11—C19—C20—C21
178.7 (3)
2.9 (3)
C19—C20—C21—C22
−0.2 (5) sup-5 Acta Cryst. supporting information Geometric parameters (Å, º)
Br1—Mn1
2.5325 (5)
C10—H5
0.9500
Mn1—C3
1.796 (3)
C11—C12
1.390 (3)
Mn1—C1
1.805 (3)
C11—C19
1.486 (3)
Mn1—C2
1.840 (3)
C12—C13
1.388 (3)
Mn1—N1
2.037 (2)
C12—H6
0.9500
Mn1—N2
2.088 (2)
C13—C14
1.496 (3)
O1—C1
1.135 (4)
C14—C15
1.375 (4)
O2—C2
1.143 (3)
C15—C16
1.388 (4)
O3—C3
1.138 (3)
C15—H7
0.9500
N1—C4
1.345 (3)
C16—C17
1.381 (4)
N1—C8
1.353 (3)
C16—H8
0.9500
N2—C9
1.355 (3)
C17—C18
1.376 (4)
N2—C13
1.355 (3)
C17—H9
0.9500
N3—C18
1.346 (4)
C18—H10
0.9500
N3—C14
1.347 (3)
C19—C24
1.374 (3)
C4—C5
1.377 (4)
C19—C20
1.391 (4)
C4—H1
0.9500
C20—C21
1.386 (4)
C5—C6
1.384 (4)
C20—H11
0.9500
C5—H2
0.9500
C21—C22
1.377 (4)
C6—C7
1.385 (4)
C21—H12
0.9500
C6—H3
0.9500
C22—C23
1.361 (4)
C7—C8
1.387 (4)
C22—H13
0.9500
C7—H4
0.9500
C23—C24
1.379 (4)
C8—C9
1.471 (3)
C23—H14
0.9500
C9—C10
1.390 (3)
C24—H15
0.9500
C10—C11
1.393 (3)
C3—Mn1—C1
87.58 (13)
C9—C10—H5
120.0
C3—Mn1—C2
86.53 (13)
C11—C10—H5
120.0
C1—Mn1—C2
90.48 (13)
C12—C11—C10
116.6 (2)
C3—Mn1—N1
95.30 (10)
C12—C11—C19
121.2 (2)
C1—Mn1—N1
95.53 (11)
C10—C11—C19
122.3 (2)
C2—Mn1—N1
173.78 (11)
C13—C12—C11
121.2 (2)
C3—Mn1—N2
172.86 (11)
C13—C12—H6
119.4
C1—Mn1—N2
96.60 (10)
C11—C12—H6
119.4
C2—Mn1—N2
99.18 (11)
N2—C13—C12
121.9 (2)
N1—Mn1—N2
78.58 (8)
N2—C13—C14
120.3 (2)
C3—Mn1—Br1
89.33 (9)
C12—C13—C14
117.7 (2)
C1—Mn1—Br1
176.28 (9)
N3—C14—C15
122.7 (2)
C2—Mn1—Br1
87.27 (9)
N3—C14—C13
116.2 (2)
N1—Mn1—Br1
86.80 (6)
C15—C14—C13
121.0 (2)
N2—Mn1—Br1
86.69 (6)
C14—C15—C16
119.1 (3)
C4—N1—C8
117.9 (2)
C14—C15—H7
120.4
C4—N1—Mn1
126.07 (17)
C16—C15—H7
120.4
C8—N1—Mn1
115.98 (16)
C17—C16—C15
118.9 (3)
C9—N2—C13
117.3 (2)
C17—C16—H8
120.6 sup-4 Acta Cryst. (2020). E76, 1139-1142 supporting information Acta Cryst. (2020). E76, 1139-1142 supporting information (2020). E76, 1139-1142 supporting information C9—C10—C11—C19
−178.0 (2)
C20—C21—C22—C23
2.7 (5)
C10—C11—C12—C13
−3.1 (4)
C21—C22—C23—C24
−3.1 (5)
C19—C11—C12—C13
177.8 (2)
C20—C19—C24—C23
1.4 (5)
C9—N2—C13—C12
5.3 (3)
C11—C19—C24—C23
−179.1 (3)
Mn1—N2—C13—C12
−163.30 (18)
C22—C23—C24—C19
1.0 (6)
C9—N2—C13—C14
−171.0 (2) Hydrogen-bond geometry (Å, º)
D—H···A
D—H
H···A
D···A
D—H···A
C7—H4···Br1i
0.95
2.83
3.754 (3)
165
C16—H8···Br1ii
0.95
2.88
3.612 (4)
135
C20—H11···Br1i
0.95
2.92
3.844 (2)
163
Symmetry codes: (i) −x+1, y−1/2, −z+3/2; (ii) −x+1, y+1/2, −z+3/2. Hydrogen-bond geometry (Å, º) cis-Bromidodicarbonyl(4′-phenyl-2,2′:6′,2′′-terpyridine-κ3N,N′,N′′)manganese(I) (II) cis-Bromidodicarbonyl(4′-phenyl-2,2′:6′,2′′-terpyridine-κ3N,N′,N′′)manganese(I) (II) Crystal data
[MnBr(C21H15N3)(CO)2]
Mr = 500.23
Monoclinic, P21/c
a = 10.497 (3) Å
b = 14.123 (5) Å
c = 13.504 (4) Å
β = 96.767 (3)°
V = 1988.0 (11) Å3
Z = 4
F(000) = 1000.00
Dx = 1.671 Mg m−3
Mo Kα radiation, λ = 0.71075 Å
Cell parameters from 5160 reflections
θ = 3.0–27.5°
µ = 2.71 mm−1
T = 93 K
Block, red
0.20 × 0.08 × 0.05 mm F(000) = 1000.00
Dx = 1.671 Mg m−3
Mo Kα radiation, λ = 0.71075 Å
Cell parameters from 5160 reflections
θ = 3.0–27.5°
µ = 2.71 mm−1
T = 93 K
Block, red
0.20 × 0.08 × 0.05 mm 4518 independent reflections
4016 reflections with F2 > 2.0σ(F2)
Rint = 0.028
θmax = 27.5°, θmin = 3.0°
h = −13→13
k = −18→18
l = −17→17 4518 independent reflections
4016 reflections with F2 > 2.0σ(F2)
Rint = 0.028
θmax = 27.5°, θmin = 3.0°
h = −13→13
k = −18→18
l = −17→17 4518 independent reflections
4016 reflections with F2 > 2.0σ(F2)
Rint = 0.028
θmax = 27.5°, θmin = 3.0°
h = −13→13
k = −18→18
l = −17→17 Refinement Secondary atom site location: difference Fourier
map
Hydrogen site location: inferred from
neighbouring sites
H-atom parameters constrained
w = 1/[σ2(Fo2) + (0.0039P)2 + 5.6867P]
where P = (Fo2 + 2Fc2)/3
(Δ/σ)max < 0.001
Δρmax = 0.83 e Å−3
Δρmin = −0.80 e Å−3 Secondary atom site location: difference Fourier
map Primary atom site location: structure-invariant
direct methods sup-6 Acta Cryst. (2020). E76, 1139-1142 sup-6 supporting information p
Refinement. Refinement was performed using all reflections. The weighted R-factor (wR) and goodness of fit (S) are
based on F2. R-factor (gt) are based on F. The threshold expression of F2 > 2.0 sigma(F2) is used only for calculating R-
factor (gt). Refinement. Refinement was performed using all reflections. The weighted R-factor (wR) and goodness of fit (S) are
based on F2. R-factor (gt) are based on F. The threshold expression of F2 > 2.0 sigma(F2) is used only for calculating R-
factor (gt). Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2)
x
y
z
Uiso*/Ueq
Occ. Acta Cryst. (2020). E76, 1139-1142 Refinement (<1)
Br1
0.39194 (6)
0.11344 (3)
0.73710 (4)
0.02535 (17)
0.807 (2)
O2
−0.0653 (5)
0.0445 (4)
0.8859 (4)
0.0351 (15)
0.807 (2)
C2
0.0332 (9)
0.0641 (9)
0.8536 (10)
0.037 (3)
0.807 (2)
Br2
−0.0197 (5)
0.0661 (4)
0.8657 (4)
0.0403 (14)
0.193 (2)
O3
0.4377 (18)
0.1006 (12)
0.7111 (13)
0.033 (4)*
0.193 (2)
C3
0.339 (3)
0.092 (3)
0.746 (3)
0.076 (12)*
0.193 (2)
Mn1
0.18158 (5)
0.08786 (4)
0.80533 (4)
0.02451 (14)
O1
0.1797 (3)
−0.10749 (19)
0.7376 (2)
0.0359 (6)
N1
0.2796 (3)
0.0760 (2)
0.9421 (2)
0.0224 (6)
N2
0.2005 (3)
0.22091 (19)
0.8440 (2)
0.0202 (6)
N3
0.0937 (3)
0.1521 (2)
0.6822 (2)
0.0219 (6)
C1
0.1807 (4)
−0.0334 (3)
0.7637 (3)
0.0310 (8)
C4
0.3125 (3)
−0.0050 (3)
0.9917 (3)
0.0275 (8)
H1
0.288826
−0.063547
0.960043
0.033*
C5
0.3784 (4)
−0.0070 (3)
1.0856 (3)
0.0298 (8)
H2
0.400366
−0.065698
1.117441
0.036*
C6
0.4125 (4)
0.0773 (3)
1.1335 (3)
0.0301 (8)
H3
0.458098
0.077281
1.198549
0.036*
C7
0.3789 (3)
0.1623 (3)
1.0847 (3)
0.0257 (7)
H4
0.400583
0.221221
1.116233
0.031*
C8
0.3132 (3)
0.1592 (2)
0.9894 (3)
0.0217 (7)
C9
0.2711 (3)
0.2443 (2)
0.9303 (3)
0.0212 (7)
C10
0.3006 (3)
0.3376 (2)
0.9548 (3)
0.0227 (7)
H5
0.351261
0.352547
1.015754
0.027*
C11
0.2548 (3)
0.4096 (2)
0.8886 (3)
0.0223 (7)
C12
0.1818 (3)
0.3838 (2)
0.7994 (3)
0.0225 (7)
H6
0.149023
0.431227
0.753384
0.027*
C13
0.1572 (3)
0.2887 (2)
0.7779 (3)
0.0208 (7)
C14
0.0918 (3)
0.2483 (2)
0.6854 (3)
0.0210 (7)
C15
0.0335 (3)
0.3016 (3)
0.6061 (3)
0.0251 (7)
H7
0.033795
0.368774
0.609582
0.030*
C16
−0.0251 (3)
0.2560 (3)
0.5220 (3)
0.0274 (8)
H8
−0.066553
0.291366
0.467603
0.033*
C17
−0.0223 (3)
0.1587 (3)
0.5187 (3)
0.0277 (8)
H9
−0.060962
0.125743
0.461531
0.033*
C18
0.0376 (3)
0.1095 (3)
0.5994 (3)
0.0262 (7)
H10
0.039210
0.042307
0.596314
0.031*
C19
0.2863 (3)
0.5109 (2)
0.9128 (3)
0.0247 (7)
C20
0.3268 (4)
0.5390 (3)
1.0100 (3)
0.0316 (8)
H11
0.333688
0.493598
1.062254
0.038* Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) Acta Cryst. (2020). E76, 1139-1142 sup-7 supporting information supporting information
C21
0.3573 (4)
0.6332 (3)
1.0315 (3)
0.0385 (10)
H12
0.385755
0.651422
1.098118
0.046*
C22
0.3466 (4)
0.7000 (3)
0.9572 (4)
0.0379 (10)
H13
0.367454
0.764157
0.972651
0.045*
C23
0.3056 (4)
0.6739 (3)
0.8598 (3)
0.0376 (10)
H14
0.297827
0.720240
0.808386
0.045*
C24
0.2757 (4)
0.5797 (3)
0.8373 (3)
0.0299 (8)
H15
0.247979
0.561819
0.770438
0.036*
Atomic displacement parameters (Å2)
U11
U22
U33
U12
U13
U23
Br1
0.0222 (3)
0.0293 (3)
0.0246 (3)
0.0054 (2)
0.0032 (2)
0.00736 (19)
O2
0.038 (3)
0.028 (3)
0.041 (3)
−0.0170 (18)
0.012 (2)
0.000 (2)
C2
0.055 (7)
0.023 (3)
0.031 (4)
0.009 (5)
−0.006 (5)
−0.006 (3)
Br2
0.060 (4)
0.0228 (17)
0.035 (2)
0.001 (3)
−0.005 (3)
−0.0077 (14)
Mn1
0.0276 (3)
0.0170 (3)
0.0275 (3)
0.0001 (2)
−0.0027 (2)
0.0030 (2)
O1
0.0504 (18)
0.0244 (14)
0.0342 (15)
0.0084 (12)
0.0102 (13)
0.0043 (12)
N1
0.0202 (14)
0.0197 (14)
0.0276 (15)
0.0010 (11)
0.0042 (12)
0.0048 (12)
N2
0.0176 (13)
0.0179 (13)
0.0250 (15)
0.0001 (10)
0.0017 (11)
0.0029 (11)
N3
0.0191 (14)
0.0215 (14)
0.0249 (15)
0.0006 (11)
0.0016 (12)
0.0015 (12)
C1
0.0266 (19)
0.037 (2)
0.029 (2)
−0.0021 (16)
0.0024 (15)
0.0106 (17)
C4
0.0248 (18)
0.0231 (17)
0.035 (2)
0.0036 (14)
0.0064 (15)
0.0077 (15)
C5
0.0276 (19)
0.0283 (19)
0.034 (2)
0.0070 (15)
0.0064 (16)
0.0115 (16)
C6
0.0286 (19)
0.039 (2)
0.0235 (18)
0.0076 (16)
0.0040 (15)
0.0082 (16)
C7
0.0239 (17)
0.0283 (18)
0.0252 (18)
0.0022 (14)
0.0041 (14)
0.0017 (14)
C8
0.0182 (16)
0.0240 (17)
0.0236 (17)
0.0012 (13)
0.0059 (13)
0.0031 (14)
C9
0.0177 (16)
0.0214 (16)
0.0249 (17)
0.0006 (13)
0.0042 (13)
0.0030 (13)
C10
0.0192 (16)
0.0247 (17)
0.0244 (18)
0.0003 (13)
0.0028 (13)
−0.0012 (14)
C11
0.0180 (16)
0.0202 (16)
0.0297 (18)
0.0012 (12)
0.0064 (14)
0.0008 (14)
C12
0.0208 (16)
0.0196 (16)
0.0270 (18)
0.0014 (13)
0.0028 (13)
0.0027 (14)
C13
0.0172 (15)
0.0224 (17)
0.0232 (17)
0.0012 (12)
0.0036 (13)
0.0041 (13)
C14
0.0173 (15)
0.0209 (16)
0.0251 (17)
0.0000 (12)
0.0037 (13)
0.0018 (13)
C15
0.0221 (17)
0.0243 (17)
0.0289 (19)
0.0016 (14)
0.0023 (14)
0.0057 (14)
C16
0.0225 (17)
0.0333 (19)
0.0261 (19)
0.0011 (15)
0.0016 (14)
0.0059 (15)
C17
0.0227 (18)
0.036 (2)
0.0241 (18)
−0.0009 (15)
0.0013 (14)
−0.0016 (15)
C18
0.0224 (17)
0.0247 (17)
0.0311 (19)
−0.0016 (14)
0.0020 (14)
−0.0005 (15)
C19
0.0181 (16)
0.0208 (17)
0.036 (2)
−0.0005 (13)
0.0065 (14)
−0.0015 (14)
C20
0.0290 (19)
0.0240 (18)
0.042 (2)
0.0000 (15)
0.0027 (17)
−0.0041 (16)
C21
0.031 (2)
0.031 (2)
0.053 (3)
−0.0024 (16)
0.0019 (19)
−0.0145 (19)
C22
0.030 (2)
0.0206 (18)
0.065 (3)
−0.0047 (15)
0.014 (2)
−0.0097 (19)
C23
0.038 (2)
0.0211 (18)
0.057 (3)
−0.0022 (16)
0.018 (2)
0.0041 (18)
C24
0.0288 (19)
0.0221 (17)
0.040 (2)
0.0013 (14)
0.0081 (16)
0.0006 (16) Atomic displacement parameters (Å2) supporting information supporting information Br1—Mn1
2.5170 (11)
C10—H5
0.9500
O2—Br2
0.654 (5)
C11—C12
1.398 (5)
O2—C2
1.201 (11)
C11—C19
1.496 (5)
C2—Br2
0.597 (8)
C12—C13
1.392 (5)
C2—Mn1
1.790 (9)
C12—H6
0.9500
O3—C3
1.194 (18)
C13—C14
1.468 (5)
Mn1—C1
1.803 (4)
C14—C15
1.390 (5)
Mn1—N2
1.954 (3)
C15—C16
1.384 (5)
Mn1—N1
2.012 (3)
C15—H7
0.9500
Mn1—N3
2.019 (3)
C16—C17
1.376 (5)
O1—C1
1.103 (5)
C16—H8
0.9500
N1—C4
1.350 (4)
C17—C18
1.380 (5)
N1—C8
1.364 (4)
C17—H9
0.9500
N2—C9
1.346 (4)
C18—H10
0.9500
N2—C13
1.351 (4)
C19—C20
1.390 (5)
N3—C18
1.343 (4)
C19—C24
1.402 (5)
N3—C14
1.360 (4)
C20—C21
1.390 (5)
C4—C5
1.371 (5)
C20—H11
0.9500
C4—H1
0.9500
C21—C22
1.371 (6)
C5—C6
1.381 (6)
C21—H12
0.9500
C5—H2
0.9500
C22—C23
1.385 (6)
C6—C7
1.395 (5)
C22—H13
0.9500
C6—H3
0.9500
C23—C24
1.393 (5)
C7—C8
1.387 (5)
C23—H14
0.9500
C7—H4
0.9500
C24—H15
0.9500
C8—C9
1.481 (5)
O3—Br1—C3
131 (4)
N2—C9—C8
111.5 (3)
Br2—O2—C2
15.7 (10)
C10—C9—C8
126.8 (3)
Br2—C2—O2
17.2 (12)
C9—C10—C11
119.2 (3)
O2—C2—Mn1
177.5 (10)
C9—C10—H5
120.4
C2—Br2—O2
147 (2)
C11—C10—H5
120.4
Br1—O3—C3
24 (2)
C12—C11—C10
118.2 (3)
Br1—C3—O3
24.3 (19)
C12—C11—C19
121.5 (3)
C2—Mn1—C1
87.9 (4)
C10—C11—C19
120.3 (3)
C2—Mn1—N2
98.6 (4)
C13—C12—C11
120.0 (3)
C1—Mn1—N2
173.57 (15)
C13—C12—H6
120.0
C2—Mn1—N1
91.3 (4)
C11—C12—H6
120.0
C1—Mn1—N1
100.98 (14)
N2—C13—C12
120.4 (3)
N2—Mn1—N1
79.05 (12)
N2—C13—C14
112.0 (3)
C2—Mn1—N3
93.0 (4)
C12—C13—C14
127.5 (3)
C1—Mn1—N3
100.66 (15)
N3—C14—C15
121.5 (3)
N2—Mn1—N3
79.05 (12)
N3—C14—C13
114.1 (3)
N1—Mn1—N3
158.08 (12)
C15—C14—C13
124.4 (3)
C2—Mn1—Br1
177.4 (4)
C16—C15—C14
119.6 (3)
C1—Mn1—Br1
89.68 (12)
C16—C15—H7
120.2
N2—Mn1—Br1
83.89 (8)
C14—C15—H7
120.2
N1—Mn1—Br1
88.42 (8)
C17—C16—C15
118.8 (3) Acta Cryst. (2020). supporting information E76, 1139-1142 sup-9 supporting information pp
g
88.26 (8)
C17—C16—H8
120.6
117.4 (3)
C15—C16—H8
120.6
126.8 (3)
C16—C17—C18
119.1 (3)
115.8 (2)
C16—C17—H9
120.4
120.4 (3)
C18—C17—H9
120.4
119.7 (2)
N3—C18—C17
123.1 (3)
119.3 (2)
N3—C18—H10
118.4
117.9 (3)
C17—C18—H10
118.4
126.7 (2)
C20—C19—C24
118.5 (3)
115.5 (2)
C20—C19—C11
121.0 (3)
179.5 (4)
C24—C19—C11
120.5 (3)
123.2 (4)
C21—C20—C19
120.5 (4)
118.4
C21—C20—H11
119.8
118.4
C19—C20—H11
119.8
119.4 (3)
C22—C21—C20
120.6 (4)
120.3
C22—C21—H12
119.7
120.3
C20—C21—H12
119.7
118.9 (3)
C21—C22—C23
120.1 (4)
120.5
C21—C22—H13
120.0
120.5
C23—C22—H13
120.0
118.8 (3)
C22—C23—C24
119.8 (4)
120.6
C22—C23—H14
120.1
120.6
C24—C23—H14
120.1
122.3 (3)
C23—C24—C19
120.5 (4)
113.7 (3)
C23—C24—H15
119.8
124.0 (3)
C19—C24—H15
119.8
121.7 (3)
−0.7 (5)
C11—C12—C13—N2
1.8 (5)
−179.2 (3)
C11—C12—C13—C14
−174.8 (3)
0.7 (6)
C18—N3—C14—C15
0.3 (5)
0.0 (5)
Mn1—N3—C14—C15
−179.4 (3)
−0.5 (5)
C18—N3—C14—C13
−179.0 (3)
0.1 (5)
Mn1—N3—C14—C13
1.3 (4)
178.8 (3)
N2—C13—C14—N3
−3.4 (4)
−178.9 (3)
C12—C13—C14—N3
173.5 (3)
−0.2 (4)
N2—C13—C14—C15
177.4 (3)
0.5 (5)
C12—C13—C14—C15
−5.7 (6)
179.4 (3)
N3—C14—C15—C16
0.5 (5)
0.7 (5)
C13—C14—C15—C16
179.7 (3)
171.8 (2)
C14—C15—C16—C17
−1.0 (5)
−177.5 (3)
C15—C16—C17—C18
0.7 (5)
−6.4 (4)
C14—N3—C18—C17
−0.6 (5)
4.1 (4)
Mn1—N3—C18—C17
179.1 (3)
−174.9 (3)
C16—C17—C18—N3
0.1 (6)
−174.0 (3)
C12—C11—C19—C20
162.1 (3)
7.0 (5)
C10—C11—C19—C20
−19.3 (5)
0.6 (5)
C12—C11—C19—C24
−18.0 (5) pp
g
N3—Mn1—Br1
88.26 (8)
C17—C16—H8
120.6
C4—N1—C8
117.4 (3)
C15—C16—H8
120.6
C4—N1—Mn1
126.8 (3)
C16—C17—C18
119.1 (3)
C8—N1—Mn1
115.8 (2)
C16—C17—H9
120.4
C9—N2—C13
120.4 (3)
C18—C17—H9
120.4
C9—N2—Mn1
119.7 (2)
N3—C18—C17
123.1 (3)
C13—N2—Mn1
119.3 (2)
N3—C18—H10
118.4
C18—N3—C14
117.9 (3)
C17—C18—H10
118.4
C18—N3—Mn1
126.7 (2)
C20—C19—C24
118.5 (3)
C14—N3—Mn1
115.5 (2)
C20—C19—C11
121.0 (3)
O1—C1—Mn1
179.5 (4)
C24—C19—C11
120.5 (3)
N1—C4—C5
123.2 (4)
C21—C20—C19
120.5 (4)
N1—C4—H1
118.4
C21—C20—H11
119.8
C5—C4—H1
118.4
C19—C20—H11
119.8
C4—C5—C6
119.4 (3)
C22—C21—C20
120.6 (4)
C4—C5—H2
120.3
C22—C21—H12
119.7
C6—C5—H2
120.3
C20—C21—H12
119.7
C5—C6—C7
118.9 (3)
C21—C22—C23
120.1 (4)
C5—C6—H3
120.5
C21—C22—H13
120.0
C7—C6—H3
120.5
C23—C22—H13
120.0
C8—C7—C6
118.8 (3)
C22—C23—C24
119.8 (4)
C8—C7—H4
120.6
C22—C23—H14
120.1
C6—C7—H4
120.6
C24—C23—H14
120.1
N1—C8—C7
122.3 (3)
C23—C24—C19
120.5 (4)
N1—C8—C9
113.7 (3)
C23—C24—H15
119.8
C7—C8—C9
124.0 (3)
C19—C24—H15
119.8
N2—C9—C10
121.7 (3)
C8—N1—C4—C5
−0.7 (5)
C11—C12—C13—N2
1.8 (5)
Mn1—N1—C4—C5
−179.2 (3)
C11—C12—C13—C14
−174.8 (3)
N1—C4—C5—C6
0.7 (6)
C18—N3—C14—C15
0.3 (5)
C4—C5—C6—C7
0.0 (5)
Mn1—N3—C14—C15
−179.4 (3)
C5—C6—C7—C8
−0.5 (5)
C18—N3—C14—C13
−179.0 (3)
C4—N1—C8—C7
0.1 (5)
Mn1—N3—C14—C13
1.3 (4)
Mn1—N1—C8—C7
178.8 (3)
N2—C13—C14—N3
−3.4 (4)
C4—N1—C8—C9
−178.9 (3)
C12—C13—C14—N3
173.5 (3)
Mn1—N1—C8—C9
−0.2 (4)
N2—C13—C14—C15
177.4 (3)
C6—C7—C8—N1
0.5 (5)
C12—C13—C14—C15
−5.7 (6)
C6—C7—C8—C9
179.4 (3)
N3—C14—C15—C16
0.5 (5)
C13—N2—C9—C10
0.7 (5)
C13—C14—C15—C16
179.7 (3)
Mn1—N2—C9—C10
171.8 (2)
C14—C15—C16—C17
−1.0 (5)
C13—N2—C9—C8
−177.5 (3)
C15—C16—C17—C18
0.7 (5)
Mn1—N2—C9—C8
−6.4 (4)
C14—N3—C18—C17
−0.6 (5)
N1—C8—C9—N2
4.1 (4)
Mn1—N3—C18—C17
179.1 (3)
C7—C8—C9—N2
−174.9 (3)
C16—C17—C18—N3
0.1 (6)
N1—C8—C9—C10
−174.0 (3)
C12—C11—C19—C20
162.1 (3)
C7—C8—C9—C10
7.0 (5)
C10—C11—C19—C20
−19.3 (5)
N2—C9—C10—C11
0.6 (5)
C12—C11—C19—C24
−18.0 (5) sup-10 Acta Cryst. Symmetry codes: (i) −x+1, −y, −z+2; (ii) x, −y+1/2, z+1/2; (iii) −x, y+1/2, −z+3/2; (iv) x, −y+1/2, z−1/2. Acta Cryst. (2020). E76, 1139-1142 supporting information (2020). E76, 1139-1142 sup-10 supporting information C8—C9—C10—C11
178.5 (3)
C10—C11—C19—C24
160.6 (3)
C9—C10—C11—C12
−0.7 (5)
C24—C19—C20—C21
−0.7 (6)
C9—C10—C11—C19
−179.4 (3)
C11—C19—C20—C21
179.2 (3)
C10—C11—C12—C13
−0.5 (5)
C19—C20—C21—C22
0.7 (6)
C19—C11—C12—C13
178.2 (3)
C20—C21—C22—C23
−0.2 (6)
C9—N2—C13—C12
−1.9 (5)
C21—C22—C23—C24
−0.3 (6)
Mn1—N2—C13—C12
−173.0 (2)
C22—C23—C24—C19
0.3 (6)
C9—N2—C13—C14
175.2 (3)
C20—C19—C24—C23
0.2 (5)
Mn1—N2—C13—C14
4.1 (4)
C11—C19—C24—C23
−179.7 (3)
Hydrogen-bond geometry (Å, º)
D—H···A
D—H
H···A
D···A
D—H···A
C5—H2···Br1i
0.95
2.84
3.528 (4)
130
C7—H4···Br1ii
0.95
2.86
3.771 (4)
162
C12—H6···Br2iii
0.95
2.75
3.688 (7)
171
C12—H6···O2iii
0.95
2.55
3.491 (7)
173
C15—H7···Br2iii
0.95
2.81
3.759 (7)
175
C15—H7···O2iii
0.95
2.50
3.447 (7)
172
C16—H8···Br2iv
0.95
2.52
3.286 (7)
138
C16—H8···O2iv
0.95
2.57
3.363 (7)
141
C20—H11···Br1ii
0.95
2.81
3.743 (4)
168
C20—H11···O3ii
0.95
2.55
3.446 (18)
158
C24—H15···Br2iii
0.95
2.84
3.611 (7)
139
Symmetry codes: (i) −x+1, −y, −z+2; (ii) x, −y+1/2, z+1/2; (iii) −x, y+1/2, −z+3/2; (iv) x, −y+1/2, z−1/2. C8—C9—C10—C11
178.5 (3)
C10—C11—C19—C24
160.6 (3)
C9—C10—C11—C12
−0.7 (5)
C24—C19—C20—C21
−0.7 (6)
C9—C10—C11—C19
−179.4 (3)
C11—C19—C20—C21
179.2 (3)
C10—C11—C12—C13
−0.5 (5)
C19—C20—C21—C22
0.7 (6)
C19—C11—C12—C13
178.2 (3)
C20—C21—C22—C23
−0.2 (6)
C9—N2—C13—C12
−1.9 (5)
C21—C22—C23—C24
−0.3 (6)
Mn1—N2—C13—C12
−173.0 (2)
C22—C23—C24—C19
0.3 (6)
C9—N2—C13—C14
175.2 (3)
C20—C19—C24—C23
0.2 (5)
Mn1—N2—C13—C14
4.1 (4)
C11—C19—C24—C23
−179.7 (3) Hydrogen-bond geometry (Å, º) Hydrogen-bond geometry (Å, º)
D—H···A
D—H
H···A
D···A
D—H···A
C5—H2···Br1i
0.95
2.84
3.528 (4)
130
C7—H4···Br1ii
0.95
2.86
3.771 (4)
162
C12—H6···Br2iii
0.95
2.75
3.688 (7)
171
C12—H6···O2iii
0.95
2.55
3.491 (7)
173
C15—H7···Br2iii
0.95
2.81
3.759 (7)
175
C15—H7···O2iii
0.95
2.50
3.447 (7)
172
C16—H8···Br2iv
0.95
2.52
3.286 (7)
138
C16—H8···O2iv
0.95
2.57
3.363 (7)
141
C20—H11···Br1ii
0.95
2.81
3.743 (4)
168
C20—H11···O3ii
0.95
2.55
3.446 (18)
158
C24—H15···Br2iii
0.95
2.84
3.611 (7)
139 sup-11 Acta Cryst. (2020). E76, 1139-1142 sup-11
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Memoria oral del pueblo Mapuche en la reforma agraria y la dictadura. Aplicación del archivo en el proyecto Dungun, instalación sonora interactiva
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Anales de historia del arte
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FORO
Anales de Historia del Arte
ISSN: 0214-6452
https://dx.doi.org/10.5209/anha.83058
Memoria oral del pueblo Mapuche en la reforma agraria y la dictadura.
Aplicación del archivo en el proyecto Dungun, instalación sonora
interactiva
Luis Urquieta Robles1
Recibido: 1 de febrero de 2022 / Aceptado: 6 de junio de 2022
Resumen. El presente escrito se enmarca dentro de una pasantía realizada durante el año 2021 en el
Museo de la Memoria y los Derechos Humanos, en Santiago de Chile. La finalidad de dicho proceso
fue la utilización del extenso archivo del que dispone esta institución, tanto escrito como audiovisual,
para trabajar y reflexionar en torno a un complejo proceso de la historia reciente de este país, las comunidades Mapuche durante la reforma agraria y la posterior dictadura perpetrada por Augusto Pinochet.
Dicha etapa se encuentra soterrada debido a los graves crímenes de lesa humanidad que sufrió todo
Chile durante esa fatídica época, relegando a un segundo plano las violaciones contra el pueblo Mapuche. El trabajo investigativo en este Museo culminó con la creación de una propuesta de una instalación
artística titulada Dungun, un proyecto interactivo sonoro que se nutre de toda la información recopilada
durante la pasantía, generando una propuesta estética sonora en torno a la memoria, el archivo y el acto
de escuchar.
Palabras clave: museo; archivo; Mapuche; memoria; instalación; arte sonoro.
[en] Oral Memory of the Mapuche People during the Agrarian Reform and the
Dictatorship. Application of the Archive in the Dungun Project, Interactive
Sound Installation
Abstract. This paper is part of an internship carried out during the year 2021 at the Museum of Memory
and Human Rights in Santiago, Chile. The purpose of this process was the use of the extensive archive
that this institution has, both written and audiovisual, to work and reflect on a complex process of the
recent history of this country, the Mapuche communities during the agrarian reform and the subsequent
dictatorship perpetrated by Augusto Pinochet. This stage is buried due to the serious crimes against humanity suffered by all Chile during that fateful time, relegating to the background the violations against
the Mapuche people. The research work in this museum culminated with the creation of an artistic
installation proposal entitled Dungun, an interactive sound project that draws on all the information
collected during the internship, generating an aesthetic sound proposal around memory, the archive and
the act of listening.
Keywords: museum; archive; Mapuche; memory; installation; sound art.
Sumario: 1. Introducción. 2. Las comunidades Mapuche y la reforma agraria. 3. Dictadura en la Araucanía. 4. La historia oral y el archivo en la preservación de la memoria. 5. Proyecto arte sonoro Dungun.
6. Referencias. 7. Instalación. 8. Conclusiones. 9. Conflicto de intereses. 10. Referencias bibliográficas.
1
Universidad Politécnica de Valencia (UPV).
E-mail: lurquieta.robles@gmail.com
An. his. arte 32 (2022): 77-97
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Cómo citar: Urquieta Robles, Luis (2022). Memoria oral del pueblo Mapuche en la reforma agraria y
la dictadura. Aplicación del archivo en el proyecto Dungun, instalación sonora interactiva, en Anales de
Historia del Arte nº 32 (2022), 77-97.
1. Introducción
El inicio de la memoria oral, abordada desde el punto de vista de la institucionalidad
historiográfica, de archivo o documental, está vinculada de manera irrestricta a los
procesos sociales de nuestra historia más reciente. Fue en 1948 cuando el concepto
de historia oral comenzó a ser utilizado. Allan Nevins, profesor de la Universidad de
Columbia, funda la Oral History Research Office en la misma institución2.
En el caso del Cono Sur, también tenemos importantes ejemplos de cómo se usó
la historia oral para preservar las memorias de personas vinculadas a colectividades
que lograron sobrevivir, como familiares de detenidos desaparecidos o torturados
políticos en Argentina, a raíz de la cruenta dictadura de Videla de 1976. Lo mismo
ocurre en el caso chileno de 1973 con Pinochet.
Sobre este último, el Museo de la Memoria y los Derechos Humanos (MMDH)
ha realizado una enorme labor de archivar, documentar y crear memoria en torno a
los crímenes de lesa humanidad ocurridos bajo la dictadura cívico-militar de Augusto
Pinochet, disponiendo de una extensa documentación de casos. Para este artículo, nos
centraremos en aquellas zonas rurales donde se vio afectada la población mapuche3.
Como veremos más adelante, este vasto archivo de memoria oral mapuche será aplicado en un caso concreto expositivo de una obra de arte sonoro. Para ello, ahondaremos
en los antecedentes históricos sobre el pueblo Mapuche, la vida y las condiciones de
las comunidades, previas y durante el golpe cívico militar. Después de ello, indagaremos en torno al archivo oral mapuche que posee el Museo y su importancia tanto en
lo concerniente a la memoria como a sus aspectos técnicos, y cómo estas cuestiones se
pueden abordar en una obra de arte, a través de un caso en concreto.
2. Las comunidades Mapuche y la reforma agraria
Hasta los primeros atisbos de la reforma agraria en Chile durante el año 19624, las
comunidades Mapuche sólo disponían de pequeñas mercedes de tierra, que les fue2
3
4
Si bien los inicios de la historia oral, como tal, se enfocaron en personalidades ilustres, como Grover Cleveland,
presidente de los Estados Unidos, con el tiempo, el enfoque usado en la memoria oral fue dando un giro hacia
la historia social, poniendo el énfasis en personas comunes, que, al dar sus testimonios, permiten mantener la
memoria de determinados hechos de colectividades más invisibilizadas. Este cambio estuvo influenciado en
gran medida por lo realizado previamente por estudios sociológicos en la Universidad de Chicago, los cuales
abordaban temáticas vinculadas a las capas marginales de la sociedad.
En este informe usaremos la palabra «Mapuche» en singular para referirnos a la comunidad en su conjunto, pues,
consideramos que, al tratarse de una palabra que significa «gente de la tierra», ya tiene de manera implícita la
pluralidad. Por otra parte, estimamos que debemos respetar el modo en que el mismo pueblo Mapuche se llama a
sí mismo, sobre todo si se trata de un acto de resistencia a partir de su lengua, tema principal de este artículo.
Nos abocaremos únicamente al proceso de la reforma agraria en adelante, sin profundizar en el conflicto previo
entre el Estado chileno y el pueblo Mapuche, durante la llamada «Pacificación de la Araucanía».
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ron entregadas en compensación a todo su terreno perdido durante la «Pacificación
de la Araucanía», reduciendo su territorio de manera drástica, no sólo en tamaño sino
también en calidad, ya que las tierras entregadas estaban en lugares cordilleranos o
muy próximos a la costa, haciendo muy difícil poder llevar a cabo una buena labor
en lo agrícola. Tras la derrota durante la Ocupación de la Araucanía, «cinco millones
de hectáreas entre el Malleco y Valdivia son rematadas»5, obligando al Mapuche a
desplazarse a otras zonas. Muy pocos pudieron quedarse en sus terrenos ancestrales,
debiendo convivir, en muchos casos, con colonos tanto chilenos como extranjeros.
«En este periodo sólo retuvieron unas 510 mil hectáreas, es decir, el 6,3% de su territorio original»6. Esta estrategia fue muy beneficiosa para el gobierno, ya que pudo
hacerse con una extensa cantidad de hectáreas, las cuales comenzaron a ser destinadas a las plantaciones agrícolas.
La situación territorial de las comunidades Mapuche no varió positivamente hasta
1962, año en que se produjo un hecho que, más allá de lo tangible en términos de
recuperación de territorios, se convirtió en un momento simbólico que propició el
empoderamiento de dichas comunidades. Durante ese año, se dio la primera reforma
agraria, impulsada por el gobierno de Alessandri en respuesta al poco aprovechamiento y abandono de la tierra, tanto en los fundos como en grandes latifundios,
además de la creciente demanda de alimentos debido a la crisis alimentaria suscitada
en ese entonces. Tras su implementación, el resultado de esta medida fue tan exiguo
que fue conocida como la «ley macetero», debido a las paupérrimas recuperaciones
de territorio para con las comunidades campesinas, alcanzando la cifra de 491 nuevos propietarios agrícolas a nivel nacional7. De éstas, tan solo una pequeña fracción
fue destinada al pueblo Mapuche.
Como señalamos anteriormente, la Ley Nº 15.020, más allá de cambios administrativos –como la creación de la Corporación de la Reforma Agraria (CORA)8– no
generó un cambio sustancial en la entrega de terrenos al campesinado, pero sirvió
para posicionar esta demanda en todo el territorio por parte de las comunidades Mapuche, que vieron aquí una gran oportunidad de exigir de manera más formal las
tierras arrebatadas tras la guerra ocurrida a mediados del siglo XIX.
Con el cambio de presidencia del país y el desplazamiento del eje político de
este gobierno, pasando de un mandato de derecha altamente conservador a una postura de centro izquierda, Eduardo Frei Montalva impulsó tempranamente la nueva
Ley de Reforma Agraria, despachándola al congreso en 1965, siendo aprobada en
19679. Previamente a la aprobación de la nueva reforma agraria, se lograron expropiar 1.209.975 hectáreas10 a través de la antigua ley, cifra que, al compararla con la
administración anterior, habla de la poca voluntad política por parte del gobierno de
5
6
7
8
9
10
Bengoa, J. (1985). Historia del Pueblo Mapuche (siglos XIX y XX). Santiago de Chile: LOM, 336.
Araya, J. (1999). Conflicto mapuche: Algunas razones y derechos en este nuevo enfrentamiento. Derechos
Humanos y trabajadores. Sobre el derecho de los ex prisioneros políticos a ser indemnizados por el Estado.
Santiago de Chile: LOM, 12.
Comisión Verdad Histórica y Nuevo Trato con los Pueblos Indígenas. (2008). Informe de la Comisión Verdad
Histórica y Nuevo Trato con los Pueblos Indígenas. Santiago de Chile: Gobierno de Chile, 403.
La Corporación de Reforma Agraria, nació en 1962 como la empresa estatal encargada de llevar a cabo lo desarrollado por la ley del mismo nombre, efectuando las subdivisiones de tierras que se iban aprobando.
Correa, M., Molina, R., y Yáñez, N. (2005). La Reforma Agraria y las tierras mapuche: Chile 1962 – 1975.
Santiago de Chile: LOM, 79.
Ibid., 80.
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Alessandri por favorecer al campesinado, dada la baja extensión de terreno entregado durante su mandato.
Con la promulgación de la Ley de Reforma Agraria Nº 16.640, se amplían las
causas para que un predio pueda ser expropiado, adoptando una postura más proactiva para con el campesinado, entregándole capacitación, promoviendo el desarrollo
económico de la comunidad y suministrando infraestructura mínima para que la familia beneficiada pueda desempeñar la normal explotación de su terreno.
En esta nueva ley, se incluye el «problema indígena» dentro de la discusión, dado
que, en la ley anterior, se hablaba únicamente del campesinado. Lamentablemente,
todo esto se realizó de manera tangencial, ya que no afrontaba la situación desde la
etnicidad de las comunidades, haciendo imposible abordar estas demandas desde lo
histórico. Pese a ello, las reivindicaciones mapuches por sus tierras siguieron su curso,
obteniendo buenos resultados en aquellas comunidades que se organizaron en torno a
Cooperativas y Comités Campesinos, incrementando la posibilidad de acceder a créditos en infraestructura, asesoría técnica, aumento de becas de estudios, etc.
Aquel impulso iniciado con la primera reforma agraria ya se hacía notar con mayor fuerza para la segunda mitad de la década de los sesenta. La acción directa cobra
relevancia en el quehacer indígena, sin dejar de lado la vía legal, siendo esta última la
que daba pie a las tomas de predios y las posteriores corridas de cerco. El número de
tomas fue creciendo a medida que las comunidades fueron viendo cómo otras agrupaciones sí estaban teniendo resultados, procediendo a expropiar predios, cada vez con
mayor frecuencia. Ejemplo de ello fue lo ocurrido en la comuna de Lumaco, donde la
Confederación Indígena Campesina de diversas zonas de la comuna se tomó el fundo
Reñico, el cual perteneció a Raimán Pichulmán y había sido usurpado.
Para finales de 1969, las tomas de terreno se extendieron en la provincia de Malleco, llegando hasta Ercilla, en el centro de la provincia, dando muestra de que el
proceso parecía no tener marcha atrás. Fue en este mismo año que comienzan a
suceder las corridas de cerco11, acto que consistía básicamente en restablecer los
límites legales existentes hasta antes de lo que había sido usurpado de los Títulos
de Merced12. Para ello, se movían los cercos perimetrales hasta el punto señalado
anteriormente.
Estas medidas insurreccionales se debían a las discrepancias existentes entre, por
un lado, las leyes de corte reformista y, por otro lado, las necesidades urgentes y la
mirada revolucionaria de muchos sectores del campesinado mapuche, sumadas a
la larga e infructuosa historia de peticiones y reclamos que no terminaban en algo
concreto. Además hicieron que, para finales de 1970, las tierras recuperadas llegaran
a las casi 100.000 hectáreas.
La llegada al poder por parte de la Unidad Popular, liderada por Salvador Allende, allanó el camino para que las tomas y corridas de cerco fueran en aumento, sobre
todo considerando que, dentro del gobierno de Allende, se contemplaba un programa
que se hacía cargo específicamente de la problemática territorial mapuche. Aquí, se
hablaba de defender la integridad de las comunidades indígenas asegurando tierras
suficientes, apoyo técnico y crediticio para su normal funcionamiento.
11
12
Las corridas de cerco fueron propiciadas en su gran mayoría por el Movimiento Campesino Revolucionario
(MCR), agrupación de corta data, que aglutinó personas provenientes del campesinado mapuche y del Movimiento de Izquierda Revolucionario (MIR).
Comisión Verdad Histórica y Nuevo Trato con los Pueblos Indígenas. (2008) op.cit., 406.
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Si bien hubo un notable apoyo por parte del gobierno de Allende respecto a las
tomas y corridas de cerco, éste mantenía su postura de realizar esta recuperación
de territorio por medio de la vía institucional. Para ello, trasladó el Ministerio de
Agricultura desde Santiago a la ciudad de Temuco, capital de la provincia de Cautín,
constituyendo la «Comisión de Restitución de Tierras Mapuche Usurpadas»13 con la
finalidad de poder trabajar en terreno y otorgar una pronta salida a aquellas comunidades donde el nivel de conflictividad era mayor.
Para poder concretar los traspasos de tierra a los pueblos originarios, Allende
promulga, el 15 de septiembre de 1972, la Ley Nº 17.729, conocida como Ley de
Indígenas, la cual tenía la particularidad de restituir las tierras mapuches que fueron
perdidas durante el proceso de la Ocupación de la Araucanía. Además, se estipulaba
que no se podrían «enajenar, gravar ni dar en arrendamiento o aparcería los goces
que posean en la reserva, ni los derechos que les correspondan en la comunidad,
excepto en favor de otro u otros miembros de la misma»14. Esta ley, sin duda fue
un avance cualitativo y sustancial, no solo en el proceso de recuperación de tierras
ancestrales, ya que por primera vez existían medios legales efectivos, sino también
en el otorgamiento de dignidad a las comunidades: al ser reconocidas como tales y
brindarles diversas ayudas, más allá de su condición de campesinos, eran entregadas
por el hecho de ser mapuche.
Lamentablemente, este hito no duró mucho. El golpe cívico-militar de Pinochet,
derribó inmediatamente estos avances para con las comunidades Mapuche. Es más,
en muchos relatos se habla que el golpe de Estado de 1973 no se originó ni en Valparaíso ni en Santiago, y tampoco un 11 de septiembre, sino en la Araucanía y durante
el mes de agosto de ese año. Esto se explica porque, debido a la Ley de Control de
Armas, se realizó una fuerte fiscalización por parte de las fuerzas armadas y de aire
en los caminos rurales de la zona y en particular contra los principales Centros de
Reforma Agraria, llevándose a cabo allanamientos y detenciones incluso con resultado de torturas y muerte15.
Entre el 4 de noviembre de 1970 y el 11 de septiembre de 1973 se expropiaron en
la Araucanía 574 fundos, con una superficie de 636.288,3 hectáreas. Los predios
expropiados a favor de comunidades mapuche o con participación mapuche fueron 138, con una superficie total de 132.115,78 hectáreas físicas16.
3. Dictadura en la Araucanía
El golpe cívico-militar impulsado por Pinochet tuvo un particular ensañamiento en la
actual Región de la Araucanía debido a todo lo ocurrido en relación a la recuperación
de tierras por parte de las comunidades Mapuche. Este clima reivindicatorio atrajo,
13
14
15
16
Navarrete, J. (2020). Movimiento Campesino Revolucionario. Lucha mapuche, política de clase y «proyecto
socialista» durante el gobierno de la Unidad Popular (Cautín, 1970-1971). En R. Austin (Eds.), La vía al socialismo chileno 50 años después (pp. 495-519). Buenos Aires: Clacso.
Chile. (1972). Ley 17.729. Propiedad intelectual. Obtenido de https://www.bcn.cl/leychile/navegar?idNorma=29250 [Consulta: 18 de octubre de 2021].
Correa, M., Molina, R., y Yáñez, N. (2005) op. cit., 244.
Comisión Verdad Histórica y Nuevo Trato con los Pueblos Indígenas. (2008) op. cit., 403.
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durante la Unidad Popular, la llegada de «organizaciones políticas como el Partido Socialista, el Partido Comunista, el MAPU-OC (Movimiento de Acción Popular
Unitaria, Obrero Campesino), y políticas-militares como el MIR y el PCR a través de
sus sub-orgánicas MCR y Netuaiñ Mapu»17. Las personas agrupadas en confederaciones, cooperativas y comités campesinos fueron las primeras en sufrir persecución,
tortura y muerte. Todo lo construido a partir de la primera reforma agraria se esfumó.
La dictadura de Pinochet acabó con las libertades colectivas eliminando de raíz todo
medio de organización, como partidos políticos, entidades públicas indígenas, consejos comunales, organizaciones sociales, cooperativas y comités.
Otra arista no menos importante fueron las consecuencias territoriales generadas
a partir del golpe de estado. La junta de gobierno encabezada por Pinochet inició una
fuerte contrarreforma agraria desmantelando todo lo avanzado en políticas públicas
de otorgamiento de tierras a las comunidades indígenas, restituyendo dichos terrenos
a sus antiguos dueños. Dicha medida, además, les despojó de sus bienes materiales
–herramientas, tractores, etc.– obligando a una gran cantidad de familias a salir de
su comunidad sin bienes, en busca de oportunidades laborales; muchas migraron a
Temuco o ciudades aledañas a sus comunidades, mientras otras se debieron instalar
en Santiago, e incluso huir a Argentina cruzando la cordillera.
En términos legislativos, la dictadura de Pinochet en 1978, por medio del Decreto
Ley Nº 2.405, disolvió la Corporación de la Reforma Agraria sepultando con esto la
reforma iniciada en 1962. En reemplazo de la CORA se constituyó la Oficina de Normalización Agraria (ODENA), la cual tendría la misión de finiquitar toda situación
pendiente de la extinta CORA, teniendo la potestad completa de su predecesora18.
En 1979 promulgó el Decreto de Ley 2.568 liquidando las comunidades Mapuche19. Hasta esa fecha, la Ley Indígena se mantuvo pero, al eliminar por completo el
Instituto de Desarrollo Indígena, la Ley 17.729 no podía ser utilizada. Desde el punto
de vista organizativo, se constituyeron entidades, como el Comité Ejecutivo Agrario
(CEA), que tenía la finalidad de restituir las tierras a los latifundistas que habían sido
expropiados.
Con los años, estas tierras arrebatadas a los Mapuche terminaron transformándose en monocultivos de pinos o eucaliptos. La CONAF20 había rematado una gran
extensión de hectáreas, las cuales fueron compradas a precios ínfimos por empresas
forestales.
Dicha aparición se ve favorecida con la aplicación del Decreto Ley Nº 701, de
1974, de fomento forestal, el que estableció́ una serie de incentivos a las plantaciones forestales, entre ellas que el Estado, bonificaba en un 75 % los costos
netos de forestación, por un periodo de 10 años, incluidos los gastos de manejo
correspondiente en que incurran las personas naturales o jurídicas de cualquier
17
18
19
20
Farías, C. (2012). Pueblo mapuche y dictadura: desde el terror hacia la nueva esperanza. Elementos configurativos para el entendimiento de la represión efectuada por el Régimen Militar hacia el pueblo mapuche. (Informe
final de práctica profesional). Santiago de Chile: Museo de la Memoria y los Derechos Humanos.
Chile. (1978). Ley 2.405. Propiedad intelectual. Obtenido de https://www.bcn.cl/leychile/navegar?idNorma=6911 [Consulta: 3 de noviembre de 2021].
Comisión Verdad Histórica y Nuevo Trato con los Pueblos Indígenas. (2008) op.cit., 415.
La Corporación Nacional Forestal es una institución del Estado creada en 1970 bajo el gobierno de Eduardo
Frei Montalva. Esta institución contempla la realización de diversas tareas tales como: administrar las áreas
silvestres del país, combatir incendios forestales y administrar las políticas del Estado en torno a lo forestal.
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naturaleza, además de declarar inexpropiables los predios afectos a dicho Decreto,
y se declaró la libertad de comercio para todos los productos del bosque. Recién
transcurridos 6 meses de vigencia de la norma sufre la primera modificación, y el
25 de marzo de 1975, se amplía a 20 años el plazo para el pago de bonificaciones
forestales, es decir las condiciones se hacen aún más beneficiosas para el negocio
forestal21.
Volviendo a lo referente a las violaciones de derechos humanos en las comunidades Mapuche, estas fueron horrendas, de un ensañamiento brutal, tanto por parte de
las fuerzas armadas y los carabineros, como de vecinos no mapuches de las distintas
comunidades. La facilidad con la que los militares localizaron a campesinos mapuches vinculados a organizaciones o colaboradores con la reforma agraria se debió al
apoyo, tanto logístico como de recursos humanos para cazar, una a una, a personalidades mapuches que tuvieron cierta vinculación en las recuperaciones de terrenos.
Los testimonios de los sobrevivientes a la tortura de estos deleznables hechos fueron
vitales para la conformación de un archivo oral respecto a sus vivencias durante la
dictadura, situación sobre la que ahondaremos en el siguiente apartado.
4. La historia oral y el archivo en la preservación de la memoria
La historia oral, como señalamos al inicio, nació de la mano de Nevins con su trabajo sobre el presidente Grover Cleveland, una investigación en torno a la vida y
obra de esta personalidad de la sociedad estadounidense. Con los años, este modo de
hacer historia fue variando, extendiéndose a otras latitudes, acuñando otras miradas
vinculadas con la historia social y sus modos de entender esta parte de las ciencias
sociales, buscando abrir espacios a aquellos grupos menos favorecidos en diversos
procesos que, por resultar perdedores en un hecho, no son considerados como efectivamente lo merecen en los libros oficiales y registros de historia. Estos antecedentes
los encontramos en hitos como el trabajo de Ronald Fraser en España, rescatando
testimonios de personas vinculadas al bando republicano, perdedores de la Guerra
Civil española; la labor realizada por Oscar Lewis en México, investigando sobre la
vida de la gente pobre de la capital durante finales de la década del cincuenta; o los
History Workshops de Raphael Samuel en Reino Unido22, los cuales buscaban construir un nuevo paradigma en torno a estudios de historia popular en los años sesenta.
La historia oral, tal como se conoce ahora, tiene por objetivo rescatar aquellas
voces que continúan con vida, que pese a ser excluidas de la historia oficial y ser
consideradas partes del medio cotidiano, deben ser estimadas por el hecho de ser testigos o protagonistas de un hecho histórico. De esa manera, es posible constituir un
proceso de construcción de fuentes que antes no eran tomadas en cuenta por la historiografía, para así establecer una memoria colectiva a partir de la memoria individual
de cada entrevistado. Permite además el desarrollo de proyectos tanto investigativos
21
22
Correa, M., y Mella, E. (2009). El territorio mapuche de Malleco: Las razones del Illkun. Santiago de Chile:
ODHPI, 122.
Yusta, M. (2002). Historia oral, historia vivida. El uso de las fuentes orales en la investigación histórica. Pandora: revue d’etudes hispaniques, (2). Obtenido de https://dialnet.unirioja.es/servlet/articulo?codigo=3160107
[Consulta: 25 de octubre de 2021].
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como prácticos, de manera interdisciplinar, aunando diversos enfoques y realizando
cruces entre variadas especialidades, otorgando mayor profundidad a un proyecto
determinado. A través de ella, se pueden recuperar testimonios de hechos históricos,
trabajando de forma paralela con la oficialidad, por medio de personajes invisibilizados por la misma. De este modo se aportan nuevos antecedentes a un mismo hecho,
a partir de las diversas miradas existentes.
En el caso de Chile, la historia oral tiene una relación directa con el golpe cívico-militar perpetrado por Pinochet que se extendió entre los años 1973 y 1990. Fue
durante esta represión y ocultamiento de la información por parte de los medios de
comunicación oficiales cuando diversas organizaciones que se encontraban resistiendo en el territorio chileno, apoyados por la iglesia y otras instituciones extranjeras de
defensa de los derechos humanos, procedieron a la toma de testimonios, no con un
afán (inicialmente) de archivar, sino de defender, proteger y denunciar al mundo las
atrocidades cometidas por las fuerzas armadas y de orden23.
En ese sentido, el MMDH ha realizado una invaluable labor en términos de archivar, preservar y visibilizar testimonios, tanto de familiares de detenidos desaparecidos (DD.DD.), ejecutadas y ejecutados políticos, como de víctimas de tortura
durante la dictadura de Pinochet, transformando dichos testimonios en patrimonio
intangible para las futuras generaciones, y lo más importante: aprender del pasado
para que desde el presente seamos conscientes de la defensa de los derechos humanos y así no cometer los mismos errores a futuro.
Sobre crímenes de lesa humanidad ocurridos en la Araucanía durante la Dictadura, el Museo cuenta con una serie de archivos audiovisuales y documentos
escritos alusivos a este fatídico periodo. Dentro de lo destacado, encontramos el
trabajo realizado por la Corporación de Promoción y Defensa de los Derechos
del Pueblo (CODEPU)24, quienes deciden iniciar un proyecto ante «la necesidad
de documentar la represión vivida por los pueblos indígenas durante el periodo
de dictadura militar comprendido entre el 11 de septiembre de 1973 y el 10 de
marzo de 1990»25. CODEPU denotó esta especificidad al referirse a lo ocurrido en
la Araucanía debido a lo complejo de la situación político-social previa al golpe
en dicha zona, pero también por lo anómalo del proceso de detenciones y torturas
vividas en estos territorios rurales, los cuales impactaron profundamente a la comunidad.
Otro punto no menor dentro de las motivaciones de la CODEPU al trabajar con las
comunidades Mapuche víctimas de tortura fue que la Comisión Nacional sobre Prisión
Política y Tortura (CNPPT), institución encargada en el año 2004 de llevar a cabo el
proceso de registro y toma de testimonio a víctimas de tortura, no contaba con los mecanismos para recibir y gestionar los antecedentes de los pueblos originarios vejados
en este tipo de crímenes. Fue así como en 2005 CODEPU se dispuso a continuar la
labor de registrar los testimonios de dichas víctimas, las cuales no habían compadecido
23
24
25
Museo de la Memoria y los Derechos Humanos. (2013). Archivo Oral Museo de la Memoria y los Derechos
Humanos. Proyecto Piloto «Maestranza de San Bernardo» [Ponencia]. V Encuentro Latinoamericano de Historia Oral, San Salvador. Obtenido de http://www.cedocmuseodelamemoria.cl/wpcontent/uploads/2013/05/
MM_Chile_SSalvador.pdf [Consulta: 28 de octubre de 2021].
CODEPU nació en la ciudad de Santiago en noviembre de 1980 con la finalidad de dar soporte y asistencia a las
víctimas y a familiares de las víctimas de violaciones de los derechos humanos.
Colipi, G. (Ed.). (2011). Mirar hacia atrás. Memoria oral del pueblo mapuche. Una expresión sobre la represión y tortura en la Comuna de Tirúa, Chile (1973-marzo 1990). Santiago de Chile: Caballo de mar, 3.
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frente a la CNPPT, quedando fuera de la Comisión Valech26 de 2004 y la Comisión
Rettig27 de 1991, imposibilitando cualquier tipo de reparación por parte del Estado.
«Se logró calificar 4.073 personas, que aportaron antecedentes probados de tortura y
prisión política, entre ellos a personas del pueblo Mapuche»28.
Las comunidades concurrentes a este llamamiento por parte de la CODEPU y
la Fundación Salvador Allende prestaron declaración de manera colectiva, siendo
esto desestimado posteriormente por las organizaciones convocantes ya que, para
continuar con lo realizado por la comisión Valech, debían regirse bajo los mismos
preceptos. Fue así como nació la propuesta de memoria oral mapuche, llevada a cabo
por CODEPU y titulada «Memoria Mapuche de las Violaciones Graves de Derechos
Humanos: Historia oral, documental, fotográfica del pueblo Mapuche de la VII y IX
Región durante la Dictadura Militar (1973-1990)»29.
A partir de este proyecto de testimonios individualizados, CODEPU propuso la
realización de entrevistas a las 46 comunidades que ya habían participado del proceso anterior, creando un archivo pormenorizado de 624 entrevistas, tanto a diversos
lonkos30, como a mujeres y hombres de estas colectividades, con la finalidad de rescatar estas memorias y sus experiencias respecto a las violaciones de DDHH sufridas, y crear un respaldo de dichas memorias para, en una segunda etapa del proyecto,
poder crear la Casa Museo de la Memoria Mapuche.
Para lograr esta recopilación de memorias, se recurrió a la realización de una
matriz de entrevistas. En palabras de Eugenia Meyer, «una entrevista puede definirse brevemente como la conversación entre dos o más personas, con una finalidad
concreta, que por otra parte está encaminada a obtener cierta información»31. En este
caso, la finalidad de dichas entrevistas era poder reflexionar en torno a las comunidades Mapuche durante el periodo comprendido entre 1973 y 1990, sensibilizar respecto a la importancia de preservar estas memorias y reconstruir el pasado individual de
cada entrevistado. Para esto, se recurrió a una serie de preguntas ya definidas, tales
como nombre, edad, grupo familiar, etc. Las siguientes preguntas guardaban relación
con la vida de las y los entrevistados previa al golpe de estado y con el modo en que
se desenvolvían con su comunidad y con la gente winka32 del sector; también con
lo ocurrido el mismo martes 11 de septiembre de 1973 en su entorno cotidiano y su
familia más cercana, con el modo en que actuaron las fuerzas armadas y de orden en
su comunidad, si fueron víctimas directas o si tuvieron algún familiar que sufriera
represión, las consecuencias físicas y psicológicas de dichos actos, el tiempo en el
que se produjeron las vejaciones, el lugar al que fueron trasladados, si hubo civiles
26
27
28
29
30
31
32
La Comisión Nacional Sobre Prisión Política y Tortura fue un organismo chileno dirigido por el sacerdote,
Monseñor Sergio Valech, con la finalidad de identificar a las personas víctimas de prisión y tortura llevadas a
cabo por agentes del Estado en el periodo comprendido entre 1973 y 1990.
La Comisión Nacional de Verdad y Reconciliación fue un organismo chileno creado por el presidente Patricio
Aylwin que estuvo presidida por Raúl Rettig entre los años 1990 y 1991. La finalidad de esta comisión era la
de esclarecer la verdad respecto a los crímenes de lesa humanidad cometidos por la dictadura de Pinochet entre
1973 y 1990.
Colipi, G. (Ed.). (2011) op.cit., 15.
Ibid., 17.
Lonko, Longko (cabeza) o Cacique, corresponde al líder de una comunidad Mapuche.
Meyer, E. (1971). La historia oral. Origen, metodología, desarrollo y perspectivas. Historia Mexicana, volumen
21 (2). Obtenido de https://www.jstor.org/stable/25134913 [Consulta: 8 de septiembre de 2021].
Winka, Wingka o Huinca, es un término mapudungun, que inicialmente se usaba para referirse a los españoles
conquistadores. Con el tiempo, comenzó a usarse para referirse a cualquier persona no mapuche.
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involucrados de manera directa o indirecta en estos hechos, si el Estado se hizo cargo
de estos crímenes al volver la democracia, etc.
Dadas las particularidades que tuvo la represión en esta zona, las entrevistas se
realizaron de manera semi estructurada, para darle al entrevistado la libertad de contar lo que estimara necesario, respetando su privacidad e intimidad.
El proyecto de CODEPU Mirar hacia atrás cuenta con un total de treinta entrevistas registradas en formato de video, donde es posible conocer en extenso las
vidas de las personas entrevistadas. Cada una de estas memorias se encuentra disponible en el MMDH, convirtiéndose en un documento audiovisual fundamental para
conocer de primera fuente las vejaciones cometidas en dictadura contra el pueblo
Mapuche, así como reconocer la labor realizada por este pueblo en la recuperación
de la democracia. Esta importancia se acentúa por medio del uso de la memoria oral,
como motor testimonial de un pueblo, que, a través de generaciones, ha construido
su cultura, traspasando conocimientos por este medio. La oralidad es vital para el
pueblo Mapuche, sin la palabra sería imposible preservar la lengua y, sin ella, desaparecería el pensamiento propio de toda una comunidad.
Cada entrevistado proporcionó su historia de vida, desde aspectos vinculados
tanto a su infancia y las carencias materiales existentes en esa etapa de su vida, como
a su adolescencia, donde muchos de ellos se incorporaron a la escuela, insertándose
en un lugar donde convivían periódicamente con niños y niñas winka, con todo lo
que esto significaba en aquella época, siendo en muchos casos, víctimas de humillaciones, tanto por sus compañeros de curso como por algunos profesores.
Durante el golpe de Estado, muchos de los entrevistados eran ya adultos, con
familia constituida y padres ya mayores. En esta etapa se siente con mayor énfasis la
dureza de lo vivido durante la dictadura. Es aquí donde la voz se torna a momentos
más frágil y el ritmo del relato cambia, haciendo de las pausas algo cada vez más
frecuentes:
La escucha atenta permite detectar –a partir de tonos, gestos, silencios, rupturaso
quiebres de discurso– sentidos actuales, vinculados al propio relato, al paso del
tiempo, a las valoraciones que de ellos se haga y al contexto cultural y epocal en
que se recuperan. Se trata de fragmentos del pasado (...) incorporados o silenciados, siempre reelaborados en función de factores ideológicos, generacionales,
culturales o históricos que al ser narrados sostienen la expresión de las voces y las
actitudes33.
La forma en que se relatan estos episodios dice mucho del modo en que padecieron esta etapa, lo extremo de estas experiencias que, en muchos casos, mantuvieron
en silencio por distintos motivos. No fue hasta la constitución de la Comisión Rettig
que «se atrevieron a contar algo de lo ocurrido»34.
La importancia de los actos de relatar lo sucedido es abordable, a nuestro parecer, desde dos importantes aristas: la primera de ellas viene dada por el componente
psicológico, en dónde muchas de las personas que prestaron testimonio no habían
comentado nunca algunos hechos debido a la vergüenza y al dolor que supone revi33
34
Diamant, A. (2012). La historia oral. Buenos Aires: BMN, 15.
Bengoa, J. (2002). Historia de un conflicto. El Estado y los mapuches en el siglo XX. Santiago de Chile: Planeta,
154.
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vir estas situaciones, o al no querer que sus hijos se enteraran de esta parte de la vida
personal de quien está relatando. El peso de cargar con estas memorias finalmente se
libera a partir del acto de hablar y contar lo ocurrido, propiciando el primer paso para
lograr la sanación personal ante estos dolorosos acontecimientos. La segunda arista
guarda relación con el profundo vínculo existente entre el acto de hablar y el pueblo
Mapuche, quienes, por una cuestión cultural y estructural intrínseca, le otorgan al
habla un valor que es difícil cuantificar para el mundo occidental. El habla da forma
y soporte al mundo conocido por ellas y ellos, es la encargada de entregar identidad
a través de cómo conciben el mundo, donde cada palabra guarda un profundo significado que se vincula con su entorno natural. Más aún si la lengua madre del pueblo
Mapuche, el mapudungun, se encuentra bajo seria amenaza de desaparecer.
Según la UNESCO, se han extinguido alrededor de 200 idiomas, de un universo
de 6.000 durante las últimas tres generaciones35. De ahí la importancia de preservar
la cosmovisión y, por ende, la lengua mapuche:
Cuando se pierde una lengua, todos nosotros perdemos el conocimiento que contienen las palabras y la gramática de esa lengua, un conocimiento que nunca podrá
recuperarse si la lengua no ha sido estudiada o grabada. No toso estos conocimientos tiene un beneficio práctico inmediato, por supuesto, pero todos son vitales
para enseñarnos diferentes formas de pensar en la vida, de guiar nuestra existencia
cotidiana en el planeta Tierra36.
5. Proyecto arte sonoro Dungun
A partir de toda la información recopilada en el MMDH en torno al archivo y las
memorias mapuches en dictadura, nos proponemos realizar un proyecto archivístico-instalativo sonoro, que por un lado contenga parte de estas memorias en formato
audiovisual y, por otra parte, contemple la realización de una instalación sonora que
ahonde en torno al mapudungun, como lengua en resistencia por la pervivencia de
esta cultura ancestral.
Dungun (nombre del proyecto propuesto) es una palabra de origen mapuche que
en su forma verbal significa hablar. Se trata de una lengua de carácter espiritualista:
en todas las ceremonias mapuches el habla es la estructura fundamental. El mapudungun se desarrolló inicialmente como lenguaje oral, mnemónico y no escrito. No
fue hasta 1982 que se creó el primer grafemario mapuche con el objetivo de mantener el idioma como símbolo de la identidad y cultura colectiva37.
La pervivencia del mapudungun se ha visto amenazada en varias ocasiones: la
primera vez fue con la llegada de los españoles en 154138 y la posterior ocupación de
la Araucanía en 1860 por parte del Estado Chileno, lo que significó la mayor pérdida
35
36
37
38
Iglesias, L. (2009). Lenguas en peligro. El correo de la UNESCO, (2). Obtenido de https://unesdoc.unesco.org/
ark:/48223/pf0000186521_spa/PDF/186521spa.pdf.multi [Consulta: 21 de septiembre de 2021].
Everett, D. (2012). Lenguage: The Cultural Tool. Londres: Pantheon Books, 303. Traducción del autor.
Álvarez, P., Forno, A., y Risco, E. (2015). Propuestas de grafemarios para la lengua mapuche: desde los fonemas
a las representaciones político-identitarias. Alpha, (40). Obtenido de https://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0718-22012015000100009 [Consulta: 3 de septiembre de 2021].
Retamal, J., (1980). Descubrimiento y conquista de Chile. Santiago de Chile: Salesiana, 14.
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de territorio por parte del pueblo Mpuche, siendo concentrados en pequeñas reservas
en el sur del país, reduciendo su territorio para finales del siglo XIX a poco más de
quinientas mil hectáreas39.
Actualmente el pueblo Mapuche se encuentra diseminado en varias regiones del
país; los resultados de la Ocupación de la Araucanía, la pérdida de sus tierras y el
empobrecimiento que ha azotado a la región histórica de los Mapuche, obligó a
muchas familias a buscar nuevos horizontes para así lograr mejorar su calidad de
vida. Según el INE, en 2017 existen alrededor 1.700.000 mapuches en Chile40, de
los cuales cerca de 615.000 están afincados en la Región Metropolitana. De ese total
nacional, cerca del 65% no habla ni entiende su lengua.
6. Referencias
El proyecto Dungun se nutre de diversos artistas para su conformación teórica y técnica; comenzando con Sebastián Calfuqueo, artista chileno de origen mapuche que
ha realizado obra en torno a sus raíces y las relaciones biopolíticas existentes entre el
mundo occidentalizado y el mundo mapuche. Cada una de sus intervenciones tiene
como eje la constante tensión existente entre la cultura mapuche y la sociedad chilena, ya sea cuestionando el tema territorial mapuche, la creciente problemática del
agua, la lengua, la identidad y la sexualidad.
Dentro de la extensa lista de obras con las que cuenta Calfuqueo, nacido en 1991,
todas poseen como hilo conductor la materialidad y la técnica, que en su gran mayoría
son instalaciones, trabajos en cerámica, performance o video. De ellas podemos destacar Palabras a las aguas de 2021, obra que busca reflexionar en torno a la importancia
de las aguas para la vida, no solo humana, sino para el ecosistema completo41, más
aún tratándose de Chile, país que lleva años atravesando por serios problemas hídricos
tanto a nivel ambiental como socioeconómico. Esta instalación está constituida por 70
metros de tela azul, 12 piezas de cerámica esmaltada azul con textos en color blanco,
3 luces de color azul y 2 canales de audio. La tela que zigzaguea a través de la sala
lleva una inscripción en letras blancas que dice «un río que no corre es solo un reflejo»,
haciendo alusión a la privatización de los ríos y napas subterráneas de nuestro territorio. Sobre la tela se posicionan las 12 piezas de cerámica azul, cada una de las cuales
contiene una inscripción que hace referencia a este elemento, pero abordado desde los
distintos intereses que cruzan la obra de Calfuqueo: el agua desde lo elemental para
el pueblo mapuche, como bien público y de acceso universal, como creadora de vida
y como elemento fluido de cuerpo líquido. Los últimos elementos a destacar de esta
instalación son los dos audios: se trata de dos poemas que suenan al mismo tiempo y
hablan de la potencia y fluidez del no binarismo que representan las aguas, así como de
la trascendencia que tienen las aguas para la vida en el planeta.
39
40
41
Almonacid, F. (2009). El problema de la propiedad de la tierra en el sur de Chile (1850-1930). Historia (Santiago), 1 (42). Obtenido de https://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-71942009000100001
[Consulta: 3 de septiembre de 2021]
Instituto Nacional de Estadísticas. (s.f). Obtenido de http://resultados.censo2017.cl/ [Consulta: 21 de septiembre de 2021]
Calfuqueo, S. (2022). Palabras a las aguas. Obtenido de https://sebastiancalfuqueo.com/2022/04/09/palabrasa-las-aguas-2021/ [Consulta: 17 de junio de 2022]
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Continuando con las referencias existentes en Dungun, encontramos el proyecto
Argentina suena del año 201642, dirigido por el investigador y docente de la Universidad Nacional de Tres de Febrero, Raúl Minsburg, quien convocó a una serie de
artistas sonoros de distintas zonas de Argentina para realizar grabaciones de campo
de sus respectivos pueblos o ciudades y, de esa manera, componer una obra. En palabras de Minsburg, «no se intentó realizar un mapa sonoro, (…) sino que se convocó a
compositores que vivieran en zonas lo más alejadas posibles entre sí»43. El resultado
del proyecto se tradujo en 14 piezas, las cuales buscan plasmar la identidad sonora
de sus respectivas regiones a través de los sonidos más característicos.
Dentro de estas propuestas, muy particulares y distintas unas de otras, quisiéramos detenernos en la realizada por Federico Barabino, artista sonoro y docente, que
realizó la pieza titulada Jujuy 441, Merlo Buenos Aires. Esta pieza se desmarca de las
demás por la presencia protagónica de la voz como relato, encontrando testimonios
de dos personas que comparaban los sonidos de sus respectivos barrios en el pasado
y en el presente. Se hace referencia a antiguos clubes de baile, los cuales colmaban
el barrio de música, o la tranquilidad que ofrecía cierto lugar al no contar con el flujo
aéreo que sí posee ahora. Todo esto acompañado de quiebres sonoros, ruidos, errores, bucles de otras grabaciones, potenciando y enriqueciendo la pieza final.
Sin duda, el ajedrez electroacústico de John Cage Reunion44 es vital en la creación
de Dungun. Cage, junto a Marcel Duchamp, realizó la primera exhibición en 1968 en
el Ryerson Theatre de Toronto, Canadá. Reunion corresponde a un ajedrez electrónico que generaba sonidos que iban variando de acuerdo con la posición de las piezas
en el tablero y los fotosensores ubicados en cada una de las 64 casillas del tablero.
La infinidad de movimientos y combinaciones que permite el ajedrez le otorgaba a
esta pieza una riqueza de posibilidades sonoras insospechadas.
Otra referencia fundamental, esta vez desde lo técnico-estético, es el proyecto
del año 2006 de los alemanes Hauert & Reichmuth, titulado Instant City45. Esta obra
consiste en una mesa interactiva que combina diversos elementos, tales como: instrumento musical digital, juego de mesa, montaje experimental psicológico, etc. Se
trata de una mesa sonora a la que uno o más jugadores pueden añadir y superponer
bloques geométricos semitransparentes para así realizar distintas composiciones de
sonidos en base a la ubicación y disposición de los bloques puestos en el tablero. Se
trata de una composición sonora realizada en un micro tablero situacionista, el cual
va cobrando vida y emitiendo sonidos a medida que la ciudad va aumentando su
volumen. Las posibilidades son innumerables ya que el sonido resultante es consecuencia del número de bloques utilizados y su disposición en el espacio de la mesa,
ya que incluso se pueden poner unos sobre otros, modificando de esta manera el tono
o ritmo de la composición.
42
43
44
45
Minsburg, R. (2016). Identidad y arte sonoro: el proyecto Suena Argentina. Listen Over , 12 (1), 44–52. https://
doi.org/10.14393/OUV18-v12n1a2016-3
Ibid. 51.
Idis. (2022). Ajedrez electroacústico Duchamp vs Cage. Obtenido de https://proyectoidis.org/ajedrez-electroacustico-duchamp-vs-cage/ [Consulta: 15 de junio de 2022]
Atelier Hauert Reichmunth. (2022). Instant city. Obtenido de http://www.hauert-reichmuth.ch/en/projekte/instant-city/ [Consulta: 15 de junio de 2022]
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7. Instalación
Dungun es una instalación interactiva constituida por dos elementos centrales.
El primero de ellos consiste en un ajedrez sonoro en el que se podrá realizar una
(o muchas) partida(s) conformada por dos equipos, las piezas blancas y las negras;
estas a su vez están vinculadas a dos audios distintos: las blancas están asociadas a
un fragmento del poema épico de Alonso de Ercilla La Araucana (primer registro
escrito que habla de la resistencia mapuche ante los españoles en 1570) en castellano, mientras que las piezas negras están vinculadas al mismo fragmento del poema,
pero recitado en mapudungun.
Al iniciar la partida, los audios simultáneos se encontrarán con el mismo nivel de
volumen, pero, a medida que un equipo vaya eliminando piezas de su oponente, estos reducirán el volumen en función del número de piezas que han sido tomadas. El
sonido predominante de la instalación se escuchará en relación al equipo que tenga
más piezas en el tablero, creando la metáfora de la utilización del territorio, determinado por el mismo tablero de ajedrez.
Figura 1. Modelado 3D de instalación.
El tablero es una metáfora del territorio que habita el pueblo mapuche, donde
buscan mantener vivas sus tradiciones a través de su lengua. Son un colectivo casi
sin presencia a nivel mediático, que salvo por situaciones ligadas a la contingencia
logran algo de visibilidad. A fecha de hoy esta visibilidad ha aumentado gracias a
la Convención Constitucional y el rol que ha jugado particularmente Elisa Loncon,
activista mapuche, al presidir dicho órgano. Aún así, es importante mantener la discusión sobre la necesidad de revitalizar el mapudungun desde lo cotidiano a través
del empoderamiento local.
La inclusión del poema de La Araucana es crucial, ya que es la primera crónica
que registra el conflicto librado entre el pueblo Mapuche y los conquistadores es-
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pañoles. Es el punto de inicio de una larga historia de lucha por la pervivencia de un
pueblo y su tierra, por ende, de su lengua, la cual es un territorio cognitivo libre desde donde se puede resistir. Esta lucha es extrapolable a cada comunidad Mapuche,
resida en el sur de Chile, en la capital o en algún cerro de la ciudad de Valparaíso.
Digo que norte sur corre la tierra,
y báñala de oeste la marina;
a la banda del este va una sierra
que el mismo rumbo mil leguas camina:
en medio es donde el punto de la guerra
por uso y ejercicio más se afina:
Venus y Amón aquí no alcanzan parte,
sólo domina el iracundo Marte.
Pikum willi püle winufküley mapu
konwe antü püle muñetuniey lhafkenh,
puwel püle müley pire winkul mapu
feichi rüpü ka inaniyey waragka legua;
ragintu mew müley kiñe küme lelffüm,
zuampeyel, mümülpeyüm, weychapeyüm:
Venus egu Amón gelay faw ta ñi müleam,
fofoillkum Marte fey mütem güneniyey46
El objetivo de este ajedrez es visibilizar por medio de la voz una problemática
sostenida a través de décadas: la constante disminución del mapudungun a lo largo
del territorio chileno debido a factores políticos, sociales y culturales.
El ajedrez es una buena metáfora de este problema, ya que este milenario juego
ahonda en torno a la disputa de territorio entre dos grupos, de cómo se desenvuelven
estos en un territorio, y de cómo conviven en un mismo espacio. En este caso, la
disputa de territorio no es solo física, es también simbólica, puesto que a medida que
los mapuches afincados en distintas comunidades van perdiendo espacio para desarrollar sus actividades, su lengua también va perdiendo protagonismo en el diario
vivir, por lo que es importante, como país, prestar atención a la participación y cabida de nuestras lenguas originarias en más sitios, como por ejemplo algunos centros
de asistencia médica primarios, que disponen de señalética en mapudungun. De esta
manera, se potencia el desarrollo del mapudungun dándole difusión e integrándose
además como parte de la herencia cultural que posee Chile.
46
Schwemberg, H., y Azocar, A. (2006). La Araucana – Ta Awkan mapu Mew. De Alonso de Ercella y Zuñiga.
Versión Castellano-Mapuzungun. Santiago de Chile: Ministerio de Educación. Gobierno de Chile, 23.
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Figura 2. Modelado 3D de instalación
El juego del ajedrez tiene reglas predeterminadas, como los turnos, los distintos
tipos de piezas y los movimientos que hace cada una de ellas, las diferentes jugadas o
posiciones existentes en una partida, también, las múltiples estrategias que se pueden
utilizar contra el oponente.
Por otra parte, al tratarse de una instalación artística, estas reglas pueden adaptarse a la conveniencia del proyecto. En este caso, también se puede usar de manera libre, sin la necesidad de seguir las reglas clásicas de una partida de ajedrez,
y usarse como una mesa sonora en donde se puede cotejar cada poema de manera
separada, o en conjunto realizando un sinfín de variaciones en torno al volumen
y cómo estas dos lenguas pueden coexistir en un mismo espacio. Esta modalidad
está pensada principalmente para niñas y niños que, sin tener nociones de cómo
jugar ajedrez, pueden igualmente interactuar con la instalación y ahondar en torno
al mapudungun.
La posibilidad de escuchar el poema épico de Ercilla en ambos idiomas de
manera simultánea, nos ofrece la oportunidad de adentrarnos en un idioma que
para el chileno medio es identificable, pero puede resultar a la vez, ininteligible.
Poder contrastar y comparar ambas frases puede convertirse en la puerta de entrada para aquellos que se quieran iniciar en el conocimiento y aprendizaje del
mapudungun.
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Figura 2. Modelado 3D de instalación
El segundo elemento importante de esta instalación es una serie de pantallas donde la memoria oral se hace presente. Aquí también se distinguen dos grupos de audiovisuales. El primer grupo consiste en dos pantallas, cada una contiene una de las
entrevistas hecha por CODEPU y archivada por el MMDH, donde un miembro de la
comunidad relata lo sucedido durante su infancia, adolescencia, el golpe de estado
y las consecuencias que este hecho tuvo para su comunidad. Las otras dos pantallas corresponden a los testimonios de dos personalidades del mundo mapuche que
hablarán sobre la importancia de la lengua en la cosmovisión mapuche, de lo vital
que es la preservación del mapudungun como puente de memoria y transmisión de
conocimientos. Dichos testimonios serán realizados de manera personal, utilizando
la pauta que CODEPU efectuó al momento de llevar a cabo sus entrevistas y que
dejó registrada en su libro.
El proyecto Dungun remite a las memorias de un pueblo que se ha mantenido en
resistencia desde que se tienen registros escritos del mismo. Comenzando con la primera aproximación que tuvo con el ejército conquistador español; en el combate de
las tropas de Cornelio Saavedra a lo largo de la ocupación de la Araucanía; durante la
dictadura de Pinochet y las violaciones de los Derechos Humanos; o en el actual conflicto forestal en las tierras ancestrales de esta comunidad, las actuales provincias de
Malleco y Cautín, donde grupos empresariales tanto extranjeros como nacionales –la
maderera japonesa Volterra, Forestal Mininco del grupo Matte, Forestal Arauco del
grupo Angelini, o Forestal Bosques Cautín47–, se han enriquecido de manera ilícita,
a la luz de la documentación entregada, a costa de las tierras que han pertenecido de
manera milenaria al pueblo Mapuche.
Esa misma resistencia se traslada al campo de las memorias ya que, como señala
Elizabeth Jelin, «encontramos una situación de luchas por las representaciones del
47
Mapuexpress. (2016). Recuperado de https://www.mapuexpress.org/2016/11/21/araucania-listado-de-empresas-consideradas-transgresoras-de-derechos-humanos-y-la-naturaleza/ [Consulta: 24 de septiembre de 2021].
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pasado, centradas en la lucha por el poder, por la legitimidad y el reconocimiento»48,
otorgándole un nombre a un hecho en base de lo que se recuerda. En ese sentido, es
fundamental mantener y reforzar dichas memorias en la disputa social, para que la
hegemonía no siga invisibilizando la resistencia de este pueblo.
8. Conclusiones
Desde el fin de la ocupación de la Araucanía, el pueblo Mapuche sufrió una notable disminución de territorio que se tradujo en movilizaciones forzadas, empobrecimiento y estigmatización, lo que ha conllevado la pérdida gradual pero constante de
su lengua. Esta situación comenzó a cambiar después de la Ley de Reforma Agraria
de 1962, de 1967 y la Ley Indígena de 1972, logrando un significativo avance en la
recuperación de las tierras que habían sido arrebatadas un siglo atrás. Lamentablemente, todo este proceso de reconocimiento, empoderamiento y trabajo colectivo
entre las distintas comunidades Mapuche se vio truncado con el golpe de Estado
perpetrado por Pinochet, desbaratando en muy poco tiempo, con ayuda de civiles de
la zona, lo construido por las diversas agrupaciones sociales, campesinas e indígenas, y las instituciones gubernamentales de la Unidad Popular, volviendo todo, por
medio de persecución, prisión, torturas y desapariciones, a un punto con las mismas
características que tenía Chile previamente a la reforma de 1962.
Con el retorno a la democracia, instituciones como CODEPU llevaron a cabo una
invaluable labor, al realizar el registro audiovisual de comuneros mapuches en las VIII
y IX regiones sobre hechos acontecidos en la comuna de Tirúa, que dieron testimonio
respecto a la represión vivida durante los años de dictadura y las consecuencias que
ésta tuvo a nivel personal y colectivo. Dicho registro se encuentra actualmente en el
MMDH así como en otros sitios afines. Documentos como estos son vitales, ya que
constituyen parte de nuestro patrimonio, son el reflejo de una actividad política y social de una época determinada en una localidad específica; son memoria, permitiendo
configurar nuestro presente y futuro a partir de un hecho pasado; también son conocimiento, dada la enorme y valiosa cantidad de información adjunta en cada testimonio,
que se puede además contrastar con otras fuentes historiográficas. Así mismo, este tipo
de documentos proporciona pautas técnicas en torno a cómo llevar a cabo, desde lo
audiovisual, una entrevista de estas características, convirtiéndose en un manual para
aquellos realizadores que se están adentrando en el tema documental.
Desde hace ya varias décadas, se ha creado una suerte de vínculo entre arte, memoria y archivo, asociado principalmente al Holocausto y horrores posteriores que
son importantes recordar. En el Cono Sur, dicho proceso ha estado marcado, al igual
que la historia oral, por la profunda huella dejada por las dictaduras de estos países, a
la que Chile no es ajeno. Fue casi una pulsión de la coyuntura aunar estas disciplinas,
una búsqueda de verdad y justicia por parte de creadores y creadoras cercanas a los
Derechos Humanos, con la convicción de que, por medio de sus obras, se pudiese
vencer el olvido.
En el caso de Dungun, proyecto expositivo surgido a raíz de la investigación en
el MMDH, la memoria se aborda desde aquella que se quiso atribuir en el poema
48
Jelin, E. (2002). Los trabajos de la memoria. Buenos Aires: Siglo XXI, 36.
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de Ercilla, asignándole categorías épicas al pueblo Mapuche, así como desde los
testimonios del trabajo de CODEPU proyectados y los relatos que hablan sobre el
acto de hablar. Se trata de un proyecto, que, desde el punto de vista de la memoria,
es una pieza «en tanto que archivo», tanto por el acto de trabajar con un documento
literario para integrarlo a una obra de arte interactiva, como por usar un archivo
como parte fundamental de la obra. «Esta recuperación de la memoria (“recordar
como una actividad vital humana define nuestros vínculos con el pasado, y las vías
por las que recordamos nos define en el presente”) rehabilita los necesarios diálogos
pasado-presente y sincronía-diacronía»49 y, ya que el pasado se encuentra en constante disputa, nos permite hacernos cargo de él trabajando con y para las memorias.
Dichas memorias, añadidas a una instalación interactiva, no sólo se abordan desde
lo lúdico, sino que también son abordables desde la trascendencia que tienen para
entender de manera cabal la experiencia humana. En ese sentido, la labor del MMDH
es crucial, no solo por proteger, archivar y visibilizar la historia de un periodo determinado de Chile, sino también por brindar espacios culturales para la creación y
reflexión en torno a su archivo, con vistas a un futuro en donde el atropello de la vida
y la aniquilación de culturas ancestrales no vuelva a ocurrir.
Desde lo sonoro, es pertinente mencionar la importancia que tiene trabajar con
voces mapuches, ya que tanto esta como muchas otras lenguas prehispánicas, se encuentran relegadas a un segundo plano debido al proceso de colonización50. En este
sentido, la dictadura cívico-militar de Pinochet se encargó de soterrar cualquier rasgo
que fragmentara la unidad de Estado nación que el mismo régimen quería impulsar, y
en ese sentido el mapudungun era disruptivo. El mapudungun, en el momento de ser
hablado, se proyecta como un acto de resistencia, ya que dicha lengua es identitaria
de un pueblo que pervive pese a los embates recibidos durante siglos. Hablar no solo
es comunicar y transmitir un mensaje a otra persona, es también hacer presente un
territorio, material y simbólico51, más aún tratándose de una lengua donde las onomatopeyas son vitales y la configuración de las palabras está en directa relación con
el sonido de sus bosques, ríos, volcanes y aves. Por este motivo, hablar es resistir;
cada vez que se habla mapudungun, se recuerda un territorio asediado, un bosque
que lucha por no ser talado, un río secado o prisionero de una empresa papelera.
De esta manera, realizar proyectos de arte que aborden la problemática mapuche
desde lo sonoro es resignificar el habla y extender sus alcances a otras áreas del conocimiento, transformando Dungun en un dispositivo que pretende reflejar la lucha
que este pueblo ha dado, desde que se tienen registros de su existencia, gracias a las
crónicas realizadas por Alonso de Ercilla.
49
50
51
Guasch, A. (2005). Los lugares de la memoria. El arte de archivar y recordar. Matèria: Revista d’ art, 5. Obtenido de https://annamariaguasch.com/es/Publicaciones/Los_lugares_de_la_memoria:_el_arte_de_archivar_y_
de_recordar [Consulta: 11 de septiembre de 2021].
García, E. (2016). Imágenes y sonidos del Wall Mapu. El proyecto de descolonización del universo visual y
sonoro del Pueblo Mapuche. Empiria. Revista De metodología De Ciencias Sociales, (35), 125–151. https://doi.
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Ninguno.
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Chile: una aproximación preliminar a partir de tres casos en la Región de Los Lagos.
Panambí. Revista De Investigaciones Artísticas, (12). https://doi.org/10.22370/panambi.2021.12.2486
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News coverage of the Church dealing with the pandemic: Spanish and Italian newspapers
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Church, Communication and Culture Church, Communication and Culture ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/rchu20 ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/rchu20 Full Terms & Conditions of access and use can be found at
https://www.tandfonline.com/action/journalInformation?journalCode=rchu20 ABSTRACT This paper focuses on media representation of the Catholic
Church during the first months of the coronavirus pandemic in
Italy and Spain. The purpose is to know when and how the
Catholic Church starred in the news; how its actions were pre-
sented, and using which frames. The work analyses the coverage
of the Church in Corriere della Sera and El Paıs between January
and May 2020 and connects the deductive process (news frames
by Semetko and Valkenburg) with the inductive process (setting
common places or topoi) in order to get a better understanding
of the news-framing. Findings show that both newspapers gave
prevalence to news about the charitable work of the Catholic
Church during the pandemic instead of about conflict with the
political authorities because of restrictive measures. When it
comes to the relationship with civil institutions, a positive image
of the Church was offered, one that portrayed her as ready to col-
laborate and apply the preventive measures recommended by
the health authorities. The positive perception is more frequent
than the negative one when there are dualities in the topoi that
are found in the interpretation of the facts. KEYWORDS
Covid-19; pandemic;
content analysis;
framing; topoi María-José Pou-Amérigo María-José Pou-Amérigo To cite this article: María-José Pou-Amérigo (2022) News coverage of the Church dealing with
the pandemic: Spanish and Italian newspapers, Church, Communication and Culture, 7:1,
154-174, DOI: 10.1080/23753234.2022.2040373 To link to this article: https://doi.org/10.1080/23753234.2022.2040373 © 2022 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
Group. Published online: 10 May 2022. Submit your article to this journal
Article views: 393
View related articles
View Crossmark data © 2022 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
Group. Published online: 10 May 2022. Submit your article to this journal
Article views: 393
View related articles
View Crossmark data Full Terms & Conditions of access and use can be found at
https://www.tandfonline.com/action/journalInformation?journalCode=rchu20 Full Terms & Conditions of access and use can be found at
https://www.tandfonline.com/action/journalInformation?journalCode=rchu20 CHURCH, COMMUNICATION AND CULTURE
2022, VOL. 7, NO. 1, 154–174
https://doi.org/10.1080/23753234.2022.2040373 News coverage of the Church dealing with the
pandemic: Spanish and Italian newspapers Marıa-Jose Pou-Amerigo
Journalism, Universidad CEU Cardenal Herrera, Valencia, Spain Marıa-Jose Pou-Amerigo Marıa-Jose Pou-Amerigo Journalism, Universidad CEU Cardenal Herrera, Valencia, Spain Journalism, Universidad CEU Cardenal Herrera, Valencia, Spain ARTICLE HISTORY
Received 30 June 2021
Revised 11 November 2021
Accepted 22 November 2021
KEYWORDS
Covid-19; pandemic;
content analysis;
framing; topoi ARTICLE HISTORY
Received 30 June 2021
Revised 11 November 2021
Accepted 22 November 2021 ARTICLE HISTORY
Received 30 June 2021
Revised 11 November 2021
Accepted 22 November 2021 CONTACT Marıa-Jose Pou-Amerigo
mpou@uchceu.es
Journalism, Universidad CEU Cardenal Herrera,
Valencia, Spain
2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
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. ARTICLE HISTORY
Received 30 June 2021
Revised 11 November 2021
Accepted 22 November 2021 Journalism, Universidad CEU Cardenal Herrera, , p
2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. 1. Introduction The most representative image of the hardest lockdown in Europe in 2020 was absence:
streets, squares, or historical monuments, that so often used to be full of neighbors and
visitors, appeared devoid of life and movement, like a static scene captured by a photo-
graphic camera. Among all of them, Saint Peter’s Square, a point of reference for all
Catholic people and a universal tourist destination, was the best example of the excep-
tional measures taken in those days. There, one of the iconic images summarized the
effects of the pandemic on social life. It was April 26, and the Pope was preparing to
impart, alone, the Urbi et Orbi blessing. This square, designed to embrace everyone,
was completely empty. mpou@uchceu.es CHURCH, COMMUNICATION AND CULTURE 155 The image travelled around the world with adjectives about the exceptional scene
(‘historical’, ‘extraordinary’ or ‘unusual’). In the same way, the development of religious
activity without the physical presence of the people transferred the meeting of com-
munities to the digital network. This was a challenge for the Catholic Church and news
for a large part of public opinion. The life of the Church did not stop, but the approach
required the mediation of the media. This paper focuses on this mediation role and analyzes the story about the work of
the Catholic Church during the first months of the coronavirus pandemic what was
published by the Italian press and the Spanish press. Our objective is to know when
and how the Catholic Church starred in the news; how its actions were presented, and
through which frames it was seen. This paper analyzes Corriere della Sera and El Paıs,
the best-selling daily newspapers in Italy and Spain. The aim is to know what image the media offered about the Church during a global
crisis. The interest in the question is related, on the one hand, to the efforts made by
religious entities, especially the Catholic Church, to present their message to society
without distortions and, on the other, to the knowledge of the keys that produce these
distortions. 2. The Church and the pandemic in Italy and Spain In Italy, the lockdown began in Lombardy, but on March 9th, Prime
Minister Conte extended it to the whole country; in Spain, on the 11th, President
Sanchez announced it for March 14th. In this country, the measures did not include
the closure of places of worship but made it difficult, in fact, to participate in religious
ceremonies and activities, especially due to the impossibility of traveling to the spaces
dedicated to worship. In Spain, the Royal Decree 463/2020, of March 14th (BOE 2020) said in article 11 Attendance at places of worship and civil and religious ceremonies, including funerals, is
conditioned on the adoption of organizational measures consisting of avoiding crowds
of people, depending on the size and characteristics of the places, in such a way that
guarantee attendees the possibility of respecting the distance between them of at least
one meter. In Italy, however, although places of worship were not closed in the Decree of the
President of the Consiglio of March 8th (Gazetta Uficciale della Repubblica Italiana
2020) religious ceremonies, including funerals were suspended by Article 1: i) the opening of places of worship is conditional on the adoption of organizational
measures that avoid gatherings of people, considering the size and characteristics of the
places, and such as to guarantee visitors the possibility of respecting the distance
between them of at least one meter as per attachment 1 letter d). Civil and religious
ceremonies, including funeral ones, are suspended. As in the case of other individual freedoms, restrictions generated protests and
reproaches. The Council of Europe (2020, 6) had to remind governments of the
requirement of proportionality in these initiatives: Effective enjoyment of all these rights and freedoms guaranteed by Articles 8, 9, 10 and
11 of the Convention is a benchmark of modern democratic societies. Restrictions on
them are only permissible if they are established by law and proportionate to the
legitimate aim pursued, including the protection of health. The significant restrictions to
usual social activities, including access to public places of worship, public gatherings and
wedding and funeral ceremonies, may inevitably lead to arguable complaints under the
above provisions. It is for the authorities to ensure that any such restriction, whether it
is based on a derogation, is clearly established by law, in compliance with relevant
constitutional guarantees and proportionate to the aim it pursues. 2. The Church and the pandemic in Italy and Spain In December 2019, the World Health Organization (WHO) admitted the existence of a
new coronavirus, SARS-CoV-2, and the disease associated with it, Covid-19, initially
located in Wuhan (China). From that moment, the EU began to take measures to
respond to this threat. Thus, on January 17th, the Health Security Committee met to
address the new coronavirus for the first time under the indications of the European
Center for Disease Prevention and Control. The risk was considered low or very low
(European Commission 2020, 2). A few days later, on January 24th, the first case was
known in Europe, in France. At that time the disease was still linked to China or to
people who had just returned from there, however, on January 28th, the first ‘native’
case was known in Europe; it was a German person who had not traveled to Wuhan. In Italy, the first cases were known on January 30th. They were two Chinese tourists
who had been in Milan on January 23rd and, later, traveled to Rome; the first non-
imported case was found in the hospital in Codogno (Lombardy) on February 21st. In
Spain, the first known patient was a German tourist on the island of La Gomera, on
January 31st, but the first local case was known on February 26th. In Europe, the first deaths occurred in France on February 15th and in Italy on
February 22nd. In Spain, the first death occurred on February 13th, but its cause was
only known later. From that moment, cases multiplied. In Italy, Lombardy became the
‘ground zero’ of the pandemic, and one of the most affected regions in Europe. Spain
would also lead with the worst statistics during the first wave, looking itself in the
Italian mirror to gain an idea from the advance of the pandemic in that country how
things might go on the Iberian Peninsula. In both countries, the authorities began to take measures as soon as the risks were
known and, above all, when the first diagnosed cases appeared. Just a few days sepa-
rated the declaration of a state of alarm in both countries: on March 8th, in Italy; on M.-J. POU-AMÉRIGO 156 March 14th, in Spain. The pandemic in Italy seemed to advance on a timeline approxi-
mately a week ahead of Spain’s. In addition, both governments approved severe measures of lockdown almost at the
same time. 2. The Church and the pandemic in Italy and Spain The religious authorities also showed their disappointment, especially when, despite
the fact that in some cases where no rules were being broken, the forces the law inter-
vened to end ceremonies and to fine those responsible. The debate was transferred to
the media, although the respective Episcopal Conferences had given instructions, from
the beginning, to obey the sanitary rules. Thus, the Executive Commission of the
Spanish Episcopal Conference (CEE 2020) published a note on March 13th, in order to
make ‘ … a call to follow the instructions of those responsible for health to avoid the
accelerated progression of the disease’ while its president, Cardinal Omella, invited
people to follow the masses live-streamed on radio and television. The Italian
Episcopal Conference (CEI 2020), in a note published on March 8th, regretted the hard CHURCH, COMMUNICATION AND CULTURE 157 measures adopted, but he accepted them for the common good: ‘The acceptance of the
decree is mediated only by the will to do, even in this juncture, one’s own part to con-
tribute to the protection of public health’. Italy and Spain share values, history, and religious tradition, although Italy’s close-
ness with Vatican City has always made it unique. However, essentially the two are
comparable, since both are countries with a Catholic majority (around 80% baptized),
but with a much lower rate of daily religious practice. What has been called a ‘Catholic
imprinting’ (Verzichelli 2007) remains in this majority, and it spreads religious values
in social life, although some public officials reproach the religious authorities for this. These values have remained stable even during the pandemic, a period in which religi-
osity has not suffered (Reeskens et al. 2021, 159), because paradoxically, ‘in a certain
sense it is precisely at the moment of greatest difficulty that the need for support in
spiritual terms becomes stronger, in the hope of finding comfort in one’s faith and
beliefs’ (Coppola et al. 2021). Despite Italy and Spain having a similar profile in terms of religious values and the
influence and the role of the Catholic Church in public life, the social debate about the
difficult balance between religious freedom and health care differed in the two coun-
tries during the first months of the pandemic. 2. The Church and the pandemic in Italy and Spain In Italy, some voices recalled the cap-
acity of Catholic Church to decide the best measures in liturgical celebrations to
prevent contagion, but in Spain this issue was hardly raised in public opinion – there
were hardly any complaints about the restrictions on religious activity, and these came
only from people critical of power. During the pandemic, misinformation grew; in fact, the development of the disease
was plagued with fake news (Perez-Dasilva, Meso-Ayerdi, and Mendiguren-Galdospın
2020). In this sense, it is necessary to know if the media also offered a misleading view
of the Church’s work during the pandemic. It is relevant to the extent that it helps us
to understand the role of the press in building the public image of the Catholic
Church. In a context of fake news and disinformation such as the one experienced, it is
necessary to analyze the process of the construction of reality. In this way, it will be
possible to delve into, on the one hand, the decision guidelines and productive routines
of the media, and on the other, the implications that the process of construction of an
‘alternative reality’ in the media may have for social organizations, especially the
Catholic Church. 3. Methodology and the field of the study In this context, the comparative analysis of Italy and Spain seems especially timely. Both countries share religious tradition (both are Catholic, with a similar percentage of
believers and religious practice; and with the presence of the Catholic Church in public
life along with other religious communities) and both experienced a simultaneous
development of Covid-19 and in the same geographical area of the south of Europe, on
the Mediterranean. However, the voice of the Church in Italy is received differently
than it is in Spain, hence the relevance of the comparison. If the objective is to know M.-J. POU-AMÉRIGO 158 whether the media discourse varies between the Italian and Spanish press, and what
influence the frames and topoi have on it, the comparison can help. Therefore, the research objectives are: 1. Quantify the presence of the Catholic Church in the Italian and Spanish quality press; uantify the presence of the Catholic Church in the Italian and Spanish quality press; 1. Quantify the presence of the Catholic Church in the Italian and Spanish quality press;
2. Identify which frames and topoi are in the news about the activities of the Church
during the pandemic; 2. Identify which frames and topoi are in the news about the activities of the Church
during the pandemic; 3. Compare the journalistic treatment of the Church and religious activity in the
Spanish and Italian press. 3. Compare the journalistic treatment of the Church and religious activity in the
Spanish and Italian press. We believe there are common features in the Spanish and Italian press regarding the
image of the Catholic Church during the pandemic. In this context, the social system
had acquired an important role, so some groups appear as ‘close collaborators of the
citizenry’ (Argi~nano and Bilbao 2020, 19). Thus, given the social role of the Church, it
is probable that its presence in the press during the pandemic was defined by its tasks
of spiritual and charitable assistance, and not by its political role in conflict with the
authorities, as might have been expected after restrictive measures on the part of
the government. Conflict and deviation-negativity (Van Dijk 1990) are very recurrent news-values in
journalistic information (Contreras 2006) and, although we wouldn’t expect it in a tra-
gedy such as the coronavirus pandemic, disappointment with political measures was
on the table. 3. Methodology and the field of the study In addition, conflict is behind much of religious news because often jour-
nalists are more interested in news when it is connected to scandals, conflicts, and
negativity (Radde-Antweiler and Zeiler 2020, 4), without forgetting that the pandemic
‘has increased polarization in an already polarized media context’ (Iranzo-Cabrera and
Lopez-Garcıa 2021, 264). Often conflict is the key to journalistic interpretation of information about the
Church because it is presented as a political entity (Pou-Amerigo, 2007), however, the
same does not happen when the story is about the Church assisting the disadvantaged. We established various research questions: Q1: How many texts about the Catholic Church in the Italian and Spanish press were
published in the selected titles during the pandemic? published in the selected titles during the pandemic? Q2: What actions are described in them? Q3: In what percentage do actions of a spiritual or assistance nature predominate? Q4: In what percentage is the Church a factor of conflict? Q5: Are there differences between the Italian and Spanish press? Thus, hypothesis 1 is regarding the frames of reference through which the role of
the Church is interpreted when the story is a global tragedy: H1. The Italian and Spanish press gave prevalence to the charitable work of the
Catholic Church during the pandemic instead of to the conflict with the political
authorities over restrictive measures. CHURCH, COMMUNICATION AND CULTURE 159 Likewise, the paper seeks to establish the keys to the journalistic construction of the
image of the Church in the context of the pandemic: What natural topoi are activated in the news about the activity of the Church? Q6. What natural topoi are activated in the news about the activity of the Chur Q7. What cultural topoi are activated in the news about the activity of the Church? Q8. Which are positive actions on the part of the Church, according to the press? Q9. Which are negative actions on the part of the Church, according to the press? Q10. What are the differences between the Italian and Spanish press? Q10. What are the differences between the Italian and Spanish press? Hypotheses 2 and 3 regard to the evaluation criteria that the media use around
the Church: H2. The Italian and Spanish press valued the spiritual and welfare work of
the Church. H2. The Italian and Spanish press valued the spiritual and welfare work of
the Church. H3. 3. Methodology and the field of the study POU-AMÉRIGO 160 hand, in the printed edition, the limitation of space and the absence of additional con-
tent from the web allow the editorial criteria to be assessed more clearly. The selection was made based on the presence of the terms ‘catholic’, ‘church’, ‘priest’,
‘bishop’ or ‘pope’, together with ‘covid’, ‘coronavirus’ or ‘virus’. The search focused on the
informative and interpretive genres (news, report, interview and chronicle) since in opin-
ion texts the author’s interpretive framework is explicit and is not imposed on the reader,
as it is a personal point of view. However, the informative texts are offering an account of
verified facts, not a questionable opinion. Thus, the unit of analysis was each unitary
informative text. Three coders – two researchers and a Ph.D. student in Communication – worked
on the coding, as well as two researchers from outside the team who acted as external
inter-judges to validate the results. The selection of the topoi was developed from a ran-
dom reading of two pieces every week from each paper throughout the period. The
process was inductive. Subsequently, the presence of the topoi was verified in the entire
sample, so that each coder analyzed two thirds of the total sample to ensure that each
third was analyzed by two different coders. Two types of data were obtained from the units of analysis: identifying data and
analytical data. The identification data is necessary to identify each piece; they are date,
headline and page or URL. The analysis data refers to the content and narrative options
of the author. They are, therefore, the frame: the kind of collaboration with the author-
ities; the type of activity and the common places or natural and cultural ‘topoi’. The ‘news frames’ are part of the journalistic story because they serve as a concep-
tual framework (Sadaba and Virgili 2007, 210) from which the journalist analyzes, gives
meaning and, finally, narrates what happened. It is the place, therefore, where the jour-
nalist’s interpretation and the ‘framing strategy’ of the media are found (Lopez-
Rabadan 2010). It is a way of approaching the mediation process through the journalis-
tic story, with which the media becomes an ‘essential actor in the process of production
and construction of public discourse, but far from the only one’ (Valera Ordaz 2016, 15). That work involves a selection process, which means accepting and rejecting. 3. Methodology and the field of the study The Italian and Spanish press criticized the Church’s opposition to government
measures, reintroducing it as an entity with a political role. H3. The Italian and Spanish press criticized the Church’s opposition to government
measures, reintroducing it as an entity with a political role. We chose the two most representative newspapers of public opinion in each of the
countries, with a significant penetration among the population. In Italy, we chose
Corriere della Sera, the most read newspaper in 2020 according to the ADS
(Accertamenti Diffusione Stampa) (Prima online 2020). In Spain, we chose El Paıs, the
general information newspaper with the greatest circulation (EGM 2020), only sur-
passed by the sports newspaper Marca. Both constitute the quality press (Casasus and
Roig 1981, 30) and they have what Imbert and Vidal Beneyto (1986, 26) call the
‘performative power’ of a newspaper that is a dominant reference, ‘power to institu-
tionalize what it says, to give a reality check to everything it publishes and, conse-
quently, to symbolically nullify what it omits’. The period included in the analysis was four months from the date of the first
known case in each country, 30 January 2020, for Italy’s Corriere della Sera and 31
January 2020, for Spain’s El Paıs. The purpose was to obtain a wide period that
included the main starting moments and first wave of the coronavirus pandemic in
both countries. In the research, we chose journalistic stories in which focus was placed
on references to the Catholic Church directly linked to the coronavirus, discarding
others about the Church, but not connected to the virus, such as the elections in the
Spanish Episcopal Conference or the death of Ernesto Cardenal. Thus, we search terms such as ‘catholic’, and ‘virus’, in the virtual archives of each
of the newspapers (El Paıs, https://elpais.com/diario/, Corriere della Sera, http://archi-
vio.corriere.it/Archivio/interface/landing.html). We didn’t include news with circum-
stantial mentions. The sample was selected through the virtual newspaper libraries of the newspapers
that collect the editions published on paper. It is, therefore, the printed edition that is
referenced, although online instruments have been used for its compilation. We con-
sider the printed reference relevant given the peculiarities of production routines in the
digital environment, that is, the fact that the implementation of SEO techniques and
the constant flow of new information conditions the selection of content. On the other M.-J. 3. Methodology and the field of the study
We will refer to the human interest when the text reflects the suffering of people,
members of the Church or not, who need comfort or assistance from the Church,
as well as when the texts provoke emotions: anger, tenderness, sadness. p
g
The economic consequences are present due the effect of the pandemic on the
economic situation of the Church.
The economic consequences are present due the effect of the pandemic on the
economic situation of the Church.
Finally, we will consider the moral approach when judging behavior. Conflict about the Church often arises linked to civil authorities and political leaders. In
this case, the imposition of restrictive measures with a direct impact on Catholic commun-
ities was fertile ground for controversy. Failure to comply with the norm, the intervention
of the police in churches or religious ceremonies, fines to some clerics or Catholic people
for breaking lockdown, the internal division between different bishops or Catholic groups
about the rules of the Episcopal Conference or the requirement of stopping the bans on
celebrations were cause for confrontation, controversy or disagreement. However, next to this negativity, the opposite behavior was the framework of
responsibility. We found this framework in the news that showed the Church comply-
ing with health regulations to avoid contagion, even going beyond those established by
the authorities; the network of coordination in parishes and dioceses to do prevention
pedagogy; the alteration of the liturgical calendar and the proposal of alternative dates
for the main festivities such as Easter; and prayers, blessings and liturgies without peo-
ple being present – an exceptionally unusual circumstance. The third frame is that of human interest. It includes the charitable behaviors of
priests or religious people: food distribution, help over the telephone; the presence and
prayer of priests in cemeteries. 3. Methodology and the field of the study As
Entman (1993, 54) says, ‘most frames are defined by what they omit as well as include,
and the omissions of potential problem definitions, explanations, evaluations, and rec-
ommendations may be as critical as the inclusions in guiding the audience’. The first
step in analyzing the building of the image of the Church during the pandemic is to
draw a map with what they include as preferred issue and what they put in
the background. In this work we connect the deductive process (Ardevol-Abreu 2015; Ballesteros
Herencia 2015, 52) from the five news frames established by Semetko and Valkenburg
(2000) as generic frames (conflict, attribution of responsibility, human interest, eco-
nomic consequences, and morality), with the inductive process, to discover the com-
mon places or topoi present in the analyzed news. They will be our specific frames. So, we can organize into categories the events narrated by the media, according to
the focus of the journalists. The objective is to be able to quantify the presence of gen-
eric frames about the Church in the pandemic: conflict (due to opposition to restric-
tions), human interest (due to assistance activity), attribution of responsibility (due to
the implementation of prevention measures), morality (due to the judgment of the CHURCH, COMMUNICATION AND CULTURE 161 actions) and the economic consequences (due to the impact of the pandemic on the
finances of the Church).
We will refer to conflict whenever there is a confrontation or disagreement (phys-
ical, verbal or ideological) between the authorities (including the police) and the
members of the Church.
We will refer to conflict whenever there is a confrontation or disagreement (phys-
ical, verbal or ideological) between the authorities (including the police) and the
members of the Church.
We will talk about attribution of responsibility, when the Church is pointed out as
being responsible for the development of the pandemic or the possible contagion
due to its action or omission, as well as when it is related to possible solutions.
We will talk about attribution of responsibility, when the Church is pointed out as
being responsible for the development of the pandemic or the possible contagion
due to its action or omission, as well as when it is related to possible solutions. 3. Methodology and the field of the study It also includes giving words of encouragement, teach-
ing and testimony urging people to accept the lockdown situation and to prepare for
the return of normality with greater sensitivity towards people who suffer; concern for
those who are alone or suffering, for those hospitalized and for the families of those
who die and cannot accompany them; tributes to health personnel; prayer to ask for
the end of the pandemic; dispensation from Lenten abstinence, and understanding for
those who have difficulties or fear after lockdown in fulfilling their religious obliga-
tions; missionary activity in a pandemic situation; care even for unbelievers in need
of comfort. The fourth frame is the economic consequences, very common in the information
on the pandemic. It is difficult to link this one to the Church except regarding finances
or the economic impact of the suppression of some religious celebrations such as Holy
Week in emblematic places such as Seville or Rome. 162
M.-J. POU-AMÉRIGO 162
M.-J. POU-AMÉRIGO 162 The last frame is linked to morality and therefore to the moral assessment of behav-
iors both by the media and by selected sources. The media are often critical of the peo-
ple in question, especially of some bishops with very personal positions on the
complicated issues. We have searched in the units of analysis for the media keys to present each event. Because of this, we have focused on the main elements in the news: headlines, summaries
and highlights. We have applied the content analysis (Krippendorff 2012) because it helps
us to show the hidden words behind the obvious, in other words the subliminal content. This is the way of trying to get an idea of the journalists’ point of view (Pi~nuel 2002). This is the connection between saying and thinking, the link between expression
and understanding as Van Dijk (1993, 147) explains: ‘the structures and meanings
[ … ] reflect the structures and contents of the specific mental model of that individual
reporter about [a] specific event’. In any case, whether the decision is conscious or
involuntary, the result is part of the message that the reader receives. This is our object-
ive. We do not seek to inquire about the motivations of the media but to show the con-
structed image to the reader. 3. Methodology and the field of the study In the analyzed texts, the events narrated explain the contribution of the Church
during the pandemic, both in a positive and negative sense. The list of positive behav-
iors is made up of the following:
compliance with sanitary regulations
establishment of guidelines to avoid contagion
making available different ways of following the liturgy
charitable activities
spiritual or emotional accompaniment
to stay when others do not, such as at funerals
recommendations and testimonies for living during the lockdown
to stay when others do not, such as at funerals
recommendations and testimonies for living during the lockdown There are also negative behaviors and some can even be ambiguous. Among
the negatives:
non-compliance with the rules
opposition to the rules of the government or recommendations of the Vatican or
the Episcopal Conference
to deny the virus
peculiar and extravagant behavior, according to the media
to deny the virus
peculiar and extravagant behavior, according to the media We have considered a behavior ambiguous when, depending to the observer, it can
be described as positive or negative. For example, the claim of religious freedom
because of the intervention of the police in a legal mass, according to the rules. For
people who accept the rule, it is legitimate; for people who consider any assembly dan-
gerous, even if it is legal, it will be interpreted as an unnecessary provocation. In any
case, the doubt disappears when we read how the media has presented the fact. Therefore, is important to focus on headlines and prominent elements of the way the
information is presented. CHURCH, COMMUNICATION AND CULTURE 163 We have analyzed two kinds of issues: the relationship between the Church and the
government, on the one hand, and the Church’s activity during the pandemic, on the
other. The relationship with the authorities is one of the most relevant elements
because the restrictive measures implied, de facto, an intervention in the life of the
Church. Truly, the context imposed restrictions in all social fields and this is why it
was interesting to know the attitude of ‘collaboration’, ‘non-collaboration’, or ‘other’
(cases non explicit in the text) on the part of the Church. 3. Methodology and the field of the study We have differentiated the
activity of the Church during the first months of the pandemic into: ‘volunteer and
social assistance tasks’; ‘Liturgy and sacraments’; ‘concern for herself’ and ‘others’,
when not specified. To differentiate a positive or negative action, we have analyzed the topoi. The topoi
or common places are ideas shared by the community, naturally or acquired, that
‘allow to establish values and hierarchies’, according to Perelman and Olbrechts-Tyteca
(1994, 146). Their analysis has been developed by the research group ‘Family and Media’
(Gonzalez Gaitano 2015) and addresses the presence of common places related to
social virtues (Perez Martınez 2016). Some works about it are published by Norberto
Gonzalez Gaitano (2015), Giovanni Tridente (2009) or Piotr Studnicki (2016). Their presence in the news is essential (Gonzalez Gaitano 2010, 76) and contributes
to developing the frames with a constellation of issues based on the different events
and the way of interpreting them. Barthes (2009, 237) called them the ‘reserve of
themes’, that are not questioned because of their extension and assumption by the
community. That is why they are effective. We can find two kind of topoi, natural and cultural (Gonzalez Gaitano 2015). The topoi
of the first group are natural to the human being, they are anthropological; the topoi of the
second group are cultural products, the result of ideologies. Since the topoi are presupposi-
tions for the journalist’s narration, and that they share with the reader to the point of
‘guiding the judgment’ (Studnicki 2016), it is possible to find more than one topos in the
same text. The topoi are related to frames and indicate the predominant frame. In relation to the action of the Church during the pandemic, we find the following
common places. We have formulated them as statements of latent content in the news. The natural topoi are:
suffering and vulnerable people are the priority
priests know how to accept death and spiritual pain
the Church consoles and accompanies in the tragedy
the pandemic is an occasion for improving our spirituality
the Church complies with prevention rules And the cultural topoi:
religion is tradition
religion is fanaticism
religion has an immutable ancestral liturgy; our astonishment at the mediatization
of religious rites is natural 164
M.-J. POU-AMÉRIGO M.-J. 3. Methodology and the field of the study POU-AMÉRIGO
religious stubbornness leads to violation of law and conflict
there are two kinds of clergy: the collaborator and the intolerant
the Church is only concerned with its finances
the Church considers the pandemic a divine punishment
religion leads to peculiar attitudes 4.1. Generic frames The sample is made up a total of 62 pieces in El Paıs and 73 in Corriere della Sera (see
Table 1). The two most relevant frames in the information published on the actions of
the Church during the first wave of covid-19 are the ‘attribution of responsibilities’ and
‘human interest’. In El Paıs the ‘attribution of responsibilities’ predominates (33.8%)
over ‘human interest’ (30.6%) while in Corriere della Sera it is the opposite, ‘human
interest’ predominates notably (61.6%) over ‘attribution of responsibilities (26%). ‘Conflict’ is the third frame, with a much lower percentage, in both newspapers
(19.3%, El Paıs, and 9.5%, Corriere della Sera) and it is the same with morality. The
‘economic consequences’ are only relevant in El Paıs (4.8%). About the attitude of the Church to the authorities, the highest percentage is for the
field ‘others’ that do not specify the degree of collaboration (see Table 2). This indicates
that it is not the priority for the media. In addition, both in El Paıs (30.6%) and in
Corriere della Sera (17.8%), the number of texts that show the willingness of the
Church to collaborate is higher, almost double, compared to those which do not (17,
7% and 8.2, respectively). Regarding the actions of the Church, the difference between El Paıs and Corriere
della Sera is striking. Thus, for El Paıs, the main activities of the Church are the liturgy,
especially the Eucharist – sometimes ‘clandestine’, or funerals (22.5%). In contrast,
assistance to the underprivileged and spiritual care remain in the background (4.8%). Corriere della Sera is very interested in publishing news about initiatives other than the
Mass, such as blessings in cemeteries, showing relics or sick calls. It has a greater num-
ber of pieces on liturgy than El Paıs (26%), however, the number of pieces about Table 1. News frames of the Church by newspapers. F
/N
El P Table 1. News frames of the Church by newspapers. Frames/Newspapers
El Paıs
Corriere della Sera
Attribution of responsibility
33.8% (21)
26% (19)
Human interest
30.6% (19)
61.6% (45)
Conflict
19.3% (12)
9.5% (7)
Morality
11.2% (7)
2.7% (2)
Economic consequences
4.8% (3)
0
Total
62
73
Source: Author’s compilation. Table 2. Attitude of the Church to the authorities. Newspapers/Attitudes
Collaboration
No collaboration
Other
El Paıs
30.6% (19)
17.7% (11)
51.6% (32)
Corriere della Sera
17.8% (13)
8.2% (6)
73.9% (54)
Source: Author’s compilation. Table 2. 4.1. Generic frames Attitude of the Church to the authorities. CHURCH, COMMUNICATION AND CULTURE 165 charitable assistance is also higher (10.9%). The only higher percentage in El Paıs is
about the lack of activity (14.5%). It is possibly one of the biggest differences between
the two newspapers. Corriere della Sera focuses on the hard work of priests and their
sacrifice in caring for the patients and families. The evidence is that Corriere della Sera
includes a lot of news about sick bishops, priests and nuns, if they are victims of
Covid-19, but El Paıs does not. Furthermore, Corriere della Sera uses the interview
genre to give religious people a voice, an initiative that is lacking in El Paıs (see
Table 3). About the frame ‘attribution of responsibility’, both newspapers show how the
Church has implemented measures to prevent contagion and, in fewer cases, point out
when it has not been done (see Table 4). Half of the pieces refer to how the Church
has taken care of prevention, hygienic measures and compliance with Health regula-
tions. In El Paıs, 42.8% do so, compared to 9.5% that address the opposite issue. In
Corriere della Sera, 58.8% have to do with following regulations, and there is no piece
that reports on the opposite phenomenon. Both newspapers emphasize the celebration of rites in solitude or the closing of
iconic churches, such as the Duomo of Milan, which offers an image of exceptionality. This percentage corresponds to the focus on liturgical actions by El Paıs compared to
the focus on assistance by Corriere della Sera. Thus, in El Paıs, 23.8% of pieces show
surprise about rites never having been celebrated in such a way before (alone or with-
out the sign of peace). Corriere della Sera also echoes this but with a lesser percentage
(11.7%). El Paıs, for example, jokes about the disappointment of the parishioners who
in 2020 were not allowed to kiss the Christ of Medinaceli on Good Friday according to
custom. The newspaper said: ‘It is not clear if the spell loses strength when the kiss is
lacking’ (Quesada 2020). Most of the cases are from Holy Week celebrations, especially
in the Vatican, with the image of the Pope in a completely empty St. Peter’s Square. 4.1. Generic frames The frame ‘conflict’ includes resistance to the measures implemented by govern-
ments or ecclesiastical authorities and their consequences as fines or sanctions. There
are incidents of police intervention and, sometimes, the journalist is at the scene and
evaluates the ‘illegal’ situation. Its funny but there is an article in which a journalist
describes an ‘illegal’ Mass and explains the rules – and the conclusion is that the rules
are not being violated, according to his own story. When we say ‘sanctions’, we refer to
fines or something like this, because, if the police had to intervene, the behavior prob-
ably will be punished. In El Paıs, non-compliance and sanctions are more frequent (50%) than in
Corriere della Sera (42%), although it differentiates as to whether there was only a
complaint or also a fine. In Corriere della Sera, on the other hand, more emphasis is
placed on the different positions taken up regarding the norms imposed by the civil
authorities, or the different sensitivities in relation to the freedom of the Church to
self-regulate its activity (57.1%). Corriere della Sera interviews bishops reluctant to
avoid the celebration of the Eucharist; something that, however, we do not find in El
Paıs (16.6%). In the latter, on the other hand, one finds more importance given to
the political use of the argument of religious freedom by some groups or parties
(25%) (see Table 5). This difference in the main conflict between the Church and the respective govern-
ments must be sought on the one hand, in the interest of the media, but also in the
reality of the country itself. In fact, El Paıs emphasizes the criticism of some Spanish
bishops for the recommendations of the Episcopal Conference but includes a descrip-
tion of the Italian situation and the role of the Pope in the relationship between the
Church and government. El Paıs, in addition, insists on presenting an image of the
Church as divided. It presents the bishop of Alcala, Mons. Reig, as a counterpoint to
the president of the council of bishops, Cardinal Omella. We can see the same in
Corriere della Sera in a piece that recounts the ins and outs of the tensions between the
Church and the Government: ‘A telephone call to Casa Santa Marta. Thus the premier
has disarmed the CEI [Italian Bishops’ Conference]’ (Franco 2020). 4.1. Generic frames Both newspapers include anecdotical news; El Paıs writes about a Detroit priest who
sprinkles holy water for blessings with a toy water pistol to keep his distance, and in
Corriere della Sera, about another American priest who celebrates mass while the
parishioners remain in the car and called it a ‘drive-in mass’ (Maniaci 2020). Table 3. Main activities of the Church. Newspapers/Activities
Assistance
Liturgy
Not activity
Other
El Paıs
4.8% (3)
22.5% (14)
14.5% (9)
58% (36)
Corriere della Sera
10.9% (8)
26% (19)
4.1% (3)
58.9% (43)
Source: Author’s compilation Table 4. Frame ‘attribution of responsibility’. Frame/Newspapers
El Paıs
Corriere della Sera
The Church implements measures to prevent contagion
42.8% (9)
58.8% (10)
No preventive measures are taken
9.5% (2)
0
Celebrations behind closed doors an unusual image of the Church
23.8% (5)
11.7% (2)
Pedagogy of prevention in parishes and dioceses
9.5% (2)
0
Later celebration of Easter
4.7% (1)
0
Alternatives to liturgical activity via electronic means
4.7% (1)
23.5% (4)
Peculiar and extravagant behaviors. in the opinion of the media
4.7% (1)
5.8% (1)
Source: Author’s compilation 166
M.-J. POU-AMÉRIGO 166 In addition, both newspapers talk about sharing the celebration of the liturgy
through electronic media, television, and the Internet. Corriere della Sera devotes more
attention to it (23.5% compared to 4.7% in El Paıs) not only because of the novelty but
especially because of Corriere della Sera’s interest in priests. The Italian paper dedicated
more pieces to the tasks of priests and among them the necessary adaptation of litur-
gical activity through new media. Regarding celebrating Easter later, the impact was
particularly noted in Seville, where there are the confraternities, groups of people
devoted to the image of Christ or the Virgin. These became the protagonists of the
news (El Paıs 4.7%). In fact, one of the colorful bits of news collected is about the sale
of a hand sanitizer with a ‘brother’s scent’. Finally, we have included news about the
task of spreading the message about prevention of contagion among the parishioners
only in El Paıs (9.5%). Furthermore, as has been said, peculiar behaviors are mentioned
but they are not very significant (4.7% in El Paıs and 5.8% in Corriere della Sera). In
other texts we can find these keys but not predominantly, so they are not included as
prevalent frames in the quantification. 4.1. Generic frames Finally, conspiracy
theories and denialism are not present in the Corriere della Sera. They are in El Paıs CHURCH, COMMUNICATION AND CULTURE 167 Table 5. Frame ‘conflict’. Frame/Newspapers
El Paıs
Corriere della Sera
non-compliance with the rules without sanction
25% (3)
0
Sanctions
25% (3)
42.8% (3)
opposition to guidelines from both the government and
the Vatican or the Episcopal Conference
16.6% (2)
57.1% (4)
Denialism
0
0
conspiracy theories appealing to the religious factor
8.3% (1)
0
Political use of religious freedom
25% (3)
0
Source: Author’s compilation. Source: Author’s compilation. Table 6. Frame ‘human interest’. Frame/Newspapers
El Paıs
Corriere della Sera
assistance activities
26.3% (5)
17.7% (8)
spiritual or emotional accompaniment
10.5% (2)
31.1% (14)
Being present when others cannot be (funerals)
15.7% (3)
28.8% (13)
Alerts, recommendations and testimonials to help people withstand lockdown
36.8% (7)
2.2% (1)
Family or social traditions
10.5% (2)
0
Victims (bishops, priests and religious)
0
20% (9)
Source: Author’s compilation. but only as a prejudice (8.3%), that is, attributing to the Church the explanation of the
pandemic as a ‘divine punishment’ when it is not true. The frame of ‘human interest’ focuses on the activity of the Church in assisting the
underprivileged with the distribution of food or direct aid; soup kitchens or the work
of Caritas, but also includes the spiritual comfort of patients or their families as well as
emotional comfort for any person addressing the Church. El Paıs devotes more atten-
tion to assistance to vulnerable, 26%, compared to 17.7% in Corriere della Sera, but the
latter focuses much more (31.1%) on the issue of spiritual and emotional accompani-
ment, especially on the part of parish priests and bishops, whereas in El Paıs this is pre-
sent in a tiny minority of pieces (10.5%) (see Table 6). In the same way, Corriere della Sera, as has been said, dedicates a lot of space
to showing the work of priests and bishops who feel close to their community,
especially when it comes to funerals or blessings in the cemetery in the absence
of loved ones (28.8% in Corriere della Sera, compared to 15.7% in El Paıs). In this field, references to death and the disposition of coffins in cemetery chapels
or in Churches until they can be buried have been included. 4.1. Generic frames The state of alarm, which establishes that you can only leave home for essential activities
such as going to work or shopping, does not prohibit the liturgy of the Eucharist, but
conditions its celebration to "avoid crowds" since it is possible to "guarantee a minimum
distance of one meter" between assistant and assistant." (Carranco 2020) Only two pieces appear in Corriere della Sera, one about the preconceived idea that
the pandemic is divine punishment and another that criticizes a priest for imposing
preventive measures. The text criticizes an ‘excessive zeal’. In summary, in El Paıs, the frame ‘attribution of responsibility’ predominates above
all. The focus is on the acceptance by the Church of sanitary rules, and its collaboration
in the prevention of contagion. The second frame is related to the Church’s activity in
caring for the vulnerable and providing spiritual assistance, and the presence of priests
at funerals. The conflict frame is related to the division of opinions regarding the gov-
ernmental measures and the resistance to the restrictions of some voices within the
Church, even outside the Church, but using the argument of religious freedom to
demand a change in the measures. Morality is present, in the appeals to topoi that asso-
ciate religion with superstition, charlatanism, denialism or conflict. Finally, the news
does not delve into the economic consequences except linked to the suppression of
Holy Week liturgies, the closing of the churches or the absence of pilgrims on the
Camino de Santiago. g
In Corriere della Sera the most relevant frame is that of ‘human interest’ because it
gives more prominence to the interview genre, and thus gives information about spe-
cific people, previously unknown, who are interviewed either because of their experi-
ence or because of their involvement in the support and care of the community. In
this sense, it is common to find the voice of bishops or priests concerned about the
faithful of their diocese or parish; engaged in assistance tasks or promoting charity,
mutual aid and greater sensitivity with others in difficult times. In El Paıs, these
words come more frequently from the important messages of the Pope, and connect
with the frame of ‘conflict’ and the division between different sectors of the Church,
which is much more prevalent in El Paıs than in Corriere della Sera. 4.1. Generic frames This testimony also joins
that of bishops, missionaries, volunteers, Catholic people and even some patients who
have found their faith strengthened after their hard experience. El Paıs has incorpo-
rated it with more dedication (36.8%) but the figure in Corriere della Sera (2.2%) can
be misleading. The reason is that this figure is separated from the one that corresponds
to the labors of priests. In that sense, it is striking that only Corriere della Sera docu-
mented the victims of the virus among bishops, priests and religious. For Corriere della
Sera, including them as protagonists, sources or voices of social relevance seems more
natural than for the Spanish newspaper. Finally, a reference to traditions in El Paıs (10.5%) has been included, because some
people spoke of consoling religious rites as part of a family tradition that is no longer
used, but that is missed. Corriere della Sera doesn’t mention it. 68
M.-J. POU-AMÉRIGO 168 Regarding the frame ‘Economic consequences’ in El Paıs there are only three pieces
that refer to the economic impact of the suppression of Holy Week, to the marketing
of a hand sanitizer with the smell of incense (in both cases it is not an activity of the
Church itself, but of some Catholic people) and another on the impact of the corona-
virus on the finances of the Churche and its repercussion on the economy of the par-
ishes. In Corriere della Sera there are no uses of this frame. Finally, the frame ‘morality’ is present mainly in opinion texts where moral evalua-
tions of behaviors can be made. In El Paıs it appears in a veiled way in a couple of
informative pieces that focused on a hidden breach of the norm (the headline of one of
them is ‘Attending mass through the back door’), however, they show in the text that
no norm prohibits what is being described: The priest, Miguel Angel Nieto, explains in a statement published on social networks that the
agents invited them to leave the Church or, otherwise, they would be sanctioned. "We were
only five parishioners and the priest, so the safety distances were more than assured," he says. 4.2. Topoi or common places The frames are complemented with the analysis of topoi or common places that delve
into the latent content of the information. Thus, for example, the attribution of respon-
sibility, with the topoi, has a different dimension. It is not a question of responsibility
as an attribution of cause-effect, but in a deeper sense, of assumption of basic princi-
ples concerning human nature and interaction with others in society. In fact, among
the topoi present in the news, we have found ‘the Church complies with prevention
measures’ or ‘the Church consoles and accompanies in the tragedy’ as an example of
‘attribution of responsibility’ and at the same time of ‘human interest’. With this other
important frame present in this paper, something similar happens: ‘human interest’
finds its roots in those common places: ‘the suffering and vulnerable are the priority’. ‘Morality’ also connects with the judgment which is the basis of admiration and grati-
tude towards priests, ‘the priest knows how to remain close in the death’, or the convic-
tion that this time will generate a spiritual renewal, ‘the pandemic is an occasion of
greater spirituality’. On the other hand, the ‘ideological’ or cultural topoi include prejudices or the point
of view from which the Church is judged, which emanates from dominant thoughts
such as: ‘the Church is tradition’, ‘the Church is fanaticism’, ‘religion has unchanging
traditional rites, so it is natural to be surprised by changes such as going online’, ‘the
Church considers the pandemic a divine punishment’ or ‘religion fosters peculiar and
strange attitudes’. In this field appear the topoi linked to economic consequences such as ‘the Church
is concerned about its finances’; or conflict, such as ‘stubbornness leads to violation of
law and conflict’ or ‘there are two types of clerics: collaborators and rebels’. The natural topoi at the basis of judgments on religious activity are shown in
Table 7. The cultural topoi at the basis of judgments on religious activity are shown in
Table 8. 4.1. Generic frames Finally, it is
striking that the Corriere della Sera offers more variety of Catholic voices but also
fewer moral judgments about the Church even when some bishops or the CEI CHURCH, COMMUNICATION AND CULTURE 169 demand a more careful treatment by the authorities regarding the religious life of
the community. Source: Author’s compilation. 4.2. Topoi or common places the Church being concerned about itself (‘the Church is concerned about its finances’),
we find that the Church is focused on those who suffer (‘the suffering and vulnerable
are the priority ‘and’ the Church consoles and accompanies in the midst of tragedy’). In this sense, the results are always favorable to the Church: the references to compli-
ance with prevention are more numerous than references to violations (12.9% com-
pared to 4.8%, in El Paıs and 5.4% compared to 4%, in Corriere della Sera). In the same
way, the Church is seen as more focused on others, especially the suffering and vulner-
able, than on itself (9.6% compared to 1.6% in El Paıs; 19% compared to 0 in Corriere
della Sera). The more unusual topoi in both newspapers are those linked to a negative view of
the Church and the role of religion in society. In El Paıs there predominates a
Manichean view of Catholics, especially the clergy (16.1%, compared to 6.8% in
Corriere della Sera) and a constant conviction about religious fanaticism (11.2% com-
pared to 0 in the Italian newspaper). In Corriere della Sera, in contrast, the work of
priests and bishops stands out; they are seen as dedicated to the needy, rather than
engaged in litigation. Finally, both newspapers share a somewhat stagnant vision of the Church; that’s
why technological developments are repeated as surprising news (14.5% in El Paıs and
19% in Corriere della Sera). The interest of the press in this is logical but focus on the
novelty and not so much on the adaptability of the Church itself is striking. 4.2. Topoi or common places In some cases, the topoi can be paired as opposites, thus, in contrast with compli-
ance (‘the Church complies with prevention’), non-compliance appears (‘stubbornness
leads to violation of law and conflict’); in contrast to the frame of the sacrificial and
self-surrendering priest (‘the priest knows how to accompany people who are dying’),
there is the frame that the clergy is divided by different opinions about the measures
against Covid (‘there are two types of clergy: collaborators and rebels’) or in contrast to Table 7. Topoi or common places: natural topoi. Natural topoi/newspapers
El Paıs
Corriere
suffering and vulnerable people are the priority
9.6% (6)
19% (14)
the priest knows how to accompany people who are dying
6.4% (4)
21.9% (16)
the Church consoles and accompanies in the midst of tragedy
3.2% (2)
13.6% (10)
the pandemic is an occasion for greater spirituality
8% (5)
8.2% (6)
the Church complies with prevention
12.9% (8)
5.4% (4)
Source: Author’s compilation. 170
M.-J. POU-AMÉRIGO 170
M.-J. POU-AMÉRIGO Table 8. Topoi or common places: cultural topoi. Cultural topoi/Newspapers
El Paıs
Corriere della Sera
Religion is tradition
4.8% (3)
0
Religion is fanaticism
11.2% (7)
0
Religion has unchanging traditional rites, so it is natural to be
surprised by changes such as going online
14.5% (9)
19% (14)
Stubbornness brings about violation of rules and conflict
4.8% (3)
4% (3)
There are two types of clerics: collaborative and rebellious
16.1% (10)
6.8% (5)
The Church is concerned about its finances
1.6% (1)
0
The Church considers the pandemic a divine punishment
1.6% (1)
1.3% (1)
Religion fosters peculiar and strange attitudes
4.8% (3)
0
Source: Author’s compilation. the Church being concerned about itself (‘the Church is concerned about its finances’),
we find that the Church is focused on those who suffer (‘the suffering and vulnerable
are the priority ‘and’ the Church consoles and accompanies in the midst of tragedy’). In this sense, the results are always favorable to the Church: the references to compli-
ance with prevention are more numerous than references to violations (12.9% com-
pared to 4.8%, in El Paıs and 5.4% compared to 4%, in Corriere della Sera). In the same
way, the Church is seen as more focused on others, especially the suffering and vulner-
able, than on itself (9.6% compared to 1.6% in El Paıs; 19% compared to 0 in Corriere
della Sera). 5. Conclusions The Catholic Church was subject of news interest during the first months of the
Covid-19 pandemic with a similar volume of information in Spain and Italy. However,
Corriere della Sera gave voice to Catholic protagonists more frequently than El Paıs,
above all, to bishops, priests and volunteers. In this sense, it showed the hard and
necessary work of priests, especially at death-beds and at lonely funerals, as well as
through the proximity of bishops and parish priests to their parishioners. Therefore, the first hypothesis is confirmed, regarding the frames of reference
through which the role of the Church is interpreted when the story is a global tragedy:
The quality press in Italy and Spain gave precedence to the charitable work of the
Catholic Church during the pandemic, as opposed to the conflict with the political
authorities over restrictive measures. Moreover, when it comes to the relationship with
civil institutions, a positive image of the Church is offered, ready to collaborate and CHURCH, COMMUNICATION AND CULTURE 171 apply the preventive measures recommended by the health authorities. Discrepancies
regarding the application of the norms on liturgical activity appear as a factor of
internal division, especially in El Paıs, but conflict is not the predominant framework. El Paıs insists on showing the Church’s disappointment with the measures, while
Corriere della Sera shows the imaginative effort to implement novel ways to broadcast
the celebrations of the liturgy through the Internet. In both cases, the surprise at this
ability to adapt to the online context is constant. In the same way, the idea of excep-
tionality accompanies the account of ceremonies and rites in the absence of
the community. 5. Conclusions The topoi or common places present in the information are, on the one hand, of a
natural or anthropological nature: ‘suffering and vulnerable people are the priority’,
‘the priest knows how to accompany people who are dying’, ‘the Church consoles and
accompanies in the midst of tragedy’, ‘the pandemic is an occasion for greater spiritual-
ity’, ‘the Church complies with prevention measures’; and on the other hand, cultural
or ideological: ‘religion is tradition’, ‘religion is fanaticism’, ‘religion has unchanging
traditional rites, so it is natural to be surprised by changes such as going online’,
‘stubbornness leads to violation of law and conflict’, ‘there are two types of clergy: col-
laborative and rebellious’, ‘the Church is concerned about its finances’, ‘the Church
considers
the
pandemic
a
divine
punishment’,
‘religion
fosters
peculiar
and
strange attitudes’. Hypotheses 2 and 3 are confirmed, since in the media analyzed both in Italy and
in Spain, the spiritual and welfare work of the Church is valued (H2) and its oppos-
ition to government measures is criticized (H3). However, the two are not compar-
able since the positive assessment predominates in the story when there are
dualities in the topoi found in the interpretation of the facts. Thus, the Church
appears more concerned about others (‘the suffering and vulnerable are the priority’
and ‘the Church consoles and accompanies in the midsttragedy’) than for itself (‘the
Church is concerned about its finances’). Likewise, it makes more effort in preven-
tion, caring for people and the common good (‘the Church complies with preven-
tion’) than in confrontation and controversy (‘stubbornness leads to violation of
law and conflict’). The negative view of the Church and the role of religion in society can also be
paired with opposite frames: in contrast to the frame of self-sacrificial priests (‘the
priest knows how to accompany people who are dying’), we read that the the clergy is
divided by the prevention measures (‘there are two types of clergy: collaborative and
rebellious’). We see this duality in both newspapers. In El Paıs Manichean prejudice
prevails over Catholic sense, especially with regard to the clergy. In Corriere della Sera,
on the other hand, the work of priests and bishops stands out: they are seen as dedi-
cated to the needy, rather than engaged in litigation. Disclosure statement No potential conflict of interest was reported by the author(s). 5. Conclusions We can therefore appreciate that the construction of the image of the Church in the
media largely depends on the weight of prejudices or socially accepted topoi on the
situation that the media is willing to accept. As seen in the global crisis of Covid-19,
the media that give voice to the protagonists enrich the story with points of view that
could not have been foreseen. However, those that ignore the news that does not cor-
respond with the pre-established patterns obscure an important part of reality. 172
M.-J. POU-AMÉRIGO 172
M.-J. POU-AMÉRIGO Notes on contributor Marıa-Jose Pou-Amerigo has a PhD in Communication and Journalism from the Complutense
University of Madrid (1999); a Bachelor’s degree in Communications and Journalism from the
Polytechnic University of Valencia-CEU San Pablo (1992); a Bachelor’s in Humanities from
UOC of Barcelona (2009), and a Master’s in Teaching Spanish as a Foreign Language from
UNED (2012). She has been Assistant Professor of Journalism (Religion Journalism and
Journalistic Writing) in the CEU Cardenal Herrera University since 1993 and professor for the
Specialization Diploma in Corporate Communication and Religion in the Digital Age at the
Blanquerna Observatory on Media, Religion and Culture in Barcelona. Three of her most rele-
vant publications are Bishops in Spanish public life (2007), Catholics in public opinion. The
Italian example (2008) and ‘Framing ’Green Pope’ Francis: newspaper coverage of the
Encyclical ’Laudato Si’ in the United States and the United Kingdom’ (2018). She has been a
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English
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Palmitic acid inhibits vascular smooth muscle cell switch to synthetic phenotype via upregulation of miR-22 expression
|
Aging
| 2,022
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cc-by
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ABSTRACT Synthetic phenotype switch of vascular smooth muscle cells (VSMCs) has been shown to play key roles in
vascular diseases. Mounting evidence has shown that fatty acid metabolism is highly associated with vascular
diseases. However, how fatty acids regulate VSMC phenotype is poorly understood. Hence, the effects of
palmitic acid (PA) on VSMC phenotype were determined in this study. The effect of the PA on VSMCs was
measured by live/dead and EdU assays, as well as flow cytometry. Migration ability of VSMCs was evaluated
using transwell assay. The underlying targets of miR-22 were predicted using bioinformatics online tools, and
confirmed by luciferase reporter assay. The RNA and protein expression of certain gene was detected by qRT-
PCR or western blot. PA inhibited VSMC switch to synthetic phenotype, as manifested by inhibiting VSMC
proliferation, migration, and synthesis. PA upregulated miR-22 in VSMCs, and miR-22 mimics exerted similar
effects as PA treatment, inhibiting VSMC switch to synthetic phenotype. Inhibition of miR-22 using miR-22
inhibitor blocked the impacts of PA on VSMC phenotype modulation, suggesting that PA modulated VSMC
phenotype through upregulation of miR-22 expression. We found that ecotropic virus integration site 1 protein
homolog (EVI1) was the target of miR-22 in regulation of VSMC phenotype. Overexpression of miR-22 or/and
PA treatment attenuated the inhibition of EVI1 on switch of VSMCs. These findings suggested that PA inhibits
VSMC switch to synthetic phenotype through upregulation of miR-22 thereby inhibiting EVI1, and correcting the
dysregulation of miR-22/EVI1 or PA metabolism is a potential treatment to vascular diseases. AGING 2022, Vol. 14, No. 19 Palmitic acid inhibits vascular smooth muscle cell switch to
synthetic phenotype via upregulation of miR-22 expression Correspondence to: Congxia Wang; email: wcxcirculation@163.com, https://orcid.org/0000-0002-3423-4644
Keywords: VSMC, palmitic acid, synthetic phenotype, miR-22
Received: August 17, 2022
Accepted: October 3, 2022
Published: October 12, 2022 Copyright: © 2022 Hu et al. This is an open access article distributed under the terms of the Creative Commons Attribution
License (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original
author and source are credited. www.aging-us.com www.aging-us.com Yanchao Hu1, Yajie Fan1, Chunyan Zhang1, Congxia Wang1,& 1Department of Cardiovascular Medicine, The Second Affiliated Hospital of Xi'an Jiaotong University, Shaanxi,
Xi'an 710004, China 1Department of Cardiovascular Medicine, The Second Affiliated Hospital of Xi'an Jiaotong University, Shaanx
Xi'an 710004, China Correspondence to: Congxia Wang; email: wcxcirculation@163.com, https://orcid.org/0000-0002-3423-4644
Keywords: VSMC, palmitic acid, synthetic phenotype, miR-22
Received: August 17, 2022
Accepted: October 3, 2022
Published: October 12, 2022 Correspondence to: Congxia Wang; email: wcxcirculation@163.com, https://orcid.org/0000-0002-3423-4644
Keywords: VSMC, palmitic acid, synthetic phenotype, miR-22
Received: August 17, 2022
Accepted: October 3, 2022
Published: October 12, 2022 INTRODUCTION VSMC phenotypic switch regulation is important in
vascular diseases. Vascular function is largely dependent on vascular
smooth muscle cells (VSMCs). Different from the
skeletal muscle cells or cardiomyocytes, VSMCs
remain possessing remarkable phenotypic plasticity in
response to multiple stimuli [1]. VSMCs switch from a
contractile state to a dedifferentiated, synthetic
phenotype, playing crucial roles in several vascular
diseases [2–4]. The synthetic phenotype induces
migration to the intima and enhances proliferation and
extracellular matrix protein synthesis, thereby resulting
in an impaired contractility of VSMC [5]. Therefore,
exploration of the underlying mechanisms involved in There are multiple environmental stimuli have been
identified as factors which lead to VSMC phenotype
switch, such as growth factors, reactive oxidative
species (ROS), and mechanical injury [6, 7]. Recent
studies have shown that metabolites were also involved
in regulation of VSMC phenotype [8]. For example,
lactate, a product of glucose metabolism, was found to
promote the synthetic phenotype of VSMCs, which
links glucose metabolism to VSMC phenotypic switch
[8]. Mounting evidence has shown that fatty acid
metabolism is abnormal in vascular diseases, which AGING 8046 www.aging-us.com fluorescence was read using a microplate reader
(FLUOstar® Omega). plays an important role in the development of
atherosclerosis and other vascular diseases [9, 10]. These advances suggest that fatty acids metabolism may
play a role in regulation of VSMC phenotype. However,
how fatty acids regulate VSMC phenotype is poorly
understood. As the most common saturated fatty acid
found in organism, palmitic acid (PA) serves as an
energy source or component of partially biochemicals
and cellular structures. The circulating level of PA is
increased in metabolic disorders and correlated with the
adverse outcomes of cardiovascular diseases [11–13]. Here, we aimed to examine the impacts and underlying
mechanism of PA on VSMC phenotype. fluorescence was read using a microplate reader
(FLUOstar® Omega). EdU assay Proliferation of VSMCs was analyzed using the Click-
it EdU kit (C10086, Invitrogen, USA). Briefly, cells
were seeded on the slides at a density of 1.0 × 103 cells
in 12-well plate each well. After treatment, cells were
incubated with 50 μmol/L EdU solution at 37°C for 2
h. Then, cells were washed with cooled PBS for twice
and fix 4% PFA at 4°C for 15 min. Following this, 100
μl Apollo reaction cocktail was added into cells
followed by nucleus staining with Hoechst 33342
according
to
the
manufacturer’s
protocol. The
fluorescence signal was then analyzed under a
fluorescence microscope. EdU incorporation (%) =
EdU positive cells/(EdU-positive cells + Hoechst-
positive cells) ×100%. VSMC phenotype switch has been widely studied in
transcriptional and epigenetic levels [14, 15]. We were
very interested in the growing evidence supporting a
critical role for miRNAs in regulating VSMC
differentiation and phenotypic switch [16, 17]. A series
of miRNAs have been reported as regulators of VSMC
phenotype, including miR-21 [18], miR-22 [16], miR-
23b [19], miR-100 [20], miR-124 [16], miR-133 [21],
miR-143/145 [22], miR-146a [23], miR-195 [24], miR-
221/222 [25] and miR-424 [26]. Here, we found that PA
inhibits VSMC switch to synthetic phenotype via
upregulation of miR-22. These results suggested that
PA plays a role in regulation of VSMC phenotype. Transwell assay Primary VSMCs were isolated from 8–10 weeks old
male SD rat (weighed 170–250 g) thoracic aorta as
reported previously [27]. Briefly, thoracic aortas were
excised followed by phosphate buffered saline (PBS)
washing for 3 times. After these, the aortic media layer
was dissected, cut into pieces, and seeded onto a 6-well
plate. Cells were maintained in DMEM supplemented
with 10% fetal bovine serum (FBS), 1% penicillin and
streptomycin at 37°C in a humidified incubator with 5%
CO2 in atmosphere for 2 weeks. All animal procedures
in this study were conducted in accordance with the
National Institutes of Health Guidelines on the Use of
Laboratory Animals, and were approved by the Xi’an
Jiaotong University Second Affiliated Hospital. VSMCs were seeded in the upper chamber of
transwell (12 µm) and placed in a 24-well plate at a
density of 1.0 × 105 cells/well in 200 μl DMEM
contained with 0.5% FBS. The lower chamber was
filled up with 600 μl DMEM contained with 10%
FBS. After incubation with for 24 h, medium was
discarded and the lower chamber membrane was fixed
with methanol at room temperature for 15 min. Subsequently, cells were stained with 0.1 crystal
violet-methanol solution for 15 min at room
temperature. Finally, the migrated cells were pictured
and calculated under a light microscope. Apoptosis determination The apoptosis of VSMCs was detected using an
Annexin V-FITC apoptosis detection kit (C1062,
Beyotime, China). Briefly, cells were collected after
treatment, washed with cooled PBS twice, resuspended
with 1 mL AnnexinV-FITC, and maintained for 10
min at room temperature according to the kit’s
protocol. Following this, cells were subjected to flow
cytometry analysis. Cell transfection MiR-22
mimics
(5′-AAGCUGCCAGUUGAAGAA
CUGU-3′), miR-23b mimics (5-′AUCACAUUGCCA
GGGAUUACCAC-3′),
miR-125b
mimics
(5′-
UCCCUGAGACCCUAACUUGUGA-3′),
negative
control mimics (NC mimics, #miR1N0000001-1-10),
miR-22
inhibitors
(5′-ACAGUUCUUCAA
CUGGCAGCUU-3′),
and
NC
inhibitors
(#miR2N0000001-1-10) were synthesized by RIBOBIO
Co., Ltd Chin (Guangzhou, China). Empty vector
(pcDNA3.1)
and
EVI1
overexpression
plasmid
(pcDNA3.1-EVI1 OE) were purchased from GeneChem
(Shanghai, China). miRNA mimics (100 nmol/L),
inhibitors
(200
nmol/L),
or
NC
(5′-
UUCUCCGAACGUGUCACGUTT-3′) (100 nmol/L)
were
transfected
using
Lipofectamine™
3000
(Invitrogen) according to manufacturer’s instruction. After 60 h post-transfection, the transfected cells were
harvested and utilized for further analyses. Dual-luciferase reporter assay VSMCs were treated with PA (0, 100, 200 or 400 μM)
for 3 d. Live/Dead assay suggested that PA treatment
decreased cell viability, and increased cell death in a
dose-dependent manner in VSMCs (Figure 1A). Moreover, EdU assay suggested that PA treatment
significantly decreased the EdU incorporation of
VSMCs in a dose-dependent manner (Figure 1B and
1C). Further analysis indicated that PA treatment
could significantly increase VSMCs apoptosis (Figure
1D and 1E). Western blot analysis presented that PA
treatment markedly increase the Bax and cleaved-
caspase-3 expression but decreased Bcl-2 expression
(Figure 1F). These findings suggested that PA may
inhibit the VSMC switch to synthetic phenotype. In
addition, PA treatment (200 μM) suppressed the
migration of VSMCs as detected by transwell assay
(Figure 1G). Furthermore, PA treatment (200 μM) for
3 d increased protein levels for markers of the
contractile phenotype, including α-SMA, calponin, and
SMMHC, and decreased protein levels of the synthetic
phenotype, including vimentin, collagen I, and
osteopontin (OPN) (Figure 1H). These results
reinforced the notion that PA inhibits the VSMC
switch to synthetic phenotype. Wt and Mt ecotropic virus integration site 1 protein
homolog (EVI1) 3′UTR sequence was acquired using
PCRmethod, and then cloned into SpeI and HindIII sites
of pMir-Report Luciferase vector (Applied Biosystems). The resulting construct was transfected (5ng) into 293T
cells with 20 nM control mimics or miR-22 mimics
using Lipofectamine-2000 (Invitrogen). After 24 h post-
transfection, luciferase activity of cells was assessed
using a Luciferase Assay System (Promega). Western blot For immunoblotting, proteins were isolated from cells
using RIPA buffer. Total protein extracts (15–50 μg)
were
separated
using
sodium
dodecyl
sulfate
polyacrylamide gel electrophoresis and transferred onto
polyvinylidene fluoride membrane. Membranes were
then probed with anti-bax (1:2000; #ab32503; Abcam),
bcl-2 (1:2000; #ab196495; Abcam), cleaved-caspase-
3/caspase-3 (1:2000; #ab184787; Abcam), SM22α
(1:2000;
#ab14106;
Abcam),
calponin
(1:500;
#ab227661; Abcam), SMMHC (1:2000; #ab125884;
Abcam),
vimentin
(1:2000;
#ab92547;
Abcam),
collagen I (1:1000; #ab270993; Abcam), osteopontin
(OPN; 1:1000; #ab63856; Abcam), LAMC1 (1:1000;
#ab233389; Abcam), EVI1 (1:1000; #SAB2100723;
Sigma),
AKT3
(1:2000;
#ab152157;
Abcam),
TP53INP1 (1:2000; #ab202026; Abcam), and β-actin
(1:2000; #ab8226; Abcam) at room temperature for Live/dead cell assay RNAiso Plus reagent (Code No.: 9108, Takara) was
used for the RNA isolation as the manufacturer
recommended. The cDNA was synthesized using the
isolated RNA (500 ng/sample) and amplification of
certain genes was performed using a SYBR Green PCR
kit (Takara) in a CFX200 (Bio-Rad) with the cycles of
95°C for 10 min and 40 cycles of 95°C for 5 s, 58°C
for 30 s, and 72°C for 10 s. The mRNA level of each The 2-color fluorescence with the LIVE/DEAD
Viability/Cytotoxicity kit (Molecular Probes) was used
to quantify the living and dead cells in this study as
directed by the manufacturer’s protocol. Briefly, cells
were harvested after treatment, washed with PBS twice,
and incubated with 300 μl of live/dead solution for
half an hour at 37°C in the dark room. Then, the AGING 8047 www.aging-us.com 1.5 h. Then, membranes were immersed with the HRP-
conjugated secondary antibody at room temperature for
1 h. Following this, the BM chemiluminescence blotting
system (Thermo Scientific) was used for detection and
protein bands were quantified using Image J software
(NIH, USA). gene was normalized to housekeeping gene, namely,
GAPDH or U6. The primer sequences are listed in
Supplementary Table 1. Statistical analysis All data are presented as mean ± standard deviation, and
comparisons were performed using one-way ANOVA
or two-way ANOVA followed by an unpaired t-test, as
appropriate. P < 0.05 was considered statistically
significant. Availability of data and materials The datasets used and/or analyzed during the present
study are available from the corresponding author upon
reasonable request. PA increased miR-22 expression in VSMCs Figure 2A, 15 miRNAs were detected, and
miRNAs, 3 miRNAs were increased and 1
miRNA were decreased in PA-tre
compared with that in untreated VSM
this, the top 3 increased miRNAs were ov
VSMCs via using transfecting with
ncreased miR-22 expression in VSMCs. (A) MiRNAs levels in VSMCs treated with PA. (B) The mRNA
miR-125b in VSMCs after transfecting with their specific miRNA mimics. (C) The protein expression of
tin, collagen I, and osteopontin in VSMCs treated with miR-22, miR-23b or miR-125b mimics. (D) Migration
h miR-122 mimics determined by transwell assay. Scale bar=100 μm. (E, F) The cell proliferation of VSMCs
cted by EdU assay. (G, H) Apoptosis of VSMCs treated with miR-22 mimics determined by flow cytometr
x, bcl-2, caspase-3, and cleaved-caspase-3 in VSMCs treated with miR-22 mimics. n = 3. *P < 0.05, **P < 0.0 miRNA were decreased in PA-treated VSMCs
compared with that in untreated VSMCs. Following
this, the top 3 increased miRNAs were overexpressed in
VSMCs via using transfecting with their specific miRNAs which are involved in alteration of VSMC
phenotype switch were screened in PA-treated VSMCs. As shown in Figure 2A, 15 miRNAs were detected, and
among these miRNAs, 3 miRNAs were increased and 1 which are involved in alteration of VSMC
switch were screened in PA-treated VSMCs. in Figure 2A, 15 miRNAs were detected, and
se miRNAs, 3 miRNAs were increased and 1
miRNA were decreased in PA-trea
compared with that in untreated VSMC
this, the top 3 increased miRNAs were ove
VSMCs via using transfecting with t
A increased miR-22 expression in VSMCs. (A) MiRNAs levels in VSMCs treated with PA. (B) The mRNA le
d miR-125b in VSMCs after transfecting with their specific miRNA mimics. (C) The protein expression of SM
entin, collagen I, and osteopontin in VSMCs treated with miR-22, miR-23b or miR-125b mimics. (D) Migration a
with miR-122 mimics determined by transwell assay. Scale bar=100 μm. (E, F) The cell proliferation of VSMCs tr
etected by EdU assay. (G, H) Apoptosis of VSMCs treated with miR-22 mimics determined by flow cytometry
f bax, bcl-2, caspase-3, and cleaved-caspase-3 in VSMCs treated with miR-22 mimics. n = 3. *P < 0.05, **P < 0.01. Figure 2. PA increased miR-22 expression in VSMCs. (A) MiRNAs levels in VSMCs treated with PA. (B) The mRNA levels of miR-22,
miR-23b, and miR-125b in VSMCs after transfecting with their specific miRNA mimics. PA increased miR-22 expression in VSMCs To test whether miRNA is involved in regulation of
VSMC phenotype switch induced by PA, the report AGING 8048 www.aging-us.com A inhibited VSMC switch to synthetic phenotype. VSMC morphologies after PA treatments (0, 100, 200 o
ead cell assay of PA-treated VSMCs. (B and C). EdU assay to detect the proliferation on VSMCs treated with PA. (D
detect the apoptosis of PA-treated VSMCs. (F) Western blot determined the expression of apoptosis associated
f PA
d (200 M) VSMC
S
l b
100
(H) E
i
l
l
f SM22
l
i
SMMHC
i Figure 1. PA inhibited VSMC switch to synthetic phenotype. VSMC morphologies after PA treatments (0, 100, 200 or 400 μM) for
3d. (A) Live/dead cell assay of PA-treated VSMCs. (B and C). EdU assay to detect the proliferation on VSMCs treated with PA. (D and E). Flow
cytometry to detect the apoptosis of PA-treated VSMCs. (F) Western blot determined the expression of apoptosis associated markers. (G)
Transwell assay of PA-treated (200 μM) VSMCs. Scale bar = 100 μm. (H) Expression levels of SM22α, calponin, SMMHC, vimentin, collagen I,
and osteopontin in PA-treated (200 μM for 3 d) VSMCs. n = 3. *P < 0.05, **P < 0.01. 8049 AGING www.aging-us.com miRNAs which are involved in alteration of VSMC
henotype switch were screened in PA-treated VSMCs. s shown in Figure 2A, 15 miRNAs were detected, and
mong these miRNAs, 3 miRNAs were increased and 1
miRNA were decreased in PA-treated VSMCs
compared with that in untreated VSMCs. Following
this, the top 3 increased miRNAs were overexpressed in
VSMCs via using transfecting with their specific
gure 2. PA increased miR-22 expression in VSMCs. (A) MiRNAs levels in VSMCs treated with PA. (B) The mRNA levels of miR-22,
iR-23b, and miR-125b in VSMCs after transfecting with their specific miRNA mimics. (C) The protein expression of SM22α, calponin,
MMHC, vimentin, collagen I, and osteopontin in VSMCs treated with miR-22, miR-23b or miR-125b mimics. (D) Migration ability of VSMCs
ansfecting with miR-122 mimics determined by transwell assay. Scale bar=100 μm. (E, F) The cell proliferation of VSMCs treated with miR-
2
i i
d
d b EdU
(G H) A
i
f VSMC
d
i h
iR 22
i i
d
i
d b fl
(I) Th
i ch are involved in alteration of VSMC
itch were screened in PA-treated VSMCs. PA treatment enhanced the effect of miR-22 on
abolishing EVI1mediated VSMC phenotype switch To test whether EVI1 contributes to the effects of PA on
VSMC phenotype switch, EVI1 and miR-22 were
overexpressed in VMSCs followed by PA treatment. The qRT-PCR demonstrated that EVI1 overexpression
did not change the levels of miR-22 in VSMCs, but PA
treatment could enhance the upregulation of miR-22
(Figure
5A). Overexpression
of
miR-22
mimic
significantly suppressed the EVI1 expression and PA
treatment further enhanced this inhibition on the
expression of EVI1 (Figure 5A). Moreover, PA
treatment promoted cell proliferation as detected by EdU
staining in VSMCs with EVI1 overexpression, while
miR-22 mimic expression aborted this upregulation and
PA treatment markedly enhanced this inhibitive effect
mediated by miR-22 (Figure 5B). Flow cytometry
analysis presented that overexpression of EVI1 had no
obvious effect on the apoptosis of VSMCs, but miR-22
and PA treatment could enhance the apoptosis of EVI1
(Figure 5C). Similarly, EVI1 had no obviously effect on
the expression of bax, bcl-2, caspase-3, and cleaved
caspase-3, while miR-22 mimic and PA treatment could
significantly promote the bax and cleaved-caspase-3 but
decreased bcl-2 expression (Figure 5D). Transwell
assays indicated that EVI1 significantly increased the
migration of VSMCs, while miR-22 and PA treatment
obviously attenuated this promotion to suppress the
migration of VSMCs (Figure 5E and 5F). In addition,
EVI1 also inhibited the contractile markers of SM22α,
calponin, and SMMHC and promoted the synthetic
markers of vimentin, collagen I, and OPN, while miR-22
and PA treatment attenuated these changes (Figure 5G). These results reinforced the notion that PA inhibits the
VSMC switch to synthetic phenotype through regulation
of miR-22/EVI1 axis. PA increased miR-22 expression in VSMCs (C) The protein expression of SM22α, calponin,
SMMHC, vimentin, collagen I, and osteopontin in VSMCs treated with miR-22, miR-23b or miR-125b mimics. (D) Migration ability of VSMCs
transfecting with miR-122 mimics determined by transwell assay. Scale bar=100 μm. (E, F) The cell proliferation of VSMCs treated with miR-
22 mimics detected by EdU assay. (G, H) Apoptosis of VSMCs treated with miR-22 mimics determined by flow cytometry. (I) The protein
expression of bax, bcl-2, caspase-3, and cleaved-caspase-3 in VSMCs treated with miR-22 mimics. n = 3. *P < 0.05, **P < 0.01. AGING 8050 www.aging-us.com miRNA mimics, respectively (Figure 2B). Following this,
the expression of synthetic and contractile markers was
detected in VSMCs. As shown in Figure 2C, miR-22,
miR-23b, and miR-125b mimics all increased the mRNA
levels for SM22α, calponin, and SMMHC, and decreased
mRNA levels of vimentin, collagen I, and OPN,
suggesting that PA may inhibit the VSMC switch to
synthetic phenotype via upregulation of these miRNAs. Specifically, miR-22 presented the most significant effect
among these miRNAs. Thus, we had chosen miR-22 for
the following investigation. Moreover, transwell assay
suggested that increased expression of miR-22 mimics
obviously inhibited the VSMCs migration (Figure 2D). Overexpression of miR-22 mimics also inhibited the
proliferation of VSMCs (Figure 2E and 2F). In addition,
flow cytometry suggested that overexpression of miR-22
mimic increased the apoptosis of VSMCs (Figure 2G and
2H). Correspondingly, the western blot analysis showed
that miR-22 mimic increased the expression of Bax and
clveaed-caspase-3, but decrease Bcl-2 expression (Figure
2I). These results suggested that PA may inhibit the
VSMC switch to synthetic phenotype via upregulation of
miR-22. candidates (Figure 4A), and 10 of them are associate
with cell proliferation, migration, or apoptosis (Figure
4B). Among these candidates, overexpression of miR-
22 reduced EVI1 mRNA levels significantly in VSMCs
(Figure 4B). Western blot array showed the miR-22
mimic
could
significantly
decrease
the
EVI1
expression, but miR-22 inhibitor largely enhanced the
EVI1 expression (Figure 4C and 4D), indicating EVI1
acted as a candidate target of miR-22. This result was
further confirmed by dual-luciferase reporter assay and
presented that miR-22 reduced luciferase activity for
EVI1 wild-type 3′UTR constructs but had no effect on
the mutated binding site (Figure 4E and 4F). In
addition, PA treatment abolished the EVI1 protein
expression, while miR-22 inhibitor attenuated these
downregulation, thereby upregulating EVI1 expression
(Figure 4G). These results suggested that EVI1 is a
target of miR-22. miR-22 inhibitor abolished the effects of PA on
VSMC phenotype switch To test whether miR-22 is involved in the PA’s effects
on VSMC phenotype switch, miR-22 inhibitor was used
to inhibit the PA-upregulated miR-22. As shown in
Figure 3A, miR-22 inhibitor decreased the miR-22
levels in VSMCs. As a result, PA treatment (200 μM)
significantly inhibited the cell viability but increased
apoptosis in VSMCs, while overexpression of miR-22
inhibitor attenuated the impacts of PA on the
proliferation and apoptosis of VSMCs (Figure 3B–3E). Correspondingly, the western blot analysis presented
that overexpression of miR-22 inhibitor attenuated the
effect of PA in increasing bax and cleaved-caspase-3
expression, and decreasing bcl-2 expression (Figure
3F). Transwell analysis showed that overexpression of
PA treatment significantly decreased the migration of
VSMCs, but miR-22 inhibitor obviously aborted this
enhancement (Figure 3G). In addition, the western blot
analyses
showed
that
PA
treatment
obviously
accumulated the expression of SM22α, calponin, and
SMMHC, but decreased the expression of vimentin,
collagen I, and OPN; while overexpression of miR-22
inhibitor attenuated the effect of PA in VSMCs (Figure
3H). These results reinforced the notion that PA inhibits
the VSMC switch to synthetic phenotype. EVI1 was the target of miR-22 The potential target genes of miR-22 were predicted by
miRDB, ENCOR1, and TargetScan. There were 50 AGING 8051 www.aging-us.com DISCUSSION atherosclerosis and other vascular diseases [9, 10]. However, how fatty acid regulates VSMC phenotype
switch has not been studied. Here, we found that PA,
the most common saturated fatty acid in circulation, us com
8052
ION
vidence has shown that disorder in fatty acid
plays a casual role in the development of
atherosclerosis and other vascular disease
However, how fatty acid regulates VSMC
switch has not been studied. Here, we foun
the most common saturated fatty acid in c
R-22 inhibitor abolished the effects of PA on VSMC phenotype switch. (A) MiR-22 inhibitor decreased
Cs. (B, C) The proliferation of VSMCs transfected with miR-22 inhibitor or/and 200 μM PA. (D, E) The apoptos
ith miR-22 inhibitor or/and 200 μM PA. (F) The expression of apoptosis associated markers in VSMCs transfected
nd 200 μM PA. (G) The migration of VSMCs transfected with miR-22 inhibitor or/and 200 μM PA determined
bar=100 μm. (H) Western blot analyses showing that expression of SM22α, calponin, SMMHC, vimentin, col
n PA-treated VSMCs transfected with miR-22 inhibitor. n = 3. *P < 0.05, **P < 0.01. Mounting evidence has shown that disorder in fatty acid
metabolism plays a casual role in the development of Figure 3. miR-22 inhibitor abolished the effects of PA on VSMC phenotype switch. (A) MiR-22 inhibitor decreased the miR-22
levels in VSMCs. (B, C) The proliferation of VSMCs transfected with miR-22 inhibitor or/and 200 μM PA. (D, E) The apoptosis of VSMCs
transfected with miR-22 inhibitor or/and 200 μM PA. (F) The expression of apoptosis associated markers in VSMCs transfected with miR-22
inhibitor or/and 200 μM PA. (G) The migration of VSMCs transfected with miR-22 inhibitor or/and 200 μM PA determined by transwell
assay. Scale bar=100 μm. (H) Western blot analyses showing that expression of SM22α, calponin, SMMHC, vimentin, collagen I, and
osteopontin in PA-treated VSMCs transfected with miR-22 inhibitor. n = 3. *P < 0.05, **P < 0.01. AGING 8052 www.aging-us.com inhibited VSMC switch to synthetic phenotype, as
manifested
by
inhibiting
VSMC
proliferation,
migration, and synthesis. Mechanistically, PA inhibits
VSMC
switch
to
synthetic
phenotype
through
upregulation of miR-22 by targeting EVI1. These
findings suggested that PA plays a role in the regulation inhibited VSMC switch to synthetic phenotype, as
manifested
by
inhibiting
VSMC
proliferation,
migration, and synthesis. EVI1 was the target of miR-22 Mechanistically, PA inhibits
VSMC
switch
to
synthetic
phenotype
through
upregulation of miR-22 by targeting EVI1. These
findings suggested that PA plays a role in the regulation of VSMC phenotype, which may contribute to vascular
health and diseases. Several studies have shown that saturated fatty acids
increase the risk of cardiovascular diseases [12, 28]. According to the previous study, the saturated fatty www aging-us com
8053
AGING
VSMC
switch
to
synthetic
phenotype
through
upregulation of miR-22 by targeting EVI1. These
findings suggested that PA plays a role in the regulation
Several studies have shown that saturated fatty acid
increase the risk of cardiovascular diseases [12, 28
According to the previous study, the saturated fat
Figure 4. EVI1 was the target of miR-22. (A) Target genes of miR-22 were predicted by miRDB, TargetScan and ENCORI. (B) MRNA leve
of target genes in VSMCs treated with miR-22 mimics. (C, D) Protein levels of SM22α, calponin, SMMHC, vimentin, collagen I, an
osteopontin in VSMCs detected by western blot. (E) The complementary sequence between 3′-UTR of EVIL1 and miR-22. (F) Dual-lucifera
reporter assay. (G) EVI1 protein level was detected by western blot in VSMCs treated with miR-22 inhibitor or PA. n = 3. *P < 0.05, **P < 0.01 Figure 4. EVI1 was the target of miR-22. (A) Target genes of miR-22 were predicted by miRDB, TargetScan and ENCORI. (B) MRNA levels
of target genes in VSMCs treated with miR-22 mimics. (C, D) Protein levels of SM22α, calponin, SMMHC, vimentin, collagen I, and
osteopontin in VSMCs detected by western blot. (E) The complementary sequence between 3′-UTR of EVIL1 and miR-22. (F) Dual-luciferase
reporter assay. (G) EVI1 protein level was detected by western blot in VSMCs treated with miR-22 inhibitor or PA. n = 3. *P < 0.05, **P < 0.01. AGING 8053 www.aging-us.com focused
on
different
biomarkers
of
risk
of
cardiovascular diseases found that not all SFAs exert
the same effect, namely, studies do not seem to serve as acids were usually regarded as a singular fatty acid
group and they might have the same effects during the
metabolism [29, 30]. However, some investigations eatment enhanced miR-22 mimic impacts on aborted the inhibitive effect of EVI1 on VSMC phenotype
ession of miR-22 and EVIL1 in VMSCs treated with EVI1 or/and miR-22 overexpression followed by PA treatment. (B) Cell
detected by EdU in VSMCs with EVI1/miR-22 overexpression followed by PA treatment. EVI1 was the target of miR-22 (C) Flow cytometry to detect the
MCs with EVI1/miR-22 overexpression followed by PA treatment. (D) Western blot to detect the apoptosis associated
s. (E, F). Cell migration was detected by transwell assay in EVI1/miR-22-overexpressed VSMCs followed by PA treatment. m. (G) Protein levels of SM22α, calponin, SMMHC, vimentin, collagen I, and osteopontin in VSMC in VSMCs with EVI1/miR-
n followed by PA treatment detected by western blot. n = 3. *P < 0.05, **P < 0.01. Figure 5. PA treatment enhanced miR-22 mimic impacts on aborted the inhibitive effect of EVI1 on
switch. (A) Expression of miR-22 and EVIL1 in VMSCs treated with EVI1 or/and miR-22 overexpression followed by PA
proliferation was detected by EdU in VSMCs with EVI1/miR-22 overexpression followed by PA treatment. (C) Flow cyto
apoptosis of VSMCs with EVI1/miR-22 overexpression followed by PA treatment. (D) Western blot to detect the a
markers in VSMCs. (E, F). Cell migration was detected by transwell assay in EVI1/miR-22-overexpressed VSMCs followe Figure 5 PA treatment enhanced miR 22 mimic impacts on aborted the inhibitive effect of EVI1 on VSMC phenotype Figure 5. PA treatment enhanced miR-22 mimic impacts on aborted the inhibitive effect of Figure 5. PA treatment enhanced miR-22 mimic impacts on aborted the inhibitive effect of EVI1 on VSMC phenotype
switch. (A) Expression of miR-22 and EVIL1 in VMSCs treated with EVI1 or/and miR-22 overexpression followed by PA treatment. (B) Cell
proliferation was detected by EdU in VSMCs with EVI1/miR-22 overexpression followed by PA treatment. (C) Flow cytometry to detect the
apoptosis of VSMCs with EVI1/miR-22 overexpression followed by PA treatment. (D) Western blot to detect the apoptosis associated
markers in VSMCs. (E, F). Cell migration was detected by transwell assay in EVI1/miR-22-overexpressed VSMCs followed by PA treatment. Scale bar = 100 μm. (G) Protein levels of SM22α, calponin, SMMHC, vimentin, collagen I, and osteopontin in VSMC in VSMCs with EVI1/miR-
22 overexpression followed by PA treatment detected by western blot. n = 3. *P < 0.05, **P < 0.01. Figure 5. PA treatment enhanced miR-22 mimic impacts on aborted the inhibitive effect of EVI1 on VSMC phenotype
switch. (A) Expression of miR-22 and EVIL1 in VMSCs treated with EVI1 or/and miR-22 overexpression followed by PA treatment. (B) Cell
proliferation was detected by EdU in VSMCs with EVI1/miR-22 overexpression followed by PA treatment. (C) Flow cytometry to detect the
apoptosis of VSMCs with EVI1/miR-22 overexpression followed by PA treatment. Abbreviations VSMC: Vascular smooth muscle cell; PA: palmitic
acid; EVI1: ecotropic virus integration site 1 protein
homolog. CONCLUSION Taken together, we found that PA inhibits VSMC
switch to synthetic phenotype through upregulation of
miR-22 expression. In addition, miR22 inhibits VSMC
switch to synthetic phenotype by targeting EVI1. These
findings suggested that PA plays a role in regulation of
VSMC
phenotype,
which
may
contribute
to
maintenance of vascular health and prevention of
vascular diseases. Recent studies support a critical role of miRNAs in
regulating VSMC differentiation and phenotype switch,
and miR-22 is one of the miRNAs which inhibits
VSMC switch to synthetic phenotype [16, 17]. miR-22
is previously demonstrated as a tumor suppressor, but
later has been concerned as a prohypertrophic miRNA
[34, 35]. A recent study documented that miR-22
playing key role in the regulation role in VSMC
biological activity [36]. In addition, it has also been
reported that miR-22 involved in VSMC phenotypic
modulation, which induces VSMC contractile gene
expression, but inhibits VSMC proliferation and
migration [17]. These findings indicated that miR-22
serves a key role in regulation of cardiovascular
function. Here, we show that PA increased miR-22
expression in VSMCs, and inhibition of miR-22
abolished the PA’s effects on modulation of VSMC
phenotype. It has been reported that transforming
growth factor-β1 (TGF-β1) transcriptionally modulates
miR-22 expression in VSMCs via a P53-dependent
mechanism [17]. Whether PA regulates miR-22
expression
through
TGF-β1
needed
further
investigation. Indeed, there is evidence that PA
treatment increases TGF-β1 in other cells [37]. These
findings suggested that PA modulate VSMC phenotype
via upregulating miR-22, which serves a crucial role
vascular function regulation. EVI1 was the target of miR-22 (D) Western blot to detect the apoptosis associated
markers in VSMCs. (E, F). Cell migration was detected by transwell assay in EVI1/miR-22-overexpressed VSMCs followed by PA treatment. Scale bar = 100 μm. (G) Protein levels of SM22α, calponin, SMMHC, vimentin, collagen I, and osteopontin in VSMC in VSMCs with EVI1/miR-
22 overexpression followed by PA treatment detected by western blot. n = 3. *P < 0.05, **P < 0.01. AGING 8054 www.aging-us.com a single role of PA in the development of
cardiovascular diseases [31]. PA slightly elevated the
LDL- and HDL-cholesterol, which is a significant
predictor for cardiovascular disease [32, 33]. Although
the role of PA in cardiovascular diseases needs to be
further examined, these advances suggest that PA’s
effects in cardio-vascular health and disease cannot be
easily identified as detrimental or beneficial. Here, we
found that PA inhibited VSMC switch to synthetic
phenotype, as manifested by inhibiting VSMC
proliferation, migration, and synthesis, suggesting
that PA may exert beneficial effects on vascular health
and diseases, which should be identified by further
studies. signaling axis plays a key role in VSMC phenotypic
switch and correcting the dysregulation of miR-22/EVI1
or PA could be a potential treatment to vascular
diseases. AUTHOR CONTRIBUTIONS Yanchao Hu and Congxia Wang conceived and
designed these experiments. Yajie Fan and Chunyan
Zhang performed these experiments. Yajie Fan and
Chunyan Zhang analyzed and interpreted the data. Yanchao Hu and Congxia Wang wrote the manuscript. All authors read and approved the final manuscript. CONFLICTS OF INTEREST All authors have completed the ICMJE uniform
disclosure form. All the authors declare that they have
no competing interests. ETHICAL STATEMENT All animal procedures in this study were conducted in
accordance with the National Institutes of Health
Guidelines on the Use of Laboratory Animals. The
study protocol was approved by the Ethics Committee
of Xi’an Jiaotong University Second Affiliated
Hospital. Animal care and study were approved by the
Institutional Animal Care and Use Committee of Xi’an
Jiaotong University Second Affiliated Hospital. Previous studies demonstrated that EVI1 functions as a
transcriptional regulator to modulate several biological
processes,
including
hematopoiesis,
apoptosis,
development, differentiation and proliferation [38, 39]. Here, we have found that EVI1 serves as a target gene
of miR-22 to modulate VSMC phenotype switch. Further analysis showed that EVI1 transcriptionally
inhibits VSMC-specific genes to modulate the VSMC
phenotype switch, including SMαA, SM22α, SRF, and
Myocd [17]. In addition, inhibiting EVI1 abolished the
effects of miR-22 and PA in modulation of VSMC
phenotype. These findings suggested that miR-22/EVI1 https://doi.org/10.1161/CIRCRESAHA.115.306361
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Dietary
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PMID:28322865 AGING 8058 www.aging-us.com SUPPLEMENTARY MATERIALS
Supplementary Table
Supplementary Table 1. The primer sequences. Gene
Primer (5′–3′)
MYH11
F: AGGCGAACCTAGAC
R: CTGGATGTTGAGAG
SM22α
F: TTCTGCCTCAACATG
R: CACCTTCACTGG CTT
Calponin
F: ATGTCCTCTGCTCAC
R: CACGTTCACCTTGTT
SMMHC
F: AGGCGAACCTAGAC
R: CTGGATGTTGAGAG
Collagen I
F: TGACGAGACCAAGA
R: GCACCATCATTTCCA
Vimentin
F: TCGTTTCGAGGTTTT
R: GACTAAAACTCGAC
Osteopontin
F: CAGCCTTCTCAGCCA
R: CAAATCACTGCAATT
MiR-22
F: AAGCTGCCAGTTGAA
R: Universal PCR Reverse
(cat. no. B532451; Sangon
miR-23b
F: ATCACATTGCCAGGG
R: Universal PCR Reverse
(cat. no. B532451; Sangon
miR-125b
F: TCCCTGAGACCCTAA
R: Universal PCR Reverse
(cat. no. B532451; Sangon
NET1
F: AAATCCCCGGATGAC
R: TGCACTGCTCCTTCT
SIRT1
F: GCTCGCCTTGCGGTG
R: GACGGCTGGAACTG
PTEN
F: AGCCTCTTGATGTGT
R: CCATTGGTAGCCAAA
ERBB3
F: CATCGTGAGGGACCG
R: TGTTGCTCGAGTCCA
LAMC1
F: GAGGCAAGATATCG
R: GTATCTCGCCTGTCC
AKT3
F: GGTGCAGAGTCCCCT
R: TTGGCGACAGCAGG
DPF2
F: TGCCTGTGACATTTG
R: TAGCCACGATCGCA
F: TCCAACTACTGGTTT Supplementary Table
Supplementary Table 1. The primer sequences. Gene
Primer (5′–3′)
MYH11
F: AGGCGAACCTAGACAAGAATAAG
R: CTGGATGTTGAGAGTGGAGATG
SM22α
F: TTCTGCCTCAACATGGCCAAC3
R: CACCTTCACTGG CTTGGATC
Calponin
F: ATGTCCTCTGCTCACTTCAAC
R: CACGTTCACCTTGTTTCCTTTC
SMMHC
F: AGGCGAACCTAGACAAGAATAAG
R: CTGGATGTTGAGAGTGGAGATG
Collagen I
F: TGACGAGACCAAGAACTGCC
R: GCACCATCATTTCCACGAGC
Vimentin
F: TCGTTTCGAGGTTTTCGCGTTAGAGAC
R: GACTAAAACTCGACCGACTCGCGA
Osteopontin
F: CAGCCTTCTCAGCCAAACG
R: CAAATCACTGCAATTCTCATGGTAGT
MiR-22
F: AAGCTGCCAGTTGAAGAACTGT
R: Universal PCR Reverse Primer
(cat. no. B532451; Sangon Biotech Co., Ltd.)
miR-23b
F: ATCACATTGCCAGGGATTACCAC
R: Universal PCR Reverse Primer
(cat. no. (Suppl); 34:S121–4.
https://doi.org/10.1007/BF02562257
PMID:10419117 B532451; Sangon Biotech Co., Ltd.)
miR-125b
F: TCCCTGAGACCCTAACTTGTGA
R: Universal PCR Reverse Primer
(cat. no. B532451; Sangon Biotech Co., Ltd.)
NET1
F: AAATCCCCGGATGACGGC
R: TGCACTGCTCCTTCTCTTGG
SIRT1
F: GCTCGCCTTGCGGTGGACTT
R: GACGGCTGGAACTGTCCGGG
PTEN
F: AGCCTCTTGATGTGTGCATT
R: CCATTGGTAGCCAAACGGAAC
ERBB3
F: CATCGTGAGGGACCGAGATG
R: TGTTGCTCGAGTCCACAGTC
LAMC1
F: GAGGCAAGATATCGCCGTGA
R: GTATCTCGCCTGTCCACTCG
AKT3
F: GGTGCAGAGTCCCCTAGAGA
R: TTGGCGACAGCAGGATCATT
DPF2
F: TGCCTGTGACATTTGTGGAA
R: TAGCCACGATCGCAGTCATC
TGFBR1
F: TCCAACTACTGGTTTACCATTGC
R: ACAGCAACTTCTTCTCCCCG
F: CGTCTGGGTACCTGAACGAG Primer (5′–3′)
F: AGGCGAACCTAGACAAGAATAAG
R: CTGGATGTTGAGAGTGGAGATG
F: TTCTGCCTCAACATGGCCAAC3
R: CACCTTCACTGG CTTGGATC
F: ATGTCCTCTGCTCACTTCAAC
R: CACGTTCACCTTGTTTCCTTTC
F: AGGCGAACCTAGACAAGAATAAG
R: CTGGATGTTGAGAGTGGAGATG
F: TGACGAGACCAAGAACTGCC
R: GCACCATCATTTCCACGAGC
F: TCGTTTCGAGGTTTTCGCGTTAGAGAC
R: GACTAAAACTCGACCGACTCGCGA
F: CAGCCTTCTCAGCCAAACG
R: CAAATCACTGCAATTCTCATGGTAGT
F: AAGCTGCCAGTTGAAGAACTGT
R: Universal PCR Reverse Primer
(cat. no. B532451; Sangon Biotech Co., Ltd.)
F: ATCACATTGCCAGGGATTACCAC
R: Universal PCR Reverse Primer
(cat. no. B532451; Sangon Biotech Co., Ltd.)
F: TCCCTGAGACCCTAACTTGTGA
R: Universal PCR Reverse Primer
(cat. no. B532451; Sangon Biotech Co., Ltd.)
F: AAATCCCCGGATGACGGC
R: TGCACTGCTCCTTCTCTTGG
F: GCTCGCCTTGCGGTGGACTT
R: GACGGCTGGAACTGTCCGGG
F: AGCCTCTTGATGTGTGCATT
R: CCATTGGTAGCCAAACGGAAC
F: CATCGTGAGGGACCGAGATG
R: TGTTGCTCGAGTCCACAGTC
F: GAGGCAAGATATCGCCGTGA
R: GTATCTCGCCTGTCCACTCG
F: GGTGCAGAGTCCCCTAGAGA
R: TTGGCGACAGCAGGATCATT
F: TGCCTGTGACATTTGTGGAA
R: TAGCCACGATCGCAGTCATC
F: TCCAACTACTGGTTTACCATTGC
R: ACAGCAACTTCTTCTCCCCG
F: CGTCTGGGTACCTGAACGAG
R: ACTTCTGTGCCCGTGAGTCT
F: GCAGACATTGCGCCTGGGGAA AGING 8059 8059 www.aging-us.com R: CTCACAGCGGTGCTGCCGTT
GAPDH
F: AACTTTGGCATTGTGGAAGG
R: ATTGGGGGTAGGAACA
U6
F: TGAGAACTGAATTCCATGGGTT
R: ACGCTTCACGAATTTGCGT GAPDH U6 AGING 8060 www.aging-us.com
|
https://openalex.org/W4229988195
|
https://zenodo.org/records/2231079/files/article.pdf
|
English
| null |
A Note-Book on Experimental Mathematics. C. Godfrey and G. M. Bell. (Arnold.)
|
The mathematical gazette/Mathematical gazette
| 1,906
|
public-domain
| 2,585
|
Review
Source:
The Mathematical Gazette, Vol. 3, No. 56 (Mar., 1906), pp. 297-298
Published by:
Mathematical Association
Stable URL:
http://www.jstor.org/stable/3603481
Accessed: 02-12-2015 05:46 UTC Review Review Source:
The Mathematical Gazette, Vol. 3, No. 56 (Mar., 1906), pp. 297-298
Published by:
Mathematical Association
Stable URL:
http://www.jstor.org/stable/3603481
Accessed: 02-12-2015 05:46 UTC Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at http://www.jstor.org/page/
info/about/policies/terms.jsp JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of content
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All use subject to JSTOR Terms and Conditions REVIEWS. REVIEWS. 297
297 logarithms, and it is a pity that the authors have not taken advantage
of it. logarithms, and it is a pity that the authors have not taken advantage
of it. logarithms, and it is a pity that the authors have not taken advantage
of it. logarithms, and it is a pity that the authors have not taken advantage
of it. In combining logarithms the vertical arrangement is more satis-
factory. For instance the writers have
In combining logarithms the vertical arrangement is more satis-
factory. For instance the writers have log x= 2-0913 + 1 6586 + 0-8318 = 4-5817. log x= 2-0913 + 1 6586 + 0-8318 = 4-5817. g
(20913
or
logx=
2-0913
This might be written log x=
1-6586
+ 1-6586
0 -8318
+ 08318
=
45817
=
4-5817
g
(20913
or
logx=
2-0913
This might be written log x=
1-6586
+ 1-6586
0 -8318
+ 08318
=
45817
=
4-5817 The theorem of Pythagoras is suggested for discovery (1) by squared
paper and the A 3, 4, 5; (2) by construction and calculation. One or two
"dissection proofs" might well have been added. In connection with
this theorem and the corresponding theorems for acute- and obtuse-
angled triangles too much is expected from the student, who is,
moreover, asked to discover for the first time that (a - b)2= a2 - 2ab + b2
from a figurecorrespondingto that of Euc. II. 7. The theorem of Pythagoras is suggested for discovery (1) by squared
paper and the A 3, 4, 5; (2) by construction and calculation. Review One or two
"dissection proofs" might well have been added. In connection with
this theorem and the corresponding theorems for acute- and obtuse-
angled triangles too much is expected from the student, who is,
moreover, asked to discover for the first time that (a - b)2= a2 - 2ab + b2
from a figurecorrespondingto that of Euc. II. 7. figure corresponding
The authors speak of the "Equation of a Circle," although they
state that it is very important to use the words circumference anil
circle in their right senses. figure corresponding
The authors speak of the "Equation of a Circle," although they
state that it is very important to use the words circumference anil
circle in their right senses. right
The chapter on Plans, Elevations, etc., is instructive, and should be
of great use. It is an important branch which is often neglected in
schools. right
The chapter on Plans, Elevations, etc., is instructive, and should be
of great use. It is an important branch which is often neglected in
schools. Density is not defined, but the term relative density is used for
specificgravity. Density is not defined, but the term relative density is used for
specificgravity. p
g
y
A chapter is given to Trigonometry. It forms a good introduction,
but perhaps more attention might have been given to the graphs of the
ratios. The examples in this chapter are not well chosen, as they do
not give sufficient prominence to practical applications. Moreover, no
instructions are given as to the use of trigonometricaltables. p
g
y
A chapter is given to Trigonometry. It forms a good introduction,
but perhaps more attention might have been given to the graphs of the
ratios. The examples in this chapter are not well chosen, as they do
not give sufficient prominence to practical applications. Moreover, no
instructions are given as to the use of trigonometricaltables. given
trigonometrical
The book concludes with a short chapter on Time and Velocity. given
trigonometrical
The book concludes with a short chapter on Time and Velocity. p
y
Tables of logarithms, antilogarithms, and of values of sin, cos, and tall
for angles of a whole number of degrees are placed in appendices at
the end. Answers to the examples are given. This content downloaded from 160.36.178.25 on Wed, 02 Dec 2015 05:46:36 UTC
All use subject to JSTOR Terms and Conditions Review p
y
Tables of logarithms, antilogarithms, and of values of sin, cos, and tall
for angles of a whole number of degrees are placed in appendices at
the end. Answers to the examples are given. examples
given. The book shows much originality, and though exception may perhaps
be taken to the order and arrangement, it well fulfils the aims for which
it was written. examples
given. The book shows much originality, and though exception may perhaps
be taken to the order and arrangement, it well fulfils the aims for which
it was written. A Note-Book on Experimental Mathematics. C. GODFREY and
G. M. BELL. (Arnold.)
A Note-Book on Experimental Mathematics. C. GODFREY and
G. M. BELL. (Arnold.) A Note-Book on Experimental Mathematics. C. GODFREY and
G. M. BELL. (Arnold.)
A Note-Book on Experimental Mathematics. C. GODFREY and
G. M. BELL. (Arnold.) A Note-Book on Experimental Mathematics.
C. GODFREY and
G. M. BELL. (Arnold.)
A Note-Book on Experimental Mathematics.
C. GODFREY and
G. M. BELL. (Arnold.) (
)
This book is arranged on the lines of Ashford's Note-Book of Practical
Physics, compiled for use in Harrow School, and is intended primarily
to cover the requirementsof the army entrance examinations. (
)
This book is arranged on the lines of Ashford's Note-Book of Practical
Physics, compiled for use in Harrow School, and is intended primarily
to cover the requirementsof the army entrance examinations. requirements
army
An list of apparatus required in the experiments is given at the
beginningof the book. This should provemost useful. requirements
army
An list of apparatus required in the experiments is given at the
beginningof the book. This should provemost useful. beginning
prove
There are 124 experiments with brief, but generally sufficient
instructions to the student. The concrete conceptions which he
should gain by working through them should be of great value to
him when studying the more theoretical portions of mathematics. It is important, however, that classes, taking practical work should
frequently discuss their work with the teacher. It is therefore
beginning
prove
There are 124 experiments with brief, but generally sufficient
instructions to the student. The concrete conceptions which he
should gain by working through them should be of great value to
him when studying the more theoretical portions of mathematics. It is important, however, that classes, taking practical work should
frequently discuss their work with the teacher. It is therefore This content downloaded from 160.36.178.25 on Wed, 02 Dec 2015 05:46:36 UTC
All use subject to JSTOR Terms and Conditions Fletcher (referred to at
the end of the book). Suggestions for further experiments are given in
Part IV. The authors hint that the time has come for the retirement
of Atwood's machine. It certainly deserves a rest from its old occu-
pation of trying to discover "g."
p
The work in this part is suggestive, and it is almost a pity that there
is not more of it, e.g. illustrations of accelerations by means of some
apparatus similar to that devised by Mr. W. C. Fletcher (referred to at
the end of the book). Suggestions for further experiments are given in
Part IV. The authors hint that the time has come for the retirement
of Atwood's machine. It certainly deserves a rest from its old occu-
pation of trying to discover "g." p
y
g
g
Elementary Practical Mathematics. H. A. STERN and W. A. TOPHAM. (G. Bell & Sons.)
p
y
g
g
Elementary Practical Mathematics. H. A. STERN and W. A. TOPHAM. (G. Bell & Sons.) (G
So s )
This contains the first nine chapters of a text book of Practical
Mathematics definitely intended to satisfy the new Army Regulations. The po.rtion given in this volume covers very well the syllabus for the
" Qualifying" Examination, and its equivalent, the " Practical Measure-
ments " of the LeavingCertificate Examination. (G
So s )
This contains the first nine chapters of a text book of Practical
Mathematics definitely intended to satisfy the new Army Regulations. The po.rtion given in this volume covers very well the syllabus for the
" Qualifying" Examination, and its equivalent, the " Practical Measure-
ments " of the LeavingCertificate Examination. Leaving
The first two chapters on contracted methods and graphs are
scarcely necessary, as most of the work is now well done in books
on arithmetic and algebra. In the graphs the writers are not uniform
in indicating the scales used. It seems desirable that in all cases the
numbers should be plainly marked on the axes. For some reason the
'cooling curve" on p. 31 is inverted. Leaving
The first two chapters on contracted methods and graphs are
scarcely necessary, as most of the work is now well done in books
on arithmetic and algebra. In the graphs the writers are not uniform
in indicating the scales used. This content downloaded from 160.36.178.25 on Wed, 02 Dec 2015 05:46:36 UTC
All use subject to JSTOR Terms and Conditions Part III. commences with instruments of more refined accuracy-the
vernier, screw-gauge, spherometer. The vernier is introduced by a
neat and convincing experiment, but the principle of a micrometer
screw might well have been learnt after experiments with simple
screws. The radius of a sphere is to be calculated from formula
.2
2R=h+-,,
r being obtained by calculating the radius of the
circumcircle of the equilateral triangle formed by the three feet of the
instrument. For approximate purposes a geometrical construction spring balances perhaps
easy
up. Part III. commences with instruments of more refined accuracy-the
vernier, screw-gauge, spherometer. The vernier is introduced by a
neat and convincing experiment, but the principle of a micrometer
screw might well have been learnt after experiments with simple
screws. The radius of a sphere is to be calculated from formula
.2
h
di
f
th
spring balances perhaps
easy
up. Part III. commences with instruments of more refined accuracy-the
vernier, screw-gauge, spherometer. The vernier is introduced by a
neat and convincing experiment, but the principle of a micrometer
screw might well have been learnt after experiments with simple
screws. The radius of a sphere is to be calculated from formula
.2
h
di
f
th 2R=h+-,,
r being obtained by calculating the radius of the
2R=h+-,,
r being obtained by calculating the radius of the circumcircle of the equilateral triangle formed by the three feet of the
instrument. For approximate purposes a geometrical construction
based on A and r (measuredat once) is sufficientlyaccurate. circumcircle of the equilateral triangle formed by the three feet of the
instrument. For approximate purposes a geometrical construction
based on A and r (measuredat once) is sufficientlyaccurate. (measured
once)
sufficiently
Part III. also contains further work in specific gravity and statics. It ends with a little dynamics-the determination of "g" by means of
a lead ball falling against an oscillating bar, and also by the simple
pendulum. (measured
once)
sufficiently
Part III. also contains further work in specific gravity and statics. It ends with a little dynamics-the determination of "g" by means of
a lead ball falling against an oscillating bar, and also by the simple
pendulum. p
The work in this part is suggestive, and it is almost a pity that there
is not more of it, e.g. illustrations of accelerations by means of some
apparatus similar to that devised by Mr. W. C. This content downloaded from 160.36.178.25 on Wed, 02 Dec 2015 05:46:36 UTC
All use subject to JSTOR Terms and Conditions This content downloaded from 160.36.178.25 on Wed, 02 Dec 2015 05:46:36 UTC
All use subject to JSTOR Terms and Conditions THE MATHEMATICAL GAZErrE. THE MATHEMATICAL GAZErrE. 298
298 advisable, though sometimes difficult to arrange, that the mathematical
master should take both the theoretical and practical work of his form. This will generally necessitate a "Practical Mathematics Room" in
addition to the Physical Laboratory. Such a room, unless the Physical
Laboratory is used for the purpose, will therefore be necessary for forms
in schools using this book. In fact at some of the larger schools a
Practical Mathematics Room has alreadybeen established. advisable, though sometimes difficult to arrange, that the mathematical
master should take both the theoretical and practical work of his form. This will generally necessitate a "Practical Mathematics Room" in
addition to the Physical Laboratory. Such a room, unless the Physical
Laboratory is used for the purpose, will therefore be necessary for forms
in schools using this book. In fact at some of the larger schools a
Practical Mathematics Room has alreadybeen established. already
The book is divided into four parts; Part I. contains simple experi-
ments and measurements connected with lengths, areas, volumes, mass
and density, and fluid pressure. Part II. is on statics, with experiments
on tensions, moments, and the equilibrium of three forces. The board used
to illustrate the parallelogram of forces is placed in a vertical plane, and
the three forces are obtained by using hanging weights. The method
with three spring-balancesis perhapsmore easy to set up. already
The book is divided into four parts; Part I. contains simple experi-
ments and measurements connected with lengths, areas, volumes, mass
and density, and fluid pressure. Part II. is on statics, with experiments
on tensions, moments, and the equilibrium of three forces. The board used
to illustrate the parallelogram of forces is placed in a vertical plane, and
the three forces are obtained by using hanging weights. The method
with three spring-balancesis perhapsmore easy to set up. spring balances perhaps
easy
up. Part III. commences with instruments of more refined accuracy-the
vernier, screw-gauge, spherometer. The vernier is introduced by a
neat and convincing experiment, but the principle of a micrometer
screw might well have been learnt after experiments with simple
screws. The radius of a sphere is to be calculated from formula
.2
2R=h+-,,
r being obtained by calculating the radius of the
circumcircle of the equilateral triangle formed by the three feet of the
instrument. For approximate purposes a geometrical construction
spring balances perhaps
easy
up. This content downloaded from 160.36.178.25 on Wed, 02 Dec 2015 05:46:36 UTC
All use subject to JSTOR Terms and Conditions It seems desirable that in all cases the
numbers should be plainly marked on the axes. For some reason the
'cooling curve" on p. 31 is inverted. g
p. The chapters on length, mass, area, and volume are very fair. Mass
and weighing are taken before areas and volumes. It is doubtfiu
g
p. The chapters on length, mass, area, and volume are very fair. Mass
and weighing are taken before areas and volumes. It is doubtfiu This content downloaded from 160.36.178.25 on Wed, 02 Dec 2015 05:46:36 UTC
All use subject to JSTOR Terms and Conditions
|
https://openalex.org/W4231506190
|
http://e-journal.metrouniv.ac.id/index.php/istinbath/article/download/951/783
|
Indonesian
| null |
FAKTOR PENGHAMBAT DALAM PENEGAKAN QANUN JINAYAT DI ACEH
| null | 2,019
|
cc-by-sa
| 8,010
|
Abstrak Tulisan ini membuktikan bahwa pelaksanaan Qanun Aceh No. 14 Tahun 2003 tentang
khalwat di Kota Subulussalam belum sepenuhnya berjalan dengan baik. Banyak kendala
yang dihadapi baik dari pelaksananya (pemerintah) maupun masyarakat sebagai objek
hukum penerapan syariat Islam itu sendiri. Faktor hukum yang merupakan salah satu
faktor utama penyebab stagnan-nya penindakan terhadap pelanggar Qanun No. 14 Tahun
2003 di Kota Subulussalam disamping faktor-faktor lainnya. Tulisan ini berusaha
menguraikan kendala dalam efektivitas penegakan hukum yang merupakan suatu indikator
penilaian terhadap penegakan suatu hukum, dalam artian untuk mengukur keberhasilan
atau target yang telah ditetapkan dalam penerapan suatu hukum khususnya terhadap
penegakan Qanun No. 14 Tahun 2003 di wilayah hukum Kota Subulussalam, Aceh. Kata kunci: Khalwat, Aceh, Syariat Islam, Jinayat, Subulussalam (Studi Kasus Penerapan Qanun No. 14 Tahun 2003 di Kota Subulussalam) Ali Geno Berutu
Sekolah Pascasarjan (SPs) UIN Syarif Hidayatullah Jakarta Ali Geno Berutu
Sekolah Pascasarjan (SPs) UIN Syarif Hidayatullah Jakarta
Email: ali_geno@ymail.com Ali Geno Berutu
Sekolah Pascasarjan (SPs) UIN Syarif Hidayatullah Jakarta
Email: ali_geno@ymail.com Keywords: Khalwat, Aceh, Syariat Islam, Jinayat, Subulussalam Abstract This paper proves that the implementation of Qanun Aceh no. 14 of 2003 on khalwat in
Subulussalam city has not fully run well. Many obstacles faced both from the executor
(government) and the community as the object of the law of the application of Islamic
Shari'a itself. The legal factor is one of the main factors causing its stagnant action against
the violation of Qanun. 14 Year 2003 in Subulussalam City in addition to other factors. This
paper attempts to outline the obstacles in the effectiveness of law enforcement which is an
indicator of the assessment of the enforcement of a law, in order to measure the success or
targets set in the application of a law especially to the enforcement of Qanun. 14 Year 2003 in
the jurisdiction of Subulussalam City, Aceh. Keywords: Khalwat, Aceh, Syariat Islam, Jinayat, Subulussalam Keywords: Khalwat, Aceh, Syariat Islam, Jinayat, Subulussalam 148 1 Haedar Nasir, Islam syariat: Reproduksi Salapiyah Idiologis di Indonesia (Bandung: Mizan 2013),
h. 336. 2 Rusjdi Ali Muhammad, Revitalisasi Syari’at Islam di Aceh: Problem, Solusi dan Implementasi,
(Ciputat: Logos Wacana Ilmu,2003), h. 49. 1 Haedar Nasir, Islam syariat: Reproduksi Salapiyah Idiologis di Indonesia (Bandung: Mizan 2013
6 3 Khamami Zada, “Sentuhan Adat Dalam Pemberlakuan Syariat Islam di Aceh (1514-1903)”,
Karsa, Vol. 20 No. 2, Desember 2012, h. 3. 4 Al Yasa’ Abu Bakar,”Islam, Hukam dan Masyarakat di Aceh Tajdid Syariat dalam Negara
Bangsa”, First International Conference of Aceh and Indian Ocean Studies, 24-27 Februari 2007, h. 2. 149
1 Haedar Nasir, Islam syariat: Reproduksi Salapiyah Idiologis di Indonesia (Bandung: Mizan 2013),
h. 336.
2 Rusjdi Ali Muhammad, Revitalisasi Syari’at Islam di Aceh: Problem, Solusi dan Implementasi,
(Ciputat: Logos Wacana Ilmu,2003), h. 49.
3 Khamami Zada, “Sentuhan Adat Dalam Pemberlakuan Syariat Islam di Aceh (1514-1903)”,
Karsa, Vol. 20 No. 2, Desember 2012, h. 3.
4 Al Yasa’ Abu Bakar,”Islam, Hukam dan Masyarakat di Aceh Tajdid Syariat dalam Negara
Bangsa”, First International Conference of Aceh and Indian Ocean Studies, 24-27 Februari 2007, h. 2. A. Pendahuluan Masyarakat Aceh memiliki corak kehidupan yang kental dengan agama Islam, hal
ini dapat dilacak akar sejarah berdirinya kerajaan–kerajan Islam yang menjadi pusat
kekuasaan politik sekaligus agama. Aceh tergolong wilayah yang memiliki sejarah panjang
dalam hal kedatangan Islam dan berdirinya kerajaan–kerajaan Islam yang dimulai dari
kerajaan Peureulak (840-1291), Samudra Pasai (1042-1427), Teumiang (840-1291),
Darussalam (1205-1530) dan Aceh Darussalam (1514-1903) dan kerajaan – kerajaan kecil
lainnya.1 Bila ditinjau dari sejarah kerajaan Aceh Darussalam (1514-1903) terdapat
pemberlakuan hukum jinayat yang dipengaruhi oleh hukum adat. Pemberlakuan hukum
jinayat ini dapat dibuktikan dengan adanya Qanun Meukuta Alam dan keterangan-
keterangan tentang praktek pemberlakuan hukum jinayat.2 Dalam Qanun Meukuta Alam ini
ditetapkan bahwa dasar kerajaan Acceh Darussalam adalah Islam dan bentuknya adalah
kerajaan yang bersuber kepada al-Qur’an, al-Hadist, Ijma’ dan Qiyas.3 Konsep ini
sesungguhnya mencerminkan bahwa Islam dan negara adalah integral (al-Isla din wa
daulah), tak berlebihan jika kemudian kerajaan Aceh Darussalam merumuskan Qanun al-Asyi
sebagai pedoman dasar dalam kehidupan bernegaraa, sosial dan hukum masyarakat. Meskipun Islam telah menjadi sumber undang-undang negara, tetapi tidak dapat
dielakkan keterpengaruhan hukum jinayat yang diberlakukan oleh adat istiadat yang
dipraktekkan di masyarakat. Dengan kata lain, pelanggaran terhadap pembunahan,
perzinaan, pencurian dan khamar dikenakan sanksi yang tegas yang di ambil dari hukum
jinayat yang bercampur dengan adat. Hal ini dapat disimpulkan bahwa adat orang Aceh
sangat berpengaruh oleh ajaran Islam, bahkan lebih dari sekedar itu, adat orang Aceh
terbentuk di atas kesadaran dan keinginan untuk menjalankan syariat Islam dalam
kehidupan sehari-hari.4 Masyarakat Aceh memiliki strukur sosial tersendiri yang diatur berdasarkan hukum
Qanun Meukuta Alam al-Asyi (Undang-undang kerajaan Aceh). Qanun tersebut menjelaskan
bahwa kekuasaan tertinggi berada di tangan sultan, sedangkan kekuasaan agama berada 149 ditangan ulama. Di bawah sultan ada Uleebalang (hulu balang) yang menguasai daerah
tertentu dengan hak otonomi luas. Uleebalang membawahi beberapa imum mukim dan
dibawah imum mukim terdapat beberapa keuchik.5 Di Aceh, syariat Islam di implementasikan secara formal setelah dikeluarkannya
Undang-undang No. 44 Tahun 19996 tentang Penyelenggaraan Keistimewaan Provinsi
Daerah Istimewa Aceh dan Undang-undang No. 18 Tahun 2001 tentang Otonomi Khusus
bagi Provinsi Daerah Istimewa Aceh7 sebagai Provinsi Nanggroe Aceh Darussalam. Adanya
legalitas dari pemerintah untuk menerapkan syariat Islam di Aceh direspon oleh pemerintah
daerah dengan mengeluarkan beberapa peraturan daerah (Perda) dalam rangka
terlaksananya syariat Islam di Aceh. 5 Hasan Basri, ”A. Hasjmy (1914-1998) Kajian Sosial-Intlektual Pemikiran Tentang Politik Islam”,
(Disertasi Sps UIN Jakarta, 2008), h. 53. Lihat juga, Asma Uddin, "Religious Freedom Implications of
Sharia Implementation in Aceh, Indonesia," University of St. Thomas Law Journal: Vol. 7: Iss. 3 (2010),
Article Available at: http://ir.stthomas.edu/ustlj/vol7/iss3/8(diakses pada tanggal 02 Mei 2016) h. 618. 9 Asma Uddin, "Religious Freedom Implications of Sharia ..., h. 632. 8 Syamsul Bahri, “Pelaksanaan Syariat Islam di Aceh Sebagai Bagian dari Wlayah NKRI”,
inamika Hukum Vol. 12 No. 2 Mei 2012. p
j
p
gg
6 Haidar Nassir, Islam Syariat Reproduksi Salafiyah Idiologis di Indonesia (Bandung: Mizan,
2013),h. 332.
7 Topo Santoso, Membumikan Hukum Pidana Islam: Penegakan Syariat dalam Wacana dan Agenda, 7 Topo Santoso, Membumikan Hukum Pidana Islam: Penegakan Syariat dalam Wacana dan Agenda,
: GIP, 2003),h. 106. ,
y
p
f y
g
(
g
,
2013),h. 332.
7 Topo Santoso, Membumikan Hukum Pidana Islam: Penegakan Syariat dalam Wacana dan Agenda,
( Jakarta: GIP, 2003),h. 106. p
j
p
gg
6 Haidar Nassir, Islam Syariat Reproduksi Salafiyah Idiologis di Indonesia (Bandung: Mizan,
2013),h. 332. 5 Hasan Basri, ”A. Hasjmy (1914-1998) Kajian Sosial-Intlektual Pemikiran Tentang Politik Islam”,
(Disertasi Sps UIN Jakarta, 2008), h. 53. Lihat juga, Asma Uddin, "Religious Freedom Implications of
Sharia Implementation in Aceh, Indonesia," University of St. Thomas Law Journal: Vol. 7: Iss. 3 (2010),
Article Available at: http://ir.stthomas.edu/ustlj/vol7/iss3/8(diakses pada tanggal 02 Mei 2016) h. 618.
6 Haidar Nassir, Islam Syariat Reproduksi Salafiyah Idiologis di Indonesia (Bandung: Mizan,
2013),h. 332.
7 Topo Santoso, Membumikan Hukum Pidana Islam: Penegakan Syariat dalam Wacana dan Agenda,
( Jakarta: GIP, 2003),h. 106.
8 Syamsul Bahri, “Pelaksanaan Syariat Islam di Aceh Sebagai Bagian dari Wlayah NKRI”,
Jurnal Dinamika Hukum Vol. 12 No. 2 Mei 2012.
9 Asma Uddin, "Religious Freedom Implications of Sharia ..., h. 632. 8 Syamsul Bahri, “Pelaksanaan Syariat Islam di Aceh Sebagai Bagian da
Jurnal Dinamika Hukum Vol. 12 No. 2 Mei 2012. 14 Wilayatul Hisbah adalah lembaga atau badan yang berwenang memberitahukan kepada
masyarakat tentang peraturan-peraturan yang sudah berlaku dan menyadarkan anggota masyarakat
tersebut, agar mematuhi aturan-aturan supaya tidak dikenakan sanksi atau denda dari peraturan-
peraturan terebut (law enforcement). Lihat, Al Yasa’ Abu Bakar, Wilayatul Hisbah, Polisi Pamong Praja
Dengan Kewenangan Khusus di Aceh, (Banda Aceh: Dinas Syari‘at Islam Aceh, 2009), h. 22. A. Pendahuluan Dari perda-perda itu selanjutnya dikembangkan lagi
menjadi peraturan-peraturan daerah yang menyangkut tata laksana syariat Islam yang pada
gilirannya melahirkan Qanun Aceh.8 Lima qanun disahkan antara tahun 2002-2004 yang berisi hukuman pidana atas
pelanggaran syariah yakni: Qanun No. 11 Tahun 2002 tentang Penerapan Syariah Dalam
Aspek Kepercayaan (aqidah), Ritual (ibadah), dan Penyebaran (syi’ar) Islam yang meliputi
persyaratan busana islami; Qanun No. 12 Tahun 2003 tentang larangan Menkonsumsi dan
Menjual Minuman ber–alkohol (khamar); Qanun No. 13 Tahun 2003 tentang larangan Judi
(maisir); Qanun No. 14 Tahun 2003 melarang “perbuatan bersunyi-sunyian” (khalwat)9 dan
Qanun No. 7 Tahun 2004 tentang Pembayaran Zakat. Dari penjelasan latar belakang di atas, maka tulisan ini akan mencoba
mengungkapkan faktor-faktor yang mempengaruhi implementasi Qanun No. 14 Tahun
2003 Tentang khalwat di Kota Subulussalam, baik foktor dari segi hukum maupun non
hukum, sehingga dapat tergambar dengan jelas apa saja yang menjadi penghalang
efektifitas Qanun No. 14 di di Kota Subulussalam dan di Aceh pada umumnya. Penelitian
ini bersumber dari penelitian lapangan (field research) dan penelitian kepustakaan (library
research) dengan metode kualitatif dan menggunakan pendekatan sosio-legal-historis. 150 10 Soerjono Soekanto, Faktor-Faktor yang Mempengaruhi Penegakan Hukum (Jakarta: PT. Raja
Grafindo Persada, 2008), h. 8. Lihat juga, Jainuddin Ali, Sosiologi Hukum (Jakarta: Sinar Grafika, 2007),
h. 62. B. Pembahasan Ada banyak faktor yang mempengaruhi dalam efektivitas penegakan Qanun
Kahalwat di Subulussalam. Merujuk kepada teori Soerjono Soekanto yang menjelaskan
bahwa masalah pokok dalam penegakan hukum adalah terletak pada hukumnya sendiri
(peraturan perundang-undangan yang berlaku), penegak hukumnya yakni pihak-pihak
yang mengawal penerapan hukum, sarana atau fasilitas yang mendukung penerapan
hukum, masyarakat di mana hukum tersebut diberlakukan dan budaya hukum dalam
masyarakatnya.10 Kelima faktor tesebut saling memiliki kaitan yang erat dikarenakan esensi
dari penegakan hukum. Kelima faktor tersebut merupakan tolak ukur daripada efektifitas
penerapan hukum. 11 Pasal 4 Qanun Nomor 14 Tahun 2003 Tentang Khalwat. Lihat juga Zulkarnain Lubis,
Menyambut Qanun Jinayah Nomor 6 Tahun 2014, https://drive. google. com/file/d/0B5UQVc
J8Df8WenBNSWNfZU VMcTQ/view? pli=1 (diakses 20 Oktober 2015). Q/
p
(
)
12 Pasal 22 Qanun Nomor 14 Tahun 2003 Tentang Khalwat. /
p
(
12 Pasal 22 Qanun Nomor 14 Tahun 2003 Tentang Khalwat. l 22 Qanun Nomor 14 Tahun 2003 Tentang Khalwat.
l 23 ayat (1) Qanun Nomor 6 Tahun 2014 Tentang Hukum Jinayat. Pasal 22 Qanun Nomor 14 Tahun 2003 Tentang Khalwat.
Pasal 23 ayat (1) Qanun Nomor 6 Tahun 2014 Tentang Hukum Jinayat. J8Df8WenBNSWNfZU VMcTQ/view? pli=1 (diakses 20 Oktober 2015).
12 Pasal 22 Qanun Nomor 14 Tahun 2003 Tentang Khalwat. 1. Faktor Undang-undang (Qanun) Bila kita mengkaji Qanun Nomor 14 Tahun 2003 tentang khalwat, maka kita akan
menemui suatu kepastian bentuk ‘uqubat bagi mereka yang melanggar ketentuan-ketentuan
khalwat di Aceh. Qanun ini tegas mengtakan bahwa perbuatan khalwat adalah haram,11 dan
barang siapa yang melanggar ketentuan tersebut dapat di kenakan ‘uqubat ta’zir berupa
cambuk paling banyak 9 (sembilan) kali cambukan.12 Begitu juga dengan Qanun Nomor 6
Tahun 2014 tenntang hukum jinayat yang merupa pembaharu terhadap Qanun Nomor 14
Tahun 2003, dikatakan bahwa hukum berkhalwat adalah haram dan setiap orang yang
melanggar ketentuan tersebut di ancam dengan ‘uqubat ta’zir berupa cambuk sebanyak 10
(sepuluh kali) cambukan.13 Sebenarnya kelemahan utama yang terdapat dalam Qanun 14 tahun 2003 adalah
tidak adanya kewenangan yang diberikan kepada wilayatul hisbah (WH)14 untuk
melakukan penahanan. WH tidak berwenang menahan dan memeriksa, sehingga
penahanan yang dilakukan WH merupakan tindakan melanggar ketentuan hukum dan 151 melampaui batas kewenangan. Kewenangan yang dimiliki WH hanya sebatas melakukan
pengawasan terhadap penegakan syariah Islam sekaligus melakukan pembinaan terhadap
para pelanggar syariah Islam yang sedang berlaku di Aceh. Dan tidak ada kewenangan WH
melakukan penahanan terhadap para pelanggar pelaku Syariah di Aceh bila berdasarkan
kepada Qanun Nomor 14 Tahun 2003.15 Apabila WH menangkap pelaku pelanggaran
khalwat akan dilimpahkan perkaranya ke pihak penyidik di kepolisian, dan pihak
kepolisianlah yang berwenang melakukan penahanan terhadap mereka.16 Setelah di sahkannya Qanun Nomor 7 Tahun 2013 Tentang Hukum Acara Jinayat
sepertinya telah memberi angin segar bagi penegakan syariat Islam di Aceh. Hal ini ditandai
dengan penambahan kewenangan yang diberikan kepada Wilayatul Hisbah dalam
melakukan tindakan yang nyata terhadap penegakan Qanun Jinayat di Aceh, dimana pada
qanun-qanu sebelumnya (Qanun 12, 13 dan 14 Tahun 2003) kewenangan WH hanya berupa
pengawsan terhadap pemberlakuan syariat Islam di Aceh. Sebagai mana di jelaskan dalam
Pasal 7 dan 9 Qanun 2013 sebagai berikut: Penyelidik merupakan Pejabat Polri dan PPNS yang telah diberi wewenang oleh
Undang-Undang dan/atau Qanun untuk melakukan penyelidikan (Pasal 6). Dalam Pasal 7 dan pasal 9 ayat (2) dijelaskan: Dalam Pasal 7 dan pasal 9 ayat (2) dijelaskan: (1) Penyelidik sebagaimana dimaksud dalam Pasal 6, karena kewajibannya berwenang:
a. menerima laporan atau pengaduan dari seseorang tentang adanya Jarimah; (1) Penyelidik sebagaimana dimaksud dalam Pasal 6, karena kewajibannya berwenang: a. menerima laporan atau pengaduan dari seseorang tentang adanya Jarimah;
b
i k t
d
b
b kti a. menerima laporan atau pengaduan dari seseorang tentang adanya Jarimah; b. mencari keterangan dan barang bukti; c. p
16 Keputusan Gubernur Aceh No. 01 tahun 2004 tentang kewenangan pembentukan
Organisasi Wilayatul Hisbah. 15 Wawancara dengan Sarkani, Kasi Ketentraman dan Ketertiban Pol PP dan WH Kota
Subulussalam, 23 April 2015. 17 Pasal 10 Qanun Nomor 7 Tahun 2013 Tentang Hukum Acara Jinayat. 1. Faktor Undang-undang (Qanun) menyuruh berhenti seseorang yang dicurigai dan menanyakan serta memeriksa
tanda pengenal diri; dan d. mengadakan tindakan lain menurut hukum secara bertanggung jawab dan
sesuai dengan prinsip-prinsip Syariat Islam. (2) Penyelidik sebagaimana dimaksud dalam Pasal 6, atas perintah penyidik dapat
melakukan tindakan: a. penangkapan, larangan meninggalkan tempat, penggeledahan dan penyitaan;
b. pemeriksaan dan penyitaan surat; a. penangkapan, larangan meninggalkan tempat, penggeledahan dan penyitaan; b. pemeriksaan dan penyitaan surat; c. mengambil sidik jari dan memotret seseorang; dan/atau c. mengambil sidik jari dan memotret seseorang; dan/atau d. membawa dan menghadapkan seseorang kepada penyidik. 15 Wawancara dengan Sarkani, Kasi Ketentraman dan Ketertiban Pol PP dan WH Kota
Subulussalam, 23 April 2015. 16 Keputusan Gubernur Aceh No. 01 tahun 2004 tentang kewenangan pembentukan 152 (3) Penyelidik membuat dan menyampaikan laporan hasil pelaksanaan tindakan
sebagaimana dimaksud pada ayat (1) dan ayat (2) kepada Penyidik. Pasal 9 Ayat (2): (2) Penyidik PPNS sebagaimana dimaksud dalam Pasal 8 ayat (1) huruf b, berwenang: a. menerima laporan atau pengaduan dari seseorang tentang adanya tindak
pidana pelanggaran Qanun dan/atau Peraturan Perundang-undangan lainnya; b. melakukan tindakan pertama di Tempat Kejadian Perkara (TKP) dan melakukan
pemeriksaan; c. menyuruh berhenti seorang Tersangka dan memeriksa tanda pengenal diri
Tersangka; d. melakukan pengeledahan, penangkapan, dan penahanan; e. melakukan penyitaan benda dan/atau surat sesuai dengan Peraturan
Perundang-undangan; f. mengambil sidik jari dan memotret seorang Tersangka; f. mengambil sidik jari dan memotret seorang Tersangka; g. memanggil orang untuk didengar dan diperiksa sebagai Tersangka atau Saksi; h. mendatangkan Saksi ahli yang diperlukan dalam hubungannya dengan
pemeriksaan perkara; i. membuat Surat Perintah Dimulainya Penyidikan (SPDP) kepada Penuntut
Umum dengan tembusan kepada korwas PPNS setelah terlebih dahulu
berkoordinasi dengan korwas; j. melakukan penghentian penyidikan setelah berkoordinasi dengan penuntut
umum bahwa tidak terdapat cukup bukti atau peristiwa tersebut bukan
merupakan tindak pidana dan tembusannya disampaikan kepada Korwas PPNS
dan selanjutnya memberitahukan hal tersebut kepada Tersangka atau kuasanya;
dan/atau k. mengadakan tindakan lain sesuai dengan Peraturan Perundang-undangan. (3) Dalam melaksanakan kewenangan sebagaimana dimaksud pada ayat (1) dan ayat (2)
penyidik wajib menjunjung tinggi nilai-nilai Syariat Islam dan Peraturan Perundang-
undangan. PPNS dalam melaksanakan kewenangan penyelidikan sebagimana dimaksud dalam
Pasal 7 dan Pasal 9 ayat (2) dibantu oleh anggota kepolisian dan polisi WH.17 Inilah yang
menjadi dasar bagi WH dalam melakukan penahan terhadap setiap orang yang melanggar PPNS dalam melaksanakan kewenangan penyelidikan sebagimana dimaksud dalam
Pasal 7 dan Pasal 9 ayat (2) dibantu oleh anggota kepolisian dan polisi WH.17 Inilah yang
menjadi dasar bagi WH dalam melakukan penahan terhadap setiap orang yang melanggar 153 ketentuan-ketentuan qanun jina>ya>t di Aceh, dimana ketentuan seperti ini sebelumya tidak
pernah di atur dalam qanun-qanun jinayat terdahulu (Qanun 12, 13 dan 14 Tahun 2003). Jadi kendala yang selama ini menjadi alasan utama bagi WH dalam menindak pelaku
jinayat di Aceh sudah terjawab dengan disahkannya Qanun No. 7 Tahun 2013 Tentang
Hukum Acara Jinayat. Kendala selanjutnya adalah adanya benturan antara qanun dalam penanganan kasus
khalwat. Permasalahan khalwat di Aceh, selain di atur dalam Qanun Nomor 14 Tahun 2003
dan ditegaskan kemabali dalam Qanun Nomor 6 Tahun 2014 juga di atur dan menjadi salah
satu kewenangan dari peradilan adat di Aceh, hal ini di jelaskan dalam Qanun Nomor 9
Tahun 2008 Tentang Pembinaan Kehidupan Adat dan Adat Istiadat. 18 Wawancara dengan Kalidin, Kasi Penegakan Syariat Islam, WH Kota Subulussalam, 23
April 2015.
19 Putri Sahadat Bancin, Penyelesaian Tindak Pidana Khalwat/Mesum Berdasarkan Qanun Nomor
14 Tahun 2003 (Suatu Penelitian Di Kota Subulussalam), (Banda Aceh: Skripsi Fak. Hukum Univ.
Syiah Kuala, 2015), h. 55. p
19 Putri Sahadat Bancin, Penyelesaian Tindak Pidana Khalwat/Mesum Berdasarkan Qanun
14 Tahun 2003 (Suatu Penelitian Di Kota Subulussalam), (Banda Aceh: Skripsi Fak. Hukum
Syiah Kuala, 2015), h. 55. 18 Wawancara dengan Kalidin, Kasi Penegakan Syariat Islam, WH Kota Subulussa
April 2015. Pasal 9 Ayat (2): Dalam Pasal 13 ayat (1)
dijelaskan bahwa khalwat merupakan salah satu dari 18 kewenangan yang di berikan
kepada mahkamah adat di Aceh. Hal inilah yang menjadi celah permasalahan baru dalam
penerapan Qanun Khalwat di Aceh dan khususnya Kota Subulussalam. Dengan adanya Qanun Nomor 9 Tahun 2008, setiap pelanggaran khalwat yang terjadi
Aceh harus diselesaikan melalui peradilan adat terlebih dahulu. Apabila bisa diselesaikan
dalam peradilan adat, maka pihak berwenang dalam hal ini WH tidak berwenang lagi
untuk menindaklanjuti kasus tersebut.18 Mengenai mekanisme penyelesaian kasus khalwat
di lembaga adat akan disesuaikan dengan kebiasaan adat di wilayah masyarakat setempat,
bararti hukum adat di Aceh tidak memiliki standar tertentu dalam mekasnisme
penyelesaiannya, hal ini dikarenakan masyarakat Aceh yang majemuk dan terdiri dari
beberapa suku yang berbeda.19 Adanya dualisme lembaga dalam peneyelesaian kasus khalwat di Aceh merupakan
kendala tersendiri bagi WH dalam menegakkan Qanu Khalwat. Satu penelitian terkait
dengan khalwat/mesum di kota Lhokseumawe yang dilakukan Abdullah, seorang dosen
Sekolah Tinggi Agama Islam Negeri (STAIN) Lhokseumawe menyebutkan, berkurangnya
penyelesaian kasus khalwat/mesum melalui WH dan mahkamah syar’iyah dikarena
penyelesaian kasus khalwat/mesum telah bergeser. Kasus khalwat/mesum saat ini lebih
mengutamakan penyelesaian secara adat ketimbang kepada jalur WH dan mahkamah
syar’iyah. 154 Hasil penelitian itu telah dipaparkan dalam Aceh Development International
Conference (ADIC) yang berlangsung pada 27 Maret 2013 di Kuala Lumpur. Apa yang
disimpulkan Abdullah, barangkali ada benarnya. Sebab sejak Majelis Adat Aceh20 (MAA)
melakukan sosialisasi penyelesaian kasus masyarakat melalui adat dalam beberapa tahun
terakhir, maka kasus-kasus yang masuk dalam kategori 18 kasus adat sesuai dengan
amanah Qanun Nomor 9 Tahun 2008, bisa diselesaikan melalui mahkamah adat di gampong
ataupun mukim.21 Untuk mengatasi hal tersebut, sudah saatnya para pemangku kepentingan untuk
duduk bersama dan merumuskan mekanisme penyelesaian kasus khalwat di Aceh,
khususnya di Subulussalam. Hal ini penting dilakukan guna mempertegas mengenai
ketentuan-ketentuan khalwat seperti apa yang menjadi wilayah kewenangan masing-masing
lembaga. Karena selama ini penangangan kasus khalwat menurun penyelesaiannya di
tingkat mahkamah syar’iyah. Disamping itu ada yang beranggapan bahwa hukuman22 yang
diberikan lembaga adat terhadap pelaku khalwat di nilai kurang adil, karena bentuk
hukumannya kurang begitu tegas dan tidak menimbulkan efek jera bagi para pelakunya. Di Kota Subulussalam sendiri penyelesaian kasus khalwat di tempuh dengan jalur
kekeluargaan. Sebagaimana yang dijelaskan Kasi Penegak Kebijakan Daearah Dan Syariat
Islam Kota Subulussalam bahwa, setiap pelanggaran kasus khalwat diwilayah hukum Kota
Subulussalam terlebih dahulu dilakukan penyelesaian dengan cara kekeluargaan dan adat
istiadat yang berlaku di Subulussalam. 20 Di Aceh disamping adanya lembaga hukum formal yang menangani pelanggaran syariat
Islam, juga terdapat lembaga informal yaitu lembaga adat yang sangat berpengaruh pada pola
kehidupan masyarakat Aceh. Lembaga adat memiliki landasan hukum yakni Qanun No. 9 Tahun
2008 tentan Pembinaan Kehidupan Adat dan Adat Istiadat serta Qanun Nomor 10 Tahun 2008
tentang Lembaga Adat. p g
(
p
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)
22 Pasal 16 (1) Qanun Nomor 9 Tahun 2008 dijelaskan bahwa jenis-jenis sanksi yang dapat
dijatuhkan dalam penyelesaian sengketa adat sebagai berikut: nasehat; teguran; pernyataan maaf;
sayam; diyat; denda; ganti kerugian; dikucilkan oleh masyarakat gampong atau nama lain;
dikeluarkan dari masyarakat gampong atau nama lain; pencabutan gelar adat; dan bentuk sanksi
lainnya sesuai dengan adat setempat. g
g
21 Teuku Muttaqin Mansur, “Penyelesaian Kasus Mesum Melalui Mahkamah Adat atau
Mahkamah Syar’iyah?”, http://aceh.tribunnews.com/2013/09/04 /peny elesaian-kasus-mesum-
melalui-ma-atau-ms?page3 (dikases pada tanggal 24 Agustus 2015). Pasal 9 Ayat (2): Setelah pihak Satpol PP dan WH memberikan
teguran dan peringatan kepada para pihak yang melanggar qanun tentang khalwat tersebut,
lalu menyerahkannya kepada geucik (kepala desa) untuk diselesaikan secara adat dan aturan
kampong tersebut, setelah dilakukan proses hukum adat tapi belum menghasilkan suatu 155 kesepakatan, baru diselesaikan secara qanun yang berlaku.23 Pada dasarnya penyelesaian
kasus khalwat ditempuh dengan jalan damai dimana kedua keluarga yang tersangka
khalwat/mesum duduk bersama untuk mencari solusi terhadap penyelesaian kasus tersebut
dengan hukumuan yang sering di capai adalah berupa pemberian nasehat, perdamaian,
pernyataan maaf, ganti rugi/denda dan di nikahkan.24 gg
p
24 Wawancara dengan H.M Layari Kombih, selaku Ketua Majelis Adat Aceh (MAA) Kota
Subulussalam, bertempat dikantor MAA Kota Subulussalam pada tanggal 27 April 2015. 23 Wawancara dengan Bapak Kalidin, Kasie Penegak Kebijakan Daearah Dan Syariat Islam Kota
Subulussalam. Kantor Satuan Polisis Pamong Praja dan Wilayatul Hisbah Kota Subulussalam, pada
tanggal 23 April 2015. , J
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,
,
,
26 Wawancara dengan Abdul Malik, selaku Kepala Satpol PP dan WH Kota Subulussalam. p
p
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25 Danial, “Efektifitas ‘Uqūbat Dalam Qanun No. 14 Tahun 2003 dan DQHR Tentang Khalw
khtilath”, Jurnal Asy-Syir’ah Vol. 45, No. I, 2011, h. 1004. 25 Danial, Efektifitas Uqūbat Dalam Qanun No. 14 Tahun 2003 dan DQHR Tentang K
dan Ikhtilath”, Jurnal Asy-Syir’ah Vol. 45, No. I, 2011, h. 1004. tanggal 23 April 2015.
24 Wawancara dengan H.M Layari Kombih, selaku Ketua Majelis Adat Aceh (MAA) Kota
Subulussalam, bertempat dikantor MAA Kota Subulussalam pada tanggal 27 April 2015.
25 Danial “Efektifitas ‘Uqūbat Dalam Qanun No 14 Tahun 2003 dan DQHR Tentang Khalwat 2. Faktor Penegak Hukum Penegak hukum dilakukan oleh institusi yang diberi wewenang untuk itu
(penegakan qanun syariat Islam), dalan hal ini adalah wilayatul hisbah. Sejak hukum itu
mengandung perintah dan pemaksaan, maka sejak itu pula hukum membutuhkan bantuan
untuk mewujudkan perintah tersebut. Hukum menjadi tidak ada artinya, bila perintahnya
tidak dapat dilaksanakan. Karena itu, diperlukan usaha dan tindakan manusia agar perintah
dan paksaan yang secara potensial ada di dalam peraturan itu menjadi nyata dalam praktek
hukum. Donald Black, sebagaimana dikutip Sadjipto Rahardjo mengatakan bahwa dimensi
keterlibatan manusia dalam penegakan hukum tersebut dinamakan dengan mobilisasi
hukum. Dalam mobilisasi hukum inilah manusia turut campur sehingga hukum tidak
hanya mengancam dan berjanji di atas kertas.25 Wilayatul Hisbah dan Sat Pol PP Kota Subulussalam saat ini memiliki 139 orang
anggota dan keseluruhan dari anggota tersebut berpusat di Kantor Pol PP dan WH Kota
Subulussalam di Subulussalam. Adapun status dari 139 orang anggota tersebut, baru 15
orang yang diangkat menjadi pegawai negeri sipil (PNS) dan sisanya masih berstatus
sebagai pegawai honorer.26 Selain itu WH Kota Subulussalam juga belum mempunyai
Penyidik Pegawai Negeri Sipil (PPNS), padahal keberadaan PPNS ini sangat di butuhkan
guna untuk proses penyelidikan dan penyidikan yang dilakukan WH terhadap pelaku
pelanggaran qanun jinayat di Kota Subulussalam. Ketersediaan sumber daya manusia yang memadai sangat mempengaruhi efektivitas
suatu hukum. Berdasarkan keterangan di atas bahwa saat ini Wilayatul Hisbah dan Pol PP
Kota Subulussalam memiliki 139 anggota dimana 15 belas di antaranya adalah PNS, sisanya
pengawai honorer yang kebayakan memiliki latar belakang pendidikan Sekolah Menengah 156 Atas (SMA).27 Pendidikan para penegak hukum ini (WH) sangat vital dalam penegakan
hukum syariah di Aceh, karena hukum yang akan di tegakkan nantinya bukanlah hukum
yang berdasarkan kepada UUD sebaggai landasar utama pembuatan hukumnya, akan
tetapi al-Qur’an dan al-Hadist. Maka untuk itu setiap anggota WH di tuntut untuk memiliki
kecapan dalam bidang hukum Islam (fikih). Inilah yang menjadi permaslahan selama ini,
dimana masyarakat menilai, para penegak hukum syariah di Kota Subulussalam belum
layak untuk dijadikan sebagai penegak qanun jinayat. Hal ini dikarenakan sikap dan
perilaku keseharian anggota WH yang belum mencerminkan nilai-nilai ke-Islaman dalam
kehidupan masyarakat sehari-hari di luar masa tugasnya.28 Memang dalam perekrutan anggota WH selam ini belum ada standar yang khusus
mengenai latar belakang pendidikan bagi anggota WH. 2. Faktor Penegak Hukum Menurut Wildan Sastra selaku
pemerhati syariat Islam dan gender kota Subulussalam, mengkritik bahwa sudah
seharusnya WH Kota Subulussalam membuat standar khusus dalam perekrutan anggota
WH dimana dinai-nilai ke-Islaman harus menjadi standar utama dalam perekerutan
tersebut, sehingga dalam penegakan qanun-qanun syariat anggota WH bisa mengetahui
dengan baik dan benar apa yang menjadi tugasnya dan begitu juga dalam kehidupan
bermasyarakat sehari-hari, anggota WH di harapkan bisa memberikan contoh yang nyata
kepada masyarakat akan perlunya menegakkan syariat Islam di Kota Subulussalam. Wildan
mengatakan, anggota WH seharusnya menjadi garda terdepan dalam penegakan dan
pencotohan syariat Islam bukan malah sebaliknya.29 g
29 Wawancara dengan Wildan Sastra, selaku pemerhati syariat Islam di Kota Subulussalam. 28 Wawancara dengan Arifin Sarbaini, selaku Ketua FPI Kota Subulussalam. 27 Wawancara dengan Bambang Aprianto, Selaku Subag TU Pol PP dan WH Kota
Subulussalam. 3. Faktor Fasilitas Penunjang Penegakan Hukum Keterpusatan WH di Kota Subulussalam sebenarnya menjadi masalah tersendiri
terhadap penegakan qanun jinayat di Subulusslam. Saat ini Kota Subulussalam memiliki 5
kecamatan yang terdiri dari Kecamatan Simpang Kiri, Penanggalan, Sultan Daulad,
Runding dan Longkib. Tiga dari lima kecamatan ini pada dasarnya jauh dari Kantor Pol PP
dan WH Kota Subulussalam yang terletak di pusat Pemerintahan Kota Subulussalam di
Kecamatan Simpang Kiri. Ketiga kecamatan tersebut adalah, Kecamatan Sultan Daulad,
Kecamatan Rundeng dan Kecamatan Longkib. Sedangkan dua lainnya yakni Kecamatan
Simpang Kiri dan Kecamatan Penanggalan relatif berdekatan. Maka tidak heran bila selama
ini kasus yang di tangani WH Kota Subulussalam berasal dari Kecamatan Simpang Kiri dan 157 Kecamatan Penanggalan, karena dua kecamatan inilah yang paling memungkinkan untuk
dilakukan pengontrolan setiap saat, karena letak dan jaraknya dari kantor Pol PP dan WH
tidak terlalu jauh. Keberadaan Kantor WH seharusnya tidak hanya berada di satu titik tertentu, tapi
harus ada di setiap kecamatan-kecamatan di Kota Subulusssalam. Hal ini dilakukan supaya
keberadaan WH dapat dirasakan langsung oleh masyarakat bukan hanya sebagai simbol
dari pelengkap lembaga-lembaga daerah di Aceh. Dalam Keputusan Gubernur No. 1 Tahun
2004 Tentang Pembentukan Organisasi dan Tata Kerja Wilayatul Hisbah disebutkan bahwa
susunan organisasi Wilayatul Hisbah, terdiri atas; Wilayatul Hisbah Tingkat Provinsi;
Wilayatul Hisbah Tingkat Kabupaten/Kota; Wilayatul Hisbah Tingkat Kecamatan, dan
Wilayatul Hisbah Tingkat Kemukiman.30 Berarti pembentukan kantor WH di setiap
kecamatan sangat mungkin untuk di bentuk karena telah mempunyai landasan hukum
yang kuat. Ketika penulis menanyakan hal tersebut, Kepala Satuan Polisi Pamong Praja dan
Wilayatul Hisbah Kota Subulussalam Bapak Abdul Malik mengatakan, hal itu mungkin saja
dilakukan tapi tidak dalam waktu dekat ini, karena menurut Abdul Malik saat ini Pol PP
dan WH masih berfokus kepada kelengkapan fasilitas penunjang lainnya yang menurutnya
sangat belum memadai/ideal untuk sebuah kantor WH tingkat kota di Aceh. Selain itu,
Kantor WH kota Subulussalam sekarang ini juga belum memiliki kantor yang permanen
tapi masih dalam bentuk sewa yang dilakukan oleh Pemerintah Kota Subulussalam. Memang inilah kendala utama mengapa WH kota Subulussalam belum membentuk kantor
WH di tingkat kecamatan, karena semua itu tentunya membutuhkan anggaran yang tidak
sedikit. Alokasi anggaran yang disediakan APBK Kota Subulussalam terhadap Kantor Satpol
PP dan WH memang sangat terbatas. Sebagai gambaran, pada tahun 2013 Pol PP dan WH
mendapat alokasi anggaran sebesar Rp. 4.215.000.000,00 (empat milyar dua ratus lima belas
juta rupiah) sedangkan pada tahun 2014 alokasi anggaran Pol PP dan WH adalah Rp. 30 Hirwan Jack, “Efektivitas Wilayatul Hisbah Dalam Pencegahan Aliran Sesat Di Aceh”,
http://bkpp.acehprov .go.id/ simp egbrr /Art ikel/Artikel05-02-2015/Wilayatul H i s bah _Aceh.pdf
,h. 2. (diakses pada tanggal 16/03/2015). 3. Faktor Fasilitas Penunjang Penegakan Hukum 4.300.000.000,00 (empat milyar tiga ratus juta rupiah) dan pada tahun 2015 alokasi anggaran
yang disediakan adalah Rp. 4.350.000.000,00 (empat milyar tiga ratus lima puluh juta
rupiah). Dengan anggaran tersebut WH dikatakan Abdul Malik belum bisa berbuat banyak
mengenai pengawasan syariat Islam di Kota Subulussalam, belum lagi wilayah 158 subulussalam yang menjadi daerah tugasnya juga luas dan berjauhan antar satu kecamatan
dengan kecamatan yang lainnya. Untuk saat ini Pol PP dan WH Kota Subulussalam
memiliki bebera fasilitas untuk keperluan dinas seperti, satu buah kenderaan mobil patroli,
16 (enam belas) kenderaan sepada motor, 8 (delapan) koputer/laptop.31 Dengan anggaran dan fasilitas seperti itu, bisa di bayangkan bagaimana proses
pengawasan yang di lakukan oleh WH Kota Subulussalam terhadap qanun-qanun syariat
Islam Kota Subulussalam, belum lagi status anggota WH yang mayoritas masih sebagai
pegawai honorer, tentu hal ini juga bisa mempengaruhi konsentrasi mereka dalam
menjalankan tugasnya sehari-hari. Memang dalam hal ini, keseriusan Pemerintahan Kota
Subulussalam
dalam
menerapkan
syariat
Islam
masih
sangat
mungkin
untuk
dipertanyakan, pemerintah seharusnya sepenuh hati dalam menerapkan syariat Islam di
kota ini dengan menyediakan alokasi anggaran untuk WH yang memadai. Maka wajar saja
kalau selama ini banyak kalangan yang menilai proses penerapan syariat Islam di Kota
Subulusssalam masih satgnan dan jalan di tempat. Belum lagi ketiadaan Mahkamah
Syar’iyah Kota Subulussalam, karena saat ini Mahkamah Syar’iyah Kota Subulussalam
masih menginduk kepada Mahkamah Syar’iyah Singkil di Kabupaten Aceh Singkil. Maka wajar-wajar saja jika selama ini kasus khalwat, maisir maupun khamar hanya di
tindak di sekitar wilayah Kecamatan Simpang Kiri dan Penanggalan, karena kedua
kecamatan tersebut merupakan berdekatan dengan kantor WH yang tersentral di Simpang
Kiri Kota Subulussalam. Hal ini dapat di lihat dari data tempat terjadinya pelanggaran
Qanun 12, 13 dan 14 Tahun 2003 yang di miliki oleh Kantor Satpoll PP dan WH Kota
Subulussalam sebagai berkut: 31 Wawancara dengan Bambang Aprianto, Selaku Subag TU Pol PP dan WH Kota
Subulussalam. Tabel Lokasi Tempat Pelanggaran Qanun Nomor 12, 13 dan 14 di Kecamatan Simpang
Kiri dan Penanggalan
31 Wawancara dengan Bambang Aprianto, Selaku Subag TU Pol PP dan WH Kota
Subulussalam. No
Kecamatan
Lokasi
Jenis Kegiatan
1
Simpang Kiri
1. Terminal
Terpadu Kota
Subulussalam
2. Cafe Remang-
Mesum,
Khamar
dan
Maisir No
Kecamatan
Lokasi
Jenis Kegiatan
1
Simpang Kiri
1. Terminal
Terpadu Kota
Subulussalam
2. Cafe Remang-
Mesum,
Khamar
dan
Maisir Jenis Kegiatan 31 Wawancara dengan Bambang Aprianto, Selaku Subag TU Pol PP dan WH Kota
Subulussalam. 31 Wawancara dengan Bambang Aprianto, Selaku Subag TU Pol PP dan WH Kota
Subulussalam. 159 remang
di
Suka
Makmu
dan
Bakal
Buah. 3. Taman
Kota
Subulussalam
di Lae Oram. Penanggalan
1. Hotel Hermes
One
2. Penginapan
Jambu Alas
3. Jalan
Unaya
Penanggalan
4. Cafe
di
Banataran Kali
Lae
Kombih
Gampong
Dasan Raja. 5. Cafe Remang-
remang
di
Cirep-rep
6. Cafe Remang-
remang di Lae
Mbersih. 7. Cafe Remang-
remang
di
Kuta Tengah
8. Cafe Remang-
remang
di
Sikelang. 9. Cafe Remang-
remang
di
Jontor. 10. Warung-
warung
di
Karoke, Mesum, Kahmar
dan Maisir remang
di
Suka
Makmu
dan
Bakal
Buah. 3. Taman
Kota
Subulussalam
di Lae Oram. Penanggalan
1. Hotel Hermes
One
2. Penginapan
Jambu Alas
3. Jalan
Unaya
Penanggalan
4. Cafe
di
Banataran Kali
Lae
Kombih
Gampong
Dasan Raja. 5. Cafe Remang-
remang
di
Cirep-rep
6. Cafe Remang-
remang di Lae
Mbersih. 7. Cafe Remang-
remang
di
Kuta Tengah
8. Cafe Remang-
remang
di
Sikelang. 9. Cafe Remang-
remang
di
Jontor. 10 W
Karoke, Mesum, Kahmar
dan Maisir 2 2 1. Hotel Hermes
One
Karoke, Mesum, Kahmar
dan Maisir 2. Penginapan
Jambu Alas 3. Jalan
Unaya
Penanggalan 4. Cafe
di
Banataran Kali
Lae
Kombih
Gampong
Dasan Raja. 5. Cafe Remang-
remang
di
Cirep-rep 6. Cafe Remang-
remang di Lae
Mbersih. 7. Cafe Remang-
remang
di
Kuta Tengah 8. Cafe Remang-
remang
di
Sikelang. 160 *Berdasarkan data dari Kantor Pol PP dan WH Kota Subulussalam tahun 2015. sekitar
Objek
Wisata
Pemandian air
terjun
Nantampuk
Mas,
SKPC
dan
Lae
Kombih
di
Sikelang. 11. Gang Barto di
Jln. Swadaya
Penanggalan. *Berdasarkan data dari Kantor Pol PP dan WH Kota Subulussalam tahun 2015. 4. Faktor Kesadaran Hukum Masyarakat Yang Masih Rendah Kesadaran hukum masyarakat secara umum masih rendah. Sebagian warga
masyarakat menganggap kewajiban taat pada syariat Islam merupakan beban yang
memberatkan. Perasaan terbebani ini di samping dipengaruhi oleh kondisi sosial ekonomi
yang sulit, juga karena sifat masyarakat lokal berlakunya qanun-qanun jinayat di
Subulussalam. Kondisi sosial ekonomi yang sulit banyak berpengaruh pada terjadinya
tindak pidana perjudian, perdagangan miras, prostitusi dan lain-lain. Keterlibatan masyarakat dalam mengawal Qanun Khalwat sangat mempengaruhi
efektivitas pemeberlakuan qanun tersebut. Dengan segala keterbatsan yang dimiliki oleh
WH Kota Subulussalam, keterlibatan aktif masyarakat sangat di butuhkan, karena
bagaimanpun juga perbuatan pelanggaran qanun khalwat dilakukan dan terjadi di tengah-
tengah kalangan masyarakat Kota Subulussalam. Mengingat pentingnya peran dan
kesadaran hukum masyarakat ini, Qanun Nomor 14 Tahun 2003 jauh-jauh hari telah
memberikan ruang berupa peran aktif masyarakat dalam mengawal pemberlakuan qanun
ini, seperti yang di jelaskan dalam BAB IV Pasal 8-12 sebagai berikut: Masyarakat berperanserta dalam membantu upaya pencegahan dan pemberantasan perbuatan
khalwat/mesum; Masyarakat wajib melapor kepada pejabat yang berwenang baik secara lisan
maupun tulisan apabila mengetahui adanya pelanggaran terhadap larangan khalwat/mesum;
Dalam hal pelaku pelanggaran tertangkap tangan oleh warga masyarakat, maka pelaku beserta
barang bukti segera diserahkan kepada pejabat yang berwenang; Pejabat yang berwenang wajib
memberikan perlindungan dan jaminan keamanan kepada pelapor sebagaimana dimaksud dalam 161 pasal 8 dan/atau orang yang menyerahkan pelaku sebagaimana dimaksud dalam pasal 9; Warga
masyarakat dapat menuntut pejabat yang berwenang sebagaimana dimaksud dalam pasal 9
apabila lalai memberikan perlindungan dan jaminan keamanan bagi pelapor dan/atau orang yang
menyerahkan pelaku; Tata cara penuntutan sebagaimana dimaksud dalam pasal 11 dilakukan
sesuai peraturan perundang-undangan yang berlaku dan diajukan ke mahkamah. Kesadaran dan pengetahuan tentang qanun inilah yang dirasa masih kurang di
tengah-tengah masyarakat. Di beberapa tempat, seperti Sultan Daulad, Runding dan
Penanggalan, ketika penulis berdiskusi dengan masyarakat dan pemuda setempat
mengatakan, tindak pelanggaran terhadap qanun jinayat, khususnya qanun 14/2003
sebenarnya sangat sering terjadi di daerah mereka masing-masing, masyarakat juga sudah
merasa bosan untuk selalu mengingatkan bahwa perbuatan mereka bertentangan dengan
qanun jinayat di Aceh. Pada akhirnya masyarakatpun cuek dan berharap petugas WH
melakukan patroli ke wilayah mereka.32 Sebenarnaya apabila kita memperhatikan Pasal 8-12 Qanun No. 14 Tahun 2003,
seharusnya tidak ada lagi keraguan bagi masyarakat untuk melaporkan setiap orang yang
melanggar ketentuan Qanun 14/2003. Karena dalam Pasal 10 dijelaskan pejabat yang
berwenang wajib memberikan perlindungan dan jaminan keamanan kepada pelapor
sebagaimana dimaksud dalam Pasal 8 dan/atau orang yang menyerahkan pelaku
sebagaimana dimaksud dalam Pasal 9. g
(
)
g
p
g
g
(
)
33 Dinas Syariat Islam adalah perangkat daerah sebagai unsur pelaksana syariat Islam di
lingkungan Pemerintah Daerah Aceh yang berada di bawah Gubernur. Pembentukan Dinas Syariat
Islam di Aceh ini merupakan suatu bentuk tindak lanjut terhadap UU No. 44 tahun 1999 tentang
Penyelenggaraan Keistimewaan Daerah Istimewa Aceh, untuk meningkatkan kelancaran
pelaksanaan tugas operasional Pemerintah Provinsi Daerah Istimewa Aceh di bidang pelaksanaan 32 Wawancara dengan S (nama disamarkan) Warga Gampong Sikelang Kecamatan
Penanggalan. HL (nama disamarkan) warga Gampong Rindeng, Kecamatan Rundeng. Wawancara
dengan HM (nama disamarkan) warga Gampong Sigrun, Kec. Sultan Daulad. (27 Mei 2015). syariat Islam yang lebih efisien dan berolehguna, maka dipandang sangat perlu untuk membentuk
susunan organisasi dan tata kerja Dinas Syariat Islam Provinsi Daerah Istimewa Aceh yang sesuai
dengan karakteristik, potensi dan kebutuhan daerah. Lihat, Konsideran Peraturan Daerah Propinsi
Daerah Istimewa Aceh No. 33 Tahun 2001 Tentang Pembentukan Susunan Organsasi Dan Tata Kerja Dinas
Syariat Islam Propinsi Daerah Istimewa Aceh, Lembaran Daerah Propinsi Daerah Istimewa Aceh Tahun
2001 No. 65. 34 Wawancara dengan Hotma Capah, selaku Sekretaris DSI Kota Subulussalam. 21 April 2015.
35 Hukum Online, “Fiksi Hukum Harus Didukung Sosialisasi Hukum”, http://www.
hukumonline.com/berita/baca/hol19115/fiksi-hukum-harus-didukung (diakses pada tanggal 25
Nov. 2015). 34 Wawancara dengan Hotma Capah, selaku Sekretaris DSI Kota Subulussalam. 21 April 2015 37 Pasal 13 Qanun Nomor 14 Tahun 2003 Tentang Khalwat. g
38 Pasal 13 Qanun Nomor 14 Tahun 2003 Tentang Khalwat. 4. Faktor Kesadaran Hukum Masyarakat Yang Masih Rendah Tapi permasalahannya kembali kepada kepedulian
dan kesadaran masyarakat juga, apakah masyarakat sudah mengetahui mengenai
peransertanya dalam penegakan qanun-qanun syariah di Aceh. Kesadaran hukum masyarakat tidak dapat dipisahkan dari intensitas disseminasi dan
penyuluhan yang dilakukan para penyelenggara pemerintah daerah (Aceh) kepada
masyarakat. Setiap penyelenggara negara berkewajiban memberikan penyuluhan hukum
sebagai bagian dari proses edukasi dan pembudayaan hukum. Penyuluhan hukum
merupakan tanggung jawab setiap penyelenggara negara. Kesadaran hukum masyarakat ini
sebenarnaya bisa dibagun melalui bentuk/kegiatan sosialisi tentang qanun-qanun jinayat
khususnya Qanun Nomor 14 Tahun 2003. Peran Kantor Dinas Syariat Islam33 (DSI) Kota 162 Subulussalam dalam hal ini sangat di harapkan kontribusinya. Hotma Capah selaku
Sekretasis DSI Kota Subulussalam menjelaskan bahwa, selama ini DSI telah berupa
semaksimal mungkin dalam mensosialisasikan qanun syariat Islam di Aceh, mulai dari
remaja-remaja masjid, tokoh masyarakat dan sekolah-sekolah. DSI Kota Subulussalam juga
menghimbau kepada seluruh da’i perbatasan yang ada di gampong-gampong untuk ikut
mensosialisasikan qanun-qanun syariat Islam di Aceh.34 Lain dengan keterangan pemerintah (DSI), masyarakat menilai bahwa sosialisasi
yang dilakukan DSI sekarang ini masih disara sangat kurang. DSI Kota Subulussalam di
nilai terkesan setengah hati dalam melakukan sosialisasi kepada masyarakat, terlebih-lebih
Parlemen Aceh telah mengesahkan Qanun No 6/2014 tentang Hukum Jinayat pada 27
September 2014, lalu dimasukkan ke dalam lembaran daerah pada 23 Oktober 2014. Qanun
itu baru bisa berlaku setelah setahun sejak pengesahan. Pemerintah diamanatkan untuk
mensosialiasikan keberadaan Qanun Pidana itu ke segala lapisan masyarakat. DSI Kota
Subulussalam dituntut untuk lebih giat lagi dalam melakukan sosialisasi semua qanun
syariat Islam di Kota Subulussalah, sehingga asas fiksi hukum yang menganggap semua
orang tahu hukum (presumptio iures de iure). Semua orang dianggap tahu hukum, tak
terkecuali petani yang tak lulus sekolah dasar, atau warga yang tinggal di pedalaman. Dalam bahasa latin dikenal pula adagium ignorantia jurist non excusat, ketidaktahuan hukum
tidak bisa dimaafkan. Seseorang tidak bisa mengelak dari jeratan hukum dengan berdalih
belum atau tidak mengetahui adanya hukum dan peraturan perundang-undangan
tertentu,35 dapat dijalankan ditengah-tegah masyarakat Kota ubulussalam. Dibutuhkan
kesadaran yang tinggi dari masyarakat Kota Subulussalam untuk melaksanakan Syariat
Islam, disamping itu juga harus adanya political will pemerintah dan peran serta stakeholders
dan tentunya dilakukan secara bertahap atau gradual. Nov. 2015). 163 36 Lihat Bab IV Pasal 8, 9 dan 10 Qanun Nomor 14 Tahun 2003. 36 Lihat Bab IV Pasal 8, 9 dan 10 Qanun Nomor 14 Tahun 2003. A. Pengawasan dan Pembinaan Terhadap Qanun Nomor 14 Tahun 2003 Untuk meningkatkan efektivitas menerapan qanun ini, peran serta masyarakat sangat
dibutuhkan, sama halnya dengan Qanun 12, 13 Tahun 2003 dimana masyarakat diberi peran
dalam membantu upaya pencegahan dan pemberantasan perbuatan khalwat dengan
kewajiban melapor kepada pejabat yang berwenang baik lisan maupun tulisan apabila
masyarakat mengetahui adanya orang atau kelompok masyarakat yang melakukan
pelanggaran terhadap larangan khalwat/mesum di Aceh. Guna untuk menjamin keamanan
masyarakat yang melaporkan tindakan perlanggaran terhadap qanun khalwat/mesum,
pejabat bewenang wajib memberikan perlindungan dan jaminan keamanan kepada
masyarakat yang melapor.36 Mengenai pengwasan dan pembinaan, penyidikan dan penuntutan sebagai mana
tertera dalam bab V dan Bab VI Qanun Nomor 14 Tahun 2003 memiliki kandungan yang
sama seperti dua qanun sebelumnya. Untuk melakukan pengawasan dan pembinaan
terhadap pelaksanaan qanun ini, Gubernur, Bupati/Walikota membentuk Wilayatul
Hisbah; Susunan dan kedudukan Wilayatul Hisbah diatur lebih lanjut dengan surat
Keputusan Gubernur dan/atau Bupati/Walikota setelah mendengar pendapat Majelis
Permusyawaratan Ulama.37 Dalam
melaksanakan
fungsi
pengawasannya,
Pejabat
Wilayatul
Hisbah
sebagaimana dimaksud dalam pasal 13 bila menemukan pelaku pelanggaran terhadap
larangan sebagaimana dimaksud dalam pasal 5 dan 6, menyampaikan laporan secara
tertulis kepada penyidik; Dalam melaksanakan fungsi pembinaannya, Pejabat Wilayatul
Hisbah yang menemukan pelaku jarimah khalwat/mesum dapat memberi peringatan dan
pembinaan terlebih dahulu kepada pelaku sebelum menyerahkannya kepada penyidik;
Pejabat Wilayatul Hisbah wajib menyampaikan laporan kepada penyidik tentang telah
dilakukan peringatan dan pembinaan sebagaimana dimaksud dalam ayat (2).38 Menurut hemat penulis, pengawasan terhadap qanun pidana di Kota Subulussalam
baik khamar, masir maupun khalwat selama ini memang belum begitu berjalan dengan baik. Hal ini bukan semata menajdi salah dari pemerintah saja, tetapi semua aspek tatanan
kehidupan sosial kemasyarakatan di Kota Subulussalam, baik pemerintah maupun
masyarakatnya. Kelemahan dalam melaukan penegakan dan pengawasan yang dilakukan
pihak berwenag selama ini, seperti yang telah teridentifikasi di atas seperti keterbatasan 164 anggota WH, sarana dan fasilitas, anggaran dan lainnya sebenarnya bisa disiasati dengan
melibatkan peran serta masyarakat dalam menanggulangi terjadinya perbuatan khalwat
(mesum) di Kota Subulussalam. Peran masyarakat inilah yang dirasakan masih kurang
selama ini dalam proses penegakan qanun-qanun jinayat di Kota Subulussalam.39 Untuk
meningkatkan pengawasan terhadap pemberlakuan Qanun Nomor 14 Tahun 2003 di Kota
Subulussalam, ada beberapa hal yang harus di perhatikan pemerintah sebagai berikut: 1. Melibatkan Peran Aktif Masyarakat di Gampong-gampong Peran aktif masyarakat memang sangat di harapkan dalam penerapan setiap qanun
di Aceh, begitu juga halnya dengan qanun khalwat yang diatur dalam Qanun 14 Tahun 2003. Pemerintah dalam hal ini Dinas Syariat Islam dan Wilayatul Hisbah sudah saatnya
meningkatkan hubungan dengan masyarakat dan khususnya para tokoh masyarakat di
tiap-tiap gampong yang ada di Kota Subulussalam dan menjadikan mereka menjadi salah
unsur pengawas utama setiap qanun di gampongnya masing-masing. Kenapa harus
menjadikan tokoh masyarakat gampong menjadi pengawas utama dalam penegakan Qanun
Nomor 14/2003? Hal ini dikarenakan para tokoh masyarakat gampong pada dasarnya
sangat disegani dan para pelaku khalwat diharapakan berpikir berulangkali ketika hendak
melakukan perbuatan yang bertentangan dengan ketentuan Qanun Nomor 14 Tahun 2003. Jika tiap-tiap gampong telah diterapkan sistem pengawasan yang seperti itu, maka prilaku
menyimpang masyarakat khususnya khalwat dapat di tekan ketitik yang paling rendah. Selama ini peran masyarakat bukan berarti tidak ada, hanya saja peran masyarakat
sangat sulit diwujudkan, karena tidak memiliki ruang yang legal dan tidak memiliki
wewenang formal yang dapat dipertanggung jawabkan.40 Dengan demikian apabila ada
kontrak kerja antara pemerintah dan tokoh masyarakat gampong, maka di harapkan peran
masyarakat tersebut dapat teraktualisasi di lapangan. 39 Wawancara dengan Ust. H. Qaharuddin Kombih, selaku Ketua MPU Kota Subulussalam
(20 April 2015).
b b k
d
“
d
b
h l
d l 40 Abubakar dan Anwar, “Strategi dan Hambatan Penerapan Qanun Khalwat dalam
Pencegahan Prilaku Khalwat pada Remaja Kota Banda Aceh”, JURNAL PENDIDIKAN SERAMBI
ILMU, 2011.68-90. 39 Wawancara dengan Ust. H. Qaharuddin Kombih, selaku Ketua MPU Kota Subulussalam
(20 April 2015).
40 Abubakar dan Anwar, “Strategi dan Hambatan Penerapan Qanun Khalwat dalam
Pencegahan Prilaku Khalwat pada Remaja Kota Banda Aceh”, JURNAL PENDIDIKAN SERAMBI
ILMU, 2011.68-90. 2. Megaktifkan Remaja Masjid Masa remaja adalah masa yang paling rentan, karena pada masa-masa seperti ini
anak yang baru menuju kepada dewasa sering mencari tau tentang jati dirinya di tengah-
tengah masyarakat. Maka untuk itu perlu adanya suatu konsep dan sistem yang 165 memusatkan pencegahan penyimpangan syariat Islam pada remaja. Remaja masjid
dipandang sebagai suatu sarana/sistem dalam membentuk karakter para para remaja,
sehingga dengan adanya kegiatan keremajaan di masjid di harapkan dapat memberi
pengetahuan yang baik tentang keislaman itu sendiri kepada setiap remaja. 4. Membuat Pos Pengamanan WH Seperti yang telah dijelaskan pada bab sebelumnya, bahwa salah satu kelemahan
WH dalam mengawasi pemberlakuan Qanun 14/2003 adalah dikarenakan keterpusatan
kantor WH yang hanya ada di pusat kota Subulussalam, walupun sebenarnya idealnya
kantor WH ada di setiap kecamatan di Kota Subulussalam, tapi karena angggaran yang
terbatas, maka untuk saat ini pemerintah seharusnya membentuk pos-pos pengawasan WH
di setiap kecamatan, mengingat antar kecamatan di Kota Subulussalam sangat berjauhan
secara geografis dan hal ini dianggap sangat efektif karena anggaran yang dibutuhkan juga
tidak terlalu besar. 3. Kerjasama Dengan Pengusaha Seperti Hotel dan Cafe Perbuatan khalwat yang terjadi di Kota Subulussalam kebanyakan terjadi di Hotel
(penginapan) dan cafe-cafe yang tersebar di Kota Subulussalam, seperti contoh kasus bahwa
pelaku khalwat di tangkap di sebuah penginapan di kawasan Penanggalan dan di salah satu
cafe di bantaran Lae Kombih. Maka untuk itu pemerintah seharusnya menjalin kerjasama
dengan para pengusaha tersebut supaya tidak memberikan fasilitas kepada mereka yang
melakukan perbuatan yang bertentangan dengan Qanun Aceh. C. Kesimpulan Dari penjelasan di atas dapat diambil satu kesimpulan besar bahwa banyak faktor
yang melatarbelakangi kurang efektifnya pemberlakuan qanun jinayat di Kota
Subulussalam khususnya terhadap Qanun No. 14 Tahun 2003 tentang khalwat, baik dari
segi faktor hukum maupun faktor non-hukum. Kedua faktor tersebut tentu saling bertalian
satu sama lain yang diharapkan dapat segera meneukan formulasi yang tepat untuk
mengatasi masalah-maslaah tersebut. Mengenai adanya dualisme dalam penanganan kasus khalwat di Aceh memang
menjadi polemik tersendiri bagi para penegak qanun syariah di Aceh (wilayatul hisbah). Sudah seharusnya para pemangku kepentingan duduk bersama untuk menentukan batasan
dan kadar khalwat seperti apa dan bagaimana yang dapat diselesaikan melalui Mahkamah 166 Adat Aceh dan Mahkamah Syar’iyah. Seperi yang sudah dijelaskan di atas bahwa tindak
pidana khalwat (mesum) tidak semuanya bisa dilimpahkan ke mahkamah syar’iyah. Ada
beberapa faktor yang mempengaruhinya, antara lain: Adat Aceh dan Mahkamah Syar’iyah. Seperi yang sudah dijelaskan di atas bahwa tindak
pidana khalwat (mesum) tidak semuanya bisa dilimpahkan ke mahkamah syar’iyah. Ada
beberapa faktor yang mempengaruhinya, antara lain: 1. Karena proses hukumnya di selesaikan secara hukum adat berdasarkan
kekeluargaan dengan cara menikahkan pelaku khalwat (mesum), membayar denda
dan perdamaian; 2. Karena adanya tuntutan dari masyarakat untuk diselesaikan secara damai melalui
hukum yang berlaku dalam masyarakat dengan cara mendidik dan membimbing
masyarakat yang telah berbuat khalwat (mesum); 3. Karena perbuatan khalwat (mesum) yang dilakukan hanya bersifat ringan dan
sedang, kecuali khalwat (mesum) yang sifatnya berat. 3. Karena perbuatan khalwat (mesum) yang dilakukan hanya bersifat ringan dan
sedang, kecuali khalwat (mesum) yang sifatnya berat. Di Aceh disamping adanya lembaga hukum formal yang menangani pelanggaran
syariat Islam, juga terdapat lembaga informal yaitu lembaga adat yang sangat berpengaruh
pada pola kehidupan masyarakat Aceh. Lembaga adat memiliki landasan hukum yakni
Qanun No. 9 Tahun 2008 tentan Pembinaan Kehidupan Adat dan Adat Istiadat serta Qanun
Nomor 10 Tahun 2008 tentang Lembaga Adat. Dengan demikian, hukum adat yang berlaku
dalam masyrakat merupakan salah satu hukum yang membentengi hukum-hukum yang
lainnya. Walaupun hukum adat tidak betentangan dengan hukum-hukum atau peraturan
perundang-rundangan yang lain yang dibuat oleh pemeintah, karena tujuan hukum adat
mendidik masyarakat dengan jalan menasehati dan membimbing masyarakat untuk
kembali kepada jalan yang benar, tapi pada kenyataannya hukum adat sedikit banyaknya
telah mempengaruhi proses penerapan hukum Islam di Aceh seperti halnya kasus khalwat. li, Jainuddin. Sosiologi Hukum. Jakarta: Sinar Grafika, 2007. D. PUSTAKA RUJUKAN Abu Bakar, Al Yasa’. ”Islam, Hukam dan Masyarakat di Aceh Tajdid Syariat dalam Negara
Bangsa”, First International Conference of Aceh and Indian Ocean Studies, 24-27
Februari 2007. Abu Bakar, Al Yasa’. ”Islam, Hukam dan Masyarakat di Aceh Tajdid Syariat dalam Negara
Bangsa”, First International Conference of Aceh and Indian Ocean Studies, 24-27
Februari 2007. Abu Bakar, Al Yasa’. Wilayatul Hisbah, Polisi Pamong Praja Dengan Kewenangan Khusus di
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Pencegahan Prilaku Khalwat pada Remaja Kota Banda Aceh”. JURNAL
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(diakses
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Grafindo Persada, 2008. 169
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English
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Microphthalmia in Texel Sheep Is Associated with a Missense Mutation in the Paired-Like Homeodomain 3 (PITX3) Gene
|
PloS one
| 2,010
|
cc-by
| 8,280
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Abstract The funders had no role in study design, data collection and analysis, decision to publish, or preparation of
the manuscript. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Cord.Droegemueller@itz.unibe.ch * E-mail: Cord.Droegemueller@itz.unibe.ch . These authors contributed equally to the work. . These authors contributed equally to the work. final shape of the eye [1,3]. Genetic studies have identified some
of the critical determinants of eye formation. A set of putative
transcription factors required for the earliest step of eye
development were identified in Drosophila. The involvement of
homologous
proteins
in
vertebrate
lens
development
was
subsequently elucidated by the characterization of mutations
that cause congenital human or murine ocular disorders and
their comparison to mutations in model organisms [1]. Analyzing
inherited isolated microphthalmia/anophthalmia in humans
revealed a total of eight genes (SOX2, PAX6, OTX2, RAX,
CHX10, FOXE3, PITX3, CRYBA4) carrying causative mutations
[4–11]. For some human non-syndromic microphthalmia cases
the underlying mutation has not yet been found [2]. The role of
the eight genes during lens development was confirmed by
studying spontaneous mouse mutants and genetically engineered
mice with more or less similar ocular phenotypes as in human
[12]. Besides CRYBA4, encoding a lens specific structural protein,
seven of these genes encode transcription factors which are
required for appropriate lens formation during eye development
[1]. Microphthalmia in Texel Sheep Is Associated with a
Missense Mutation in the Paired-Like Homeodomain 3
(PITX3) Gene Doreen Becker1., Jens Tetens2., Adrian Brunner1., Daniela Bu¨ rstel3, Martin Ganter3, James Kijas4 for the
International Sheep Genomics Consortium, Cord Dro¨ gemu¨ ller1* 1 Institute of Genetics, Vetsuisse Faculty, University of Berne, Berne, Switzerland, 2 Institute for Animal Breeding and Husbandry, Christian-Albrechts-University Kiel, Kiel,
Germany, 3 Clinic for Pigs and Small Ruminants, Forensic Medicine and Ambulatory Service Small Animals, University of Veterinary Medicine Hannover, Hannover,
Germany, 4 Commonwealth Scientific and Industrial Research Organisation Livestock Industries, St Lucia, Brisbane, Queensland, Australia Abstract Microphthalmia in sheep is an autosomal recessive inherited congenital anomaly found within the Texel breed. It is
characterized by extremely small or absent eyes and affected lambs are absolutely blind. For the first time, we use a
genome-wide ovine SNP array for positional cloning of a Mendelian trait in sheep. Genotyping 23 cases and 23 controls
using Illumina’s OvineSNP50 BeadChip allowed us to localize the causative mutation for microphthalmia to a 2.4 Mb interval
on sheep chromosome 22 by association and homozygosity mapping. The PITX3 gene is located within this interval and
encodes a homeodomain-containing transcription factor involved in vertebrate lens formation. An abnormal development
of the lens vesicle was shown to be the primary event in ovine microphthalmia. Therefore, we considered PITX3 a positional
and functional candidate gene. An ovine BAC clone was sequenced, and after full-length cDNA cloning the PITX3 gene was
annotated. Here we show that the ovine microphthalmia phenotype is perfectly associated with a missense mutation
(c.338G.C, p.R113P) in the evolutionary conserved homeodomain of PITX3. Selection against this candidate causative
mutation can now be used to eliminate microphthalmia from Texel sheep in production systems. Furthermore, the
identification of a naturally occurring PITX3 mutation offers the opportunity to use the Texel as a genetically characterized
large animal model for human microphthalmia. Citation: Becker D, Tetens J, Brunner A, Bu¨rstel D, Ganter M, et al. (2010) Microphthalmia in Texel Sheep Is Associated with a Missense Mutation in the Paired-Like
Homeodomain 3 (PITX3) Gene. PLoS ONE 5(1): e8689. doi:10.1371/journal.pone.0008689 Editor: Alfred Lewin, University of Florida, United States of America Received August 19, 2009; Accepted December 14, 2009; Published January 13, 2010 Received August 19, 2009; Accepted December 14, 2009; Published January 13, 2010 Copyright: 2010 Becker et al. This is an open-access article distributed under the terms of the Creative Commons Attributi
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ker et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
tion, and reproduction in any medium, provided the original author and source are credited. Funding: This study was financed by research grants of the Swiss National Science Foundation (SNSF), Berne, Switzerland (grant no. 3100A0-116247), and the
H.W. Schaumann Stiftung, Hamburg, Germany, to CD. PLoS ONE | www.plosone.org January 2010 | Volume 5 | Issue 1 | e8689 Introduction Human microphthalmia, characterized by small eyes and
other ocular abnormalities in newborns, is highly variable with
the most severe cases anophthalmic [1,2]. Anophthalmia and
microphthalmia cause congenital blindness and affect up to 30
per 100,000 people worldwide [2]. Both anophthalmia and
microphthalmia may occur in isolation or as part of a syndrome,
as in one-third of cases [2]. Morphological studies showed
that impaired lens formation seems to be the major cause of
anophthalmia and microphthalmia, although the precise path-
ogenesis of these phenotypes remains unknown [3]. Lens
development is a critical embryonic period in vertebrate eye
development during which many inductive signals are exchanged
between the optic vesicle and surface ectoderm [1,3]. This stage
is characterized by formation of the lens placode, a thickening of
the surface ectoderm that comes into contact with the optic
vesicle [1,3]. Coordinated invagination of the lens placode and
the optic vesicle results in the formation of the lens vesicle and a
double-layered optic cup and provides the first indication of the PLoS ONE | www.plosone.o January 2010 | Volume 5 | Issue 1 | e8689 January 2010 | Volume 5 | Issue 1 | e8689 1 PLoS ONE | www.plosone.org PLoS ONE | www.plosone.org PITX3 Mutation in Sheep Isolated congenital microphthalmia occurs in various mamma-
lian species [1,3,13] including the Texel breed of sheep (Figure 1)
[14–20]. In Texel sheep microphthalmia behaves as a monogenic
autosomal recessive trait [16,20]. An abnormal development of the
lens vesicle was shown to be the primary event [19], but so far the
underlying genetic defect has not been elucidated. In an initial
analysis, we performed a partial genome scan and observed
genetic linkage to microsatellite markers on sheep chromosome 23
[21]. After significant extension of the available material we were
not able to confirm these results. from our experimentally established pedigree [20,21]. Parents of
affected offspring were classified as obligate carriers (n = 133). Partial pedigree records were available and allowed us to establish
73
two-generation
spanning
sheep
families
segregating
for
microphthalmia containing 254 of the individuals collected (Figure
S1). Due to incomplete pedigree records, it was impossible to
determine if affected sheep from the Texel breeding population
share common ancestors and trace back to a single common
founder. Besides the experimental mating of an affected male to
known disease carriers [20,21], the parents of all available cases
were healthy (Figure S1). Sample Collection We collected samples from 134 microphthalmia affected lambs
and 269 unaffected sheep from different sheep farms with Texel
purebred or Texel/Whiteheaded mutton crossbred sheep, and Figure 1. Microphthalmia phenotype in Texel sheep. (A) Normal
newborn lamb. (B) Microphthalmia affected lamb. doi:10.1371/journal.pone.0008689.g001 Subsequently, we applied a homozygosity mapping approach to
narrow the region containing the microphthalmia mutation. Based
on the reported occurrence of microphthalmia some generations
after the introgression of Texel sheep from the Netherlands we
hypothesized that the affected sheep most likely were inbred to one
single founder animal. Under this scenario the affected lambs were
expected to be identical by descent (IBD) for the causative
mutation and flanking chromosomal segments. We analyzed
the cases for extended regions of homozygosity with simultaneous
allele sharing. Only one genome region fulfilled our search
criteria (Table S1). On OAR 22 all 23 affected genotyped sheep
were homozygous and shared identical alleles over 39 SNP
markers corresponding to a 2.4 Mb interval from 24.5–26.9 Mb
(Figure 2C). In order to further examine the critical interval defined using
SNP data, we genotyped three microsatellite markers derived from
the surrounding virtual genome sequence of OAR 22 (Table S2). The analysis of the microsatellite genotypes confirmed an
increased homozygosity within the microphthalmia affected lambs
compared to the controls. The observed microsatellite heterozy-
gosity in the cases ranged from 3–52% compared to 84–92% in
the controls (Table S2). A total of 130 out of 134 microphthalmia
affected lambs showed homozygosity at microsatellite INRA81
located at 24.9 Mb on the virtual genome map of OAR 22 (Table
S1). The obtained LOD score of 10.5 conclusively confirmed the
linkage of microphthalmia to the candidate gene region in the
family material. Mapping of the Causative Mutation We genotyped approximately 50’000 evenly spaced SNPs
from 23 microphthalmia affected lambs and 23 control sheep. A
genome-wide significant association was shown for SNPs on sheep
chromosome 22 (OAR 22) (Figure 2A). Of the remaining 44,865
SNPs with a genotyping rate .99% and minor allele frequency
.5%, eights SNPs, over the region 24,529,089 to 28,147,610 on
OAR 22, showed strongest association with the microphthalmia
phenotype, with a genome-wide corrected p,0.01 (Figure 2B). No
other region in the genome showed genome-wide associated SNPs. The OAR 22 SNP at position 24,952,721 showed strongest
association with the microphthalmia phenotype, with an asymp-
totic raw p of 7.6610211 and a genome-wide corrected of
9.961025. Introduction The pedigrees were consistent with a
monogenic autosomal recessive inheritance. Therefore,
we
hypothesized
that
a
more
comprehensive
genome-wide mapping strategy may lead to the identification of
the microphthalmia locus in Texel sheep. The ability to assay
50,000 evenly spaced SNP across the sheep genome was recently
made
possible
by
development
of
Illumina’s
OvineSNP50
BeadChip. This study demonstrates the effectiveness of using this
SNP array for the finescale mapping of an inherited defect when
testing only a modest number of cases and controls. Identification and Characterization of Functional
Candidate Genes As the resolution of the virtual sheep genome annotation is
still far from perfect [22], we inferred the gene annotation of
the mapped interval from the corresponding human interval
(Figure 2D). The sheep microphthalmia interval corresponds to a
segment from 102.2–104.8 Mb on HSA 10. This human interval
contains 46 annotated genes and 5 hypothetical loci (NCBI
MapViewer, build 36.3). A careful inspection of these genes and Figure 1. Microphthalmia phenotype in Texel sheep. (A) Normal
newborn lamb. (B) Microphthalmia affected lamb. doi:10.1371/journal.pone.0008689.g001 January 2010 | Volume 5 | Issue 1 | e8689 PLoS ONE | www.plosone.org 2 PITX3 Mutation in Sheep Figure 2 Genome wide association mapping of microphthalmia (A) Case control whole genome association analysis finds si
PITX3 Mutation Figure 2. Genome-wide association mapping of microphthalmia. (A) Case-control whole genome association analysis finds significa
association to SNPs on chromosome 22. (B) Single SNP association statistic across OAR 22. (C) Homozygosity mapping of the microphthalm
mutation. The analysis of SNP genotypes from affected sheep indicated that they had extended overlapping homozygous regions on OAR 2
(indicated as black blocks). Thus – assuming that it resides on the common haplotype block – the causative mutation is located within a 2.4 M
interval on OAR 22 (indicated as red box). All 23 affected sheep had homozygous identical by state intervals with shared alleles between 24.5 Mb an
26.9 Mb. (D) Gene content of the corresponding human chromosome 10 segment. doi:10.1371/journal.pone.0008689.g002
PL S ONE |
l
3
J
2010 | V l
5 | I
1 |
868 Figure 2. Genome-wide association mapping of microphthalmia. (A) Case-control whole genome association analysis finds significant
association to SNPs on chromosome 22. (B) Single SNP association statistic across OAR 22. (C) Homozygosity mapping of the microphthalmia
mutation. The analysis of SNP genotypes from affected sheep indicated that they had extended overlapping homozygous regions on OAR 22
(indicated as black blocks). Thus – assuming that it resides on the common haplotype block – the causative mutation is located within a 2.4 Mb
interval on OAR 22 (indicated as red box). All 23 affected sheep had homozygous identical by state intervals with shared alleles between 24.5 Mb and
26.9 Mb. (D) Gene content of the corresponding human chromosome 10 segment. doi:10.1371/journal.pone.0008689.g002 Figure 2. Genome-wide association mapping of microphthalmia. (A) Case-control whole genome association analysis finds significant
association to SNPs on chromosome 22. Identification and Characterization of Functional
Candidate Genes (B) Single SNP association statistic across OAR 22. (C) Homozygosity mapping of the microphthalmia
mutation. The analysis of SNP genotypes from affected sheep indicated that they had extended overlapping homozygous regions on OAR 22
(indicated as black blocks). Thus – assuming that it resides on the common haplotype block – the causative mutation is located within a 2.4 Mb
interval on OAR 22 (indicated as red box). All 23 affected sheep had homozygous identical by state intervals with shared alleles between 24.5 Mb and
26.9 Mb. (D) Gene content of the corresponding human chromosome 10 segment. doi:10.1371/journal.pone.0008689.g002 January 2010 | Volume 5 | Issue 1 | e8689 PLoS ONE | www.plosone.org 3 PITX3 Mutation in Sheep database searches of their presumed function revealed PAX2 and
PITX3 as possible functional candidate genes within the critical
interval at 102.5 Mb and 103.9 Mb on HSA 10, respectively. The
paired box gene 2 (PAX2) is a transcription factor expressed during
embryonic eye development and mutations in the human and
mouse homologs are accompanied by eye malformations [1]. PITX3 encodes the paired-like homeodomain 3 (PITX3) tran-
scription factor, expressed during early vertebrate lens develop-
ment [23]. Deletions in the promoter of this gene cause abnormal
lens development in the aphakia mouse mutant, which has only
rudimentary lenses [24,25]. An abnormal development of the lens
vesicle was shown to be the primary event in ovine microphthal-
mia as well [19]. Therefore, we initially investigated whether
mutations in the ovine PITX3 gene might be responsible for the
microphthalmia phenotype. As neither mRNA nor genomic
sequence data of ovine PITX3 was publicly available, we
determined the complete sequence from an ovine BAC clone
containing the PITX3 gene. In order to further evaluate PITX3 as
a positional candidate, we analyzed its expression in head tissue of
d30 sheep fetuses because PITX3 shows a lens specific expression
in human, mice, and cattle embryos, respectively. We detected
three transcripts, which were verified by direct DNA-sequencing
of the RT-PCR products. Full-length PITX3 cDNAs obtained by
RACE differed in their 59 untranslated regions, whereas they
shared a common open reading frame (Figure 3A). These cDNA
sequences were used for comparison with the genomic sequence. These analyses indicated that the ovine PITX3 gene consists of five
exons separated by four introns. Identification and Characterization of Functional
Candidate Genes In accordance to the human gene
annotation, we numbered the two alternatively spliced 59 exons 1
and 1A and the three coding exons 2–4 (Figure 3A). All splice
donor/splice acceptor sites conform to the GT/AG rule. The
experimentally verified existence of the alternatively spliced exon
1A is in agreement with an identified cattle 59-EST sequence
(GenBank: EG705801). The coding sequence of ovine PITX3
displays 97%, 90% and 88% similarity to the bovine, human and
murine homologue, respectively. Ovine PITX3 encodes a protein
of 302 amino acids containing a DNA binding homeobox domain
and, like in other paired-like homeodomain containing proteins, an
otp, aristaless, and rax (OAR) domain of 14 amino acids within the
C-terminal region. PITX3 along with PITX1 and PITX2 form the
PITX/RIEG sub-family of the Paired-like class of homeobox
proteins which are highly conserved across species [26,27]. In
addition, we obtained cDNA sequence of ovine PAX2 by
sequencing RT-PCR products of head tissue of d30 sheep fetuses. We detected a single PAX2 transcript with an open reading frame
of 1182 bp encoding a protein of 393 amino acids containing a N-
terminal paired box (PAX) domain which is 98% identical to
human PAX2 isoform b. within a population sample of Texel sheep was 2.2%. The mutant
C-allele was absent from 115 control sheep from 14 diverse sheep
breeds. RT-PCR on cDNA and subsequent sequencing confirmed
that the PITX3 RNA is normally spliced in d30 fetuses of both
available genotypes, homozygous C/C and heterozygous G/C,
respectively. The c.338G.C substitution is predicted to result in
an exchange of an arginine to a proline in the homeodomain of
PITX3 protein sequence (p.R113P). This suggests that the G.C
SNP is located in a functionally important region. The potential
impact
of
this
microphthalmia
associated
substitution
was
evaluated by multiple species alignment of PITX3 homeodomain
(Figure 3C). PITX3 belongs to the paired-like homeodomain
containing transcription factor group and a bicoid-like subgroup
[25]. The bicoid-like homeodomains are characterized by a lysine
at position 50 in the homeodomain, which is known to selectively
recognize the 39 CC dinucleotide adjacent to the TAAT core
DNA binding sequence. The microphthalmia associated p.R113P
substitution is located at the perfectly conserved position 52 of
the homeodomain. To evaluate possible functional consequences
we searched for experimentally determined protein structures of
PITX family members. Identification and Characterization of Functional
Candidate Genes The structure of the human PITX2
homeodomain-DNA complex showed that the p.R113P mutation
is indeed likely to affect the DNA-binding function of PITX3 [27]. The mutant proline shows an uncharged side chain and it cannot
act as a hydrogen bond donor (Figure S2). Therefore, as proline
disrupts alpha helixes in general, it may act as a structural
disruptor in the middle of third alpha helix of the PITX3
homeodomain. Furthermore, the putative consequences of the
polymorphisms on the modified proteins were analyzed using two
independent computer based amino acid conservation analysis
software. These programs are sequence homology-based tools that
sort intolerant from tolerant amino acid substitutions and predict
whether an amino acid substitution in a protein has a possible
phenotypic effect. According to PolyPhen, the PITX3 p.R113P
substitution is damaging with a high probability (score 2.6) and the
PMut calculation predicts a highly reliable pathological effect. To test for possible PAX2 mutations we sequenced RT-PCR
products of the experimentally derived d30 sheep fetuses. No size
difference or alternative splicing was detected and analyzing the
ORF of four individuals, two homozygous and two heterozygous
for the associated INRA81 microsatellite allele, respectively,
revealed no polymorphisms affecting the amino acid sequence of
PAX2. Discussion Historically, the development of genomic tools for the sheep
genome has lagged behind those of other major livestock species
such as the cattle and chicken. This has limited the ability to
identify genes controlling specific traits of interest [28]. The
development of low density microsatellite based linkage maps [29]
have lead to the mapping of Mendelian diseases [30–32] and
subsequent discovery of mutations underlying at least three genetic
diseases
in
sheep
[32–34],
however
many
others
remain
uncharacterized [35]. The recent development of a set of SNP
markers distributed across the sheep genome has changed the
prerequisites for such gene mapping projects. For the first time, we
demonstrate the use of a genome-wide ovine SNP array for
efficient positional cloning of a Mendelian trait in sheep. The
result illustrates the power of genome-wide association analysis in
domestic animals for the genetic dissection of trait loci [36]. PLoS ONE | www.plosone.org Mutation Analysis We designed PCR primers for the amplification of a 14.5 kb
segment containing the entire PITX3 gene and determined the
genomic sequence of four microphthalmia affected and four
healthy control sheep. This analysis revealed just a single sequence
polymorphism (Figure 3A). This non-synonymous SNP located in
PITX3 exon 4 (c.338G.C; Figure 3B) showed perfect association
to the microphthalmia phenotype (Table 1). All 134 affected sheep
were homozygous C/C and all 133 known carriers were
heterozygous G/C. Twelve out of 47 healthy full- and half-sibs
of microphthalmia affected sheep were also heterozygous G/C. Testing a population sample of 89 healthy Texels which had
unknown relatedness revealed none carried the homozygous C/C
genotype, while four were presumed carriers with the G/C
genotype. Thus the allele frequency of the deleterious C-allele Twenty-three affected and 23 controls were selected for
genome-wide association and were genotyped using Illumina’s
OvineSNP50 BeadChip. Both, case-control association analysis PLoS ONE | www.plosone.org January 2010 | Volume 5 | Issue 1 | e8689 January 2010 | Volume 5 | Issue 1 | e8689 4 PITX3 Mutation in Sheep Figure 3. PITX3 mutation analysis. (A) Ovine PITX3 gene structure. (B) Electropherograms of the PITX3 c. 338G.C mutation. Representative
sequence traces of PCR products amplified from genomic DNA of three sheep with the different genotypes are shown. (C) Multispecies alignment of
the evolutionary conserved homeodomain of the PITX3 protein sequence. The p.R113P mutation in PITX3is indicated by an arrow. It affects an
arginine residue, which is perfectly conserved from human to zebrafish across all investigated species. The sequences for the alignment were taken
from the following accessions: (sheep, NP_005020 (human), XP_589431 (cattle), XP_001499185 (horse), EDL41981 (mouse), NP_062120 (rat),
XP_421631 (chicken), NP_001082023 (Xenopus laevis), NP_991238 (Danio rerio). doi:10.1371/journal.pone.0008689.g003 Figure 3. PITX3 mutation analysis. (A) Ovine PITX3 gene structure. (B) Electropherograms of the PITX3 c. 338G.C mutation. Representative
sequence traces of PCR products amplified from genomic DNA of three sheep with the different genotypes are shown. (C) Multispecies alignment of
the evolutionary conserved homeodomain of the PITX3 protein sequence. The p.R113P mutation in PITX3is indicated by an arrow. It affects an
arginine residue, which is perfectly conserved from human to zebrafish across all investigated species. The sequences for the alignment were taken
from the following accessions: (sheep, NP_005020 (human), XP_589431 (cattle), XP_001499185 (horse), EDL41981 (mouse), NP_062120 (rat),
XP_421631 (chicken), NP_001082023 (Xenopus laevis), NP_991238 (Danio rerio). doi:10.1371/journal.pone.0008689.g003 Figure 3. PITX3 mutation analysis. Figure 3. PITX3 mutation analysis. (A) Ovine PITX3 gene structure. (B) Electropherograms of the PITX3 c. 338G.C mutation. Representative
sequence traces of PCR products amplified from genomic DNA of three sheep with the different genotypes are shown. (C) Multispecies alignment of
the evolutionary conserved homeodomain of the PITX3 protein sequence. The p.R113P mutation in PITX3is indicated by an arrow. It affects an
arginine residue, which is perfectly conserved from human to zebrafish across all investigated species. The sequences for the alignment were taken
from the following accessions: (sheep, NP_005020 (human), XP_589431 (cattle), XP_001499185 (horse), EDL41981 (mouse), NP_062120 (rat),
XP_421631 (chicken), NP_001082023 (Xenopus laevis), NP_991238 (Danio rerio).
doi:10.1371/journal.pone.0008689.g003 Mutation Analysis wildtype Arg113 is located in the middle of an alpha helix and
binds to the DNA that is highly likely not possible with the imino
acid proline as possible helix disruptor (Figure S2). Thus it is
conceivable that this mutation affects the proper folding and
stability of the native conformation, possibly inactivating the
transcription factor significantly. Apparently, one copy of the
PITX3 wildtype allele is sufficient for regulation an undisturbed
embryonic lens development, because no visible eye phenotype
has been reported in heterozygous carriers of the microphthalmia
mutation [19,20]. The p.R113P mutation in microphthalmia
affected
sheep
probably
does
not
affect
skeletal
muscular
development in newborn lambs, although Pitx3 expression during
myogenesis has been reported [41]. In mice it was speculated that
the lacking PITX3 function in muscles is perhaps completely
compensated by the maintenance of PITX2 expression [41]. A
microRNA was identified that regulates the maturation and
function of midbrain dopaminergic neurons within a negative
feedback circuit including the transcription factor PITX3 [42]. The authors propose a role for this feedback circuit in the fine-
tuning of dopaminergic behaviors such as locomotion. We found
no clinical evidence for conspicuous behavior in microphthalmia
affected Texel sheep [20]. p
[
]
The PITX3 gene was the most compelling functional candidate
in the 2.4 Mb critical interval (Figure 2C). It was not, however, the
only plausible positional candidate as the paired box gene 2 (PAX2)
is located within the region. While it has been associated with eye
malformations in human and mouse, these congenital phenotypes
are usually accompanied by kidney anomalies [1] which have not
been observed in the ovine form of the disease [20,21]. Sequencing
of the coding sequence of PAX2 showed no evidence for disease
causing mutations in sheep. However, DNA sequencing revealed a
non-synonymous mutation in the PITX3 gene, which is perfectly
associated with the microphthalmia phenotype in Texel sheep. We
confirmed the presence of this mutation on the genomic DNA and
mRNA level. Although we cannot provide functional proof of the
causality of the mutation at this time, the wealth of functional data,
which are available for the PITX3 gene, strongly supports the
hypothesis that p.R113P is indeed the causative mutation. Human
patients with point mutations in PITX3 demonstrate congenital
cataracts along with anterior segment defects (ASD) in some cases
when one allele is affected and microphthalmia with brain
malformations when both copies are mutated [10,37]. Mutation Analysis ASD
includes a spectrum of developmental abnormalities of the cornea
(Peters anomaly), iris (Axenfeld-Rieger syndrome), iridocorneal
angle, and ciliary body, which are caused by mutations in the
homeodomain of PITX2, encoding a closely related transcription
factor [27,38]. A morphological description of microphthalmia in
Texel sheep showed an abnormal development of the lens vesicle
[19]. These authors also concluded that hereditary microphthal-
mia in Texel sheep closely resembles autosomal recessive lens
aplasia in mice [39]. A similar phenotype is also the key feature in
the Pitx3 loss of function aphakia mouse, where the lens begins to
form, but its development is abnormal [24,25]. Morpholino-
induced knockdown of pitx3 at early embryonic stages in zebrafish
resulted in a lens and retinal phenotype similar to the one seen in
the aphakia mouse [40]. Many inherited diseases of domestic animals are analogous
to human hereditary disorders and have proven to be valuable
for the investigation of the pathogenesis and therapeutic trials
of rare human phenotypes with identical molecular basis [43]. Recently, a mutation causing an autosomal recessive inherited
disorder
characterized
by
dysplasia
of
the
lens,
retinal
detachment, persistence of the hyaloid artery, and microph-
thalmia in cattle was identified [44]. This large animal model
demonstrated the essential role of WFDC1, a small secretory
protein specifically expressed in the lens, retina, and optic
nerves of embryonic and adult mouse eyes, in mammalian eye
development for the first time. Our finding of a PITX3
p.R113P mutation in sheep with microphthalmia provides a
valuable large animal model for human medicine and confirms
PITX3 as a microphthalmia gene. Our study indicates that
coding mutations of the PITX3 gene might also be responsible
for rare recessive forms of human isolated microphthalmia. The naturally occurring microphthalmia sheep model may
represent a better model for human microphthalmia than
aphakia mice because of its eye size and structure and the
resulting similarity to the human situation. In addition, the
longer life expectancy of sheep allows for investigations over a
longer time period. Domestic production animals have the
additional advantages of being economic to maintain and
having been bred for easy management. Moreover, a high level
of expertise in reproductive technology and veterinary care is
available for them. The known PITX3 mutations in mouse and humans don’t affect
the homeodomain, which is different from that seen in other
homeodomain proteins including its close family member, PITX2
[26]. Mutation Analysis (A) Ovine PITX3 gene structure. (B) Electropherograms of the PITX3 c. 338G.C mutation. Representative
sequence traces of PCR products amplified from genomic DNA of three sheep with the different genotypes are shown. (C) Multispecies alignment of
the evolutionary conserved homeodomain of the PITX3 protein sequence. The p.R113P mutation in PITX3is indicated by an arrow. It affects an
arginine residue, which is perfectly conserved from human to zebrafish across all investigated species. The sequences for the alignment were taken
from the following accessions: (sheep, NP_005020 (human), XP_589431 (cattle), XP_001499185 (horse), EDL41981 (mouse), NP_062120 (rat),
XP_421631 (chicken), NP_001082023 (Xenopus laevis), NP_991238 (Danio rerio). doi:10.1371/journal.pone.0008689.g003 confirmed an increased homozygosity within a total of 134
microphthalmia affected lambs compared to 212 controls. In
addition, the availability of segregating families allowed us to confirmed an increased homozygosity within a total of 134
microphthalmia affected lambs compared to 212 controls. In
addition, the availability of segregating families allowed us to with
genome-wide
significance
and
homozygosity
mapping
identified the same OAR 22 region for microphthalmia in Texel
sheep. The analysis of three flanking OAR 22 microsatellites January 2010 | Volume 5 | Issue 1 | e8689 January 2010 | Volume 5 | Issue 1 | e8689 PLoS ONE | www.plosone.org 5 PITX3 Mutation in Sheep Table 1. Association of the PITX3 mutation with the microphthalmia phenotype. Texel or Texel/Whiteheaded mutton crossbred sheep
Other breeds
PITX3
microphthalmia
affected 1 (n = 134)
microphthalmia
carrier 2 (n = 133)
Control, related 3
(n = 47)
Control, unknown
relationship (n = 89)
Controls (n = 115)
c.338G.C
GG
12
85
115
(p.R113P)
GC
133
35
4
CC
134
1Thirty not closely related cases and 104 affected lambs from the family material. 2Thirty not closely related sheep recorded as parent of affected lambs and 103 parents from the family material. 3Healthy littermates of affected lambs. doi:10.1371/journal.pone.0008689.t001 Texel or Texel/Whiteheaded mutton crossbred sheep 1Thirty not closely related cases and 104 affected lambs from the family material. 2Thirty not closely related sheep recorded as parent of affected lambs and 103 parents from the family material. 3Healthy littermates of affected lambs. doi:10.1371/journal.pone.0008689.t001 confirm the mapping on OAR 22 by linkage analysis. Taken
together, the presented statistical support showed that the
previously published genetic linkage to microsatellite markers on
sheep chromosome 23 using a subset of the family material now
did not prove to be correct [21]. PLoS ONE | www.plosone.org Fetal Tissue Tissues were collected in accordance with the animal care
and use protocols approved by the Lower Saxony (Germany)
governmental animal rights protection authorities (Ref. No. 509.6-
42502/3-04/851). A total of six d30 fetuses were surgically
obtained after targeted mating of an affected male to two known
disease carriers (Figure S1). Upon collection, fetuses were divided
in front (head) and back section and stored in RNAlater (Qiagen). For mutation analysis, PCR products were amplified from four
microphthalmia affected and four healthy control sheep using
AmpliTaq Gold 360 Master Mix (Applied Biosystems). The
sequences of the primers are listed in Table S3. The subsequent re-
sequencing of the PCR products was performed after rAPid
alkaline phosphatase (Roche) and exonuclease I (New England
Biolabs) treatment using both PCR primers with the ABI BigDye
Terminator Sequencing Kit 3.1 (Applied Biosystems) on an
ABI 3730. Sequence data were analyzed with Sequencher 4.9
(GeneCodes). DNA and RNA extraction. Genomic DNA was isolated from
blood or tissue using the Nucleon Bacc2 kit (GE Healthcare). Total
RNA
was isolated
using
Trizol reagent
according
to
the
manufacturer’s instructions (Invitrogen). Mapping of the microphthalmia
mutation. Genomic
DNA from 23 cases and 23 controls, mostly selected as discordant
sib-pairs, was genotyped using Illumina’s OvineSNP50 BeadChip
(49,034 SNPs) [47]. Two small nuclear families segregating for
microphthalmia were included to check SNPs for Mendelian
inheritance. The results were analyzed with PLINK [48]. After
removing 5 SNPs with low genotyping success (failed calls .0.1) the
average genotyping rate per individual was 99.9%. A total of 4,164
SNPs had a minor allele frequency (MAF),0.05. A case-control
analysis using the options –assoc was applied. Genome-wide
corrected
empirical
p-values
were determined
applying
the
max(T) permutation procedure implemented in PLINK with
10,000 permutations. To identify extended homozygous regions
with allele sharing across all affected animals the options –homozyg-
group and –homozyg-match were applied. All given positions
correspond to the virtual sheep genome v 2 [22]. The corresponding
human chromosome segment was identified by BLASTN searches
of ovine SNP flanking sequences to the human genome sequence. Mapping of the microphthalmia
mutation. Genomic
DNA from 23 cases and 23 controls, mostly selected as discordant
sib-pairs, was genotyped using Illumina’s OvineSNP50 BeadChip
(49,034 SNPs) [47]. Two small nuclear families segregating for
microphthalmia were included to check SNPs for Mendelian
inheritance. The results were analyzed with PLINK [48]. Mutation Analysis The reported p.R113P mutation in ovine microphthalmia
lies within the conserved homeodomain and the wildtype arginine
is conserved across all PITX3 sequences (Figure 3C). Therefore, it
is likely that the mutation p.R113P results in an impaired lens
development. In addition, the arginine residue at the similar
position of PITX2 was found to be mutated to cysteine in patients
with Axenfeld-Rieger syndrome [26,27]. Furthermore, in the PLoS ONE | www.plosone.org January 2010 | Volume 5 | Issue 1 | e8689 January 2010 | Volume 5 | Issue 1 | e8689 6 PITX3 Mutation in Sheep In conclusion, we have identified the p.R113P mutation in
the ovine PITX3 gene as the candidate causative mutation for
microphthalmia in Texel sheep. In comparison to the commer-
cially available DNA test which relies on linked markers [45]
this result allows direct genetic testing and improved power to
eradicate this common genetic disease from the worldwide Texel
breeding population. Our study also provides a defined animal
model for similar human hereditary diseases and confirms PITX3
critical function for eye development. 4.0 software (Applied Biosystems). Twopoint parametric linkage
analysis under the assumption of microphthalmia segregating as a
biallelic autosomal recessive trait with complete penetrance was
performed with Merlin software version 1.1.2 [49]. The frequency of
the mutant allele in the considered population was unknown and
there were no data available that would have made it possible to
estimate the frequency in a reliable manner. For the calculations a
frequency of 0.001 for the mutant allele was assumed. The LOD
score test statistic was used to estimate the proportion of linked
families and the corresponding maximum heterogeneity LOD score. Within the available family material, a maximum LOD score of
11.512 would have been possible. Animals Later, the exact ovine genomic structure
was determined using the experimentally derived ovine mRNA
sequences. Animals Analysis
of
the
ovine
PITX3
gene
and
mutation
identification. The BAC clone CH243-315I22 containing the
ovine PITX3 gene was identified by BLASTN searches of ovine
BAC end sequences to the HSA 10 sequence [22]. The BAC DNA
was prepared using the Qiagen Midi plasmid kit according to the
modified protocol for BAC clones (Qiagen). The insert sequence of
127 kb of the CH243-315I22 BAC clone has been determined by
generating 1,149,790 read pairs using the Chrysalis 36cycles v 2.0
kit on a Genome Analyzer II (Illumina) and de novo assembly with
Velvet [50]. Remaining gaps were closed by a primer walking
strategy and an ABI 3730 capillary sequencer (Applied Biosystems)
until both strands were completely sequenced. The obtained
sequence was submitted under accession FN432136 to the EMBL
nucleotide database. We collected samples from 134 microphthalmia affected lambs
(59 male, 75 female), their available healthy siblings (n = 47), sires
(n = 10), and dams (n = 93) from different sheep farms with Texel
purebred or Texel/Whiteheaded mutton crossbred sheep and our
experimentally established pedigree [20,21]. In addition, we
collected 30 Texel sheep recorded as parents of microphthalmia
affected offspring. We also collected 89 healthy Texel sheep with
unknown relatedness resulting in a total of 403 samples. Furthermore,
we
sampled
unrelated
control
sheep
from
different breeds (Whiteheaded mutton (n = 50), Swiss White
Alpine (n = 24), Bu¨ndner Oberla¨nder (n = 4), Engadine Red
(n = 4),
Swiss
Black-Brown
Mountain
(n = 4),
Swiss
Mirror
(n = 4), Valais Blacknose (n = 4), Valais Red (n = 4), Suffolk
(n = 1)) and 16 founder animal of the International Mapping
Flock (Texel, Coopworth, Perendale, Romney, Merino) [46] for
the re-sequencing of PITX3 exon 4. The human reference PITX3 mRNA (GenBank: NM_005029)
was used as query in cross-species BLAST searches identifying
corresponding
bovine
EST
entries
(GenBank:
EG705801,
EG707045, EG707660). Initially, the putative ovine genomic
structure was determined by alignment of bovine PITX3 EST and
human PITX3 mRNA to the determined ovine genomic sequence
using Spidey [51]. Later, the exact ovine genomic structure
was determined using the experimentally derived ovine mRNA
sequences. The human reference PITX3 mRNA (GenBank: NM_005029)
was used as query in cross-species BLAST searches identifying
corresponding
bovine
EST
entries
(GenBank:
EG705801,
EG707045, EG707660). Initially, the putative ovine genomic
structure was determined by alignment of bovine PITX3 EST and
human PITX3 mRNA to the determined ovine genomic sequence
using Spidey [51]. References 18. Roe WD, West DM, Walshe MT, Jolly RD (2003) Microphthalmia in Texel
lambs. NZ Vet J 51: 194–195. 1. Graw J (2003) The genetic and molecular basis of congenital eye defects. Nat
Rev Genet 4: 876–888. 2. Verma AS, Fitzpatrick DR (2007) Anophthalmia and microphthalmia. Orphanet J Rare Dis 2: 47. 19. van der Linde-Sipman JS, van den Ingh TS, Vellema P (2003) Morphology and
morphogenesis of hereditary microphthalmia in Texel sheep. J Comp Pathol
128: 269–275. 3. Chow RL, Lang RA (2001) Early eye development in vertebrates. Annu Rev
Cell Dev Biol 17: 255–296. 20. Tetens J, Bu¨rstel D, Ganter M, Do¨pke C, Mu¨ller G, et al. (2007)
Untersuchungen zur kongenitalen Mikrophthalmie beim Texelschaf. Tiera¨rztl
Prax 35(G): 211–218. 4. Fantes J, Ragge NK, Lynch SA, McGill NI, Collin JR, et al. (2003) Mutations in
SOX2 cause anophthalmia. Nat Genet 33: 461–462. 21. Tetens J, Ganter M, Mu¨ller G, Dro¨gemu¨ller C (2007) Linkage mapping of ovine
microphthalmia to chromosome 23, the sheep orthologue of human chromo-
some 18. Invest Ophthalmol Vis Sci 48: 3506–3515. 5. Glaser T, Jepeal L, Edwards JG, Young SR, Favor J, et al. (1994) PAX6 gene
dosage effect in a family with congenital cataracts, aniridia, anophthalmia and
central nervous system defects. Nat Genet 7: 463–471. 6. Ragge NK, Brown AG, Poloschek CM, Lorenz B, Henderson RA, et al. (2005)
Heterozygous mutations of OTX2 cause severe ocular malformations. Am J Hum
Genet 76: 1008–1022. 22. Dalrymple BP, Kirkness EF, Nefedov M, McWilliam S, Ratnakumar A, et al. (2007) Using comparative genomics to reorder the human genome sequence into
a virtual sheep genome. Genome Biol 8: R152. 23. Medina-Martinez O, Shah R, Jamrich M (2009) Pitx3 controls multiple aspects
of lens development. Developmental Dynamics, early view DOI 10.1002/
dvdy.21924. 7. Voronina VA, Kozhemyakina EA, O’Kernick CM, Kahn ND, Wenger SL,
et al. (2004) Mutations in the human RAX homeobox gene in a patient with
anophthalmia and sclerocornea. Hum Mol Genet 13: 315–322. 24. Semina EV, Murray JC, Reiter R, Hrstka RF, Graw J (2000) Deletion in the
promoter region and altered expression of Pitx3 homeobox gene in aphakia
mice. Hum Mol Genet 9: 1575–1585. 8. Percin E, Ploder LA, Yu JJ, Arici K, Horsford DJ, et al. (2000) Human
microphthalmia associated with mutations in the retinal homeobox gene
CHX10. Nat Genet 25: 397–401. 25. Acknowledgments The authors are grateful that Hauke Peters and all other sheep breeders
donated samples and shared pedigree data. The authors would like to
thank Brigitta Colomb for expert technical assistance and Ulrich Baumann
for helpful discussions. Supporting Information Figure S1
Pedigrees of families in study. Filled symbols represent
microphthalmia affected sheep, open symbols represent normal
sheep. DNA samples were available from numbered sheep. Two
females, which appear as mothers in different families, are marked
with a rectangle. Sheep that were used for the initial whole
genome association study are marked with an asterisk. The
genotypes for the PITX3 c.338G.C mutation are given below the
symbols. A single affected and two healthy offspring from the
experimental family 73 were used to obtain d30 fetuses (E4-E6). Found
at:
doi:10.1371/journal.pone.0008689.s001
(0.04
MB
PDF) Figure S1
Pedigrees of families in study. Filled symbols represent
microphthalmia affected sheep, open symbols represent normal
sheep. DNA samples were available from numbered sheep. Two
females, which appear as mothers in different families, are marked
with a rectangle. Sheep that were used for the initial whole
genome association study are marked with an asterisk. The
genotypes for the PITX3 c.338G.C mutation are given below the
symbols. A single affected and two healthy offspring from the
experimental family 73 were used to obtain d30 fetuses (E4-E6). Found
at:
doi:10.1371/journal.pone.0008689.s001
(0.04
MB
PDF) Table S1
Results of homozygosity mapping. Found
at:
doi:10.1371/journal.pone.0008689.s003
(1.66
MB
PDF) Table S2
Microsatellites. Found
at:
doi:10.1371/journal.pone.0008689.s004
(0.01
MB
PDF) Table S3
Primer sequences for the amplification of ovine PITX3
gene. g
Found
at:
doi:10.1371/journal.pone.0008689.s005
(0.02
MB
PDF) Figure S2
Impact of the microphthalmia associated PITX3
substitution. (A) Alignment of human PITX3 (NP_005020) and
human PITX2 isoform a, b, and c (NP_700476; NP_700475;
NP_000316) protein sequences. The conserved domains (homeo-
domain and the C-terminal OAR domain) are indicated in bold
face type. The mutated arginine at PITX3 position 113 is shown in
red. (B) Structure of the PITX2 homeodomain-DNA complex
(Code 1YZ8 taken from PDB [54]). The tertiary structure of the
PITX2 homeodomain (96% identical to sheep PITX3 homeodo-
main) is composed of three alpha helices (shown in light grey). The
wildtype PITX3 Arg113 equivalent PITX2 Arg90 (shown with
carbon atoms in pink) is located in the third alpha helix and binds
with its positively charged capped guanidinium group (shown with
blue nitrogen atoms) via two hydrogen bonds (indicated in black)
to the DNA backbone (oxygens are shown in red, nitrogen atoms
in blue, and carbon atoms in green and light blue). Figure S2
Impact of the microphthalmia associated PITX3
substitution. Fetal Tissue After
removing 5 SNPs with low genotyping success (failed calls .0.1) the
average genotyping rate per individual was 99.9%. A total of 4,164
SNPs had a minor allele frequency (MAF),0.05. A case-control
analysis using the options –assoc was applied. Genome-wide
corrected
empirical
p-values
were determined
applying
the
max(T) permutation procedure implemented in PLINK with
10,000 permutations. To identify extended homozygous regions
with allele sharing across all affected animals the options –homozyg-
group and –homozyg-match were applied. All given positions
correspond to the virtual sheep genome v 2 [22]. The corresponding
human chromosome segment was identified by BLASTN searches
of ovine SNP flanking sequences to the human genome sequence. RT-PCR. Aliquots
of
1 mg
total
RNA
were
reverse
transcribed into cDNA using 20 pmol (T)24V primer and
Superscript III reverse transcriptase (Invitrogen). Two microliters
of the cDNA were used as a template in PCR. RT-PCR reactions
were performed as described above and primer sequences
are given in Table S4. The human reference PAX2 mRNA
(GenBank: NM_003987) was used as query in cross-species Spidey
alignment to identifying corresponding cattle sequences. Primers
for RT-PCR in sheep were derived from the bovine PAX2 59UTR
and 39UTR sequence. Isolation of full length cDNA for the
ovine PITX3 gene was achieved by a rapid amplification of
cDNA ends (RACE) protocol with the FirstChoice RNA ligase-
mediated (RLM)-RACE kit (Applied Biosystems). The ovine
cDNA sequences were deposited in the EMBL nucleotide
database (PITX3: FN432137, FN432138, and FN432139; PAX2:
FN600706). Linkage
analysis
in
candidate
genes. Microsatellite
markers were amplified using the Multiplex PCR Kit (Qiagen) and
fragment size analyses were determined on an ABI 3730 capillary
sequencer (Applied Biosystems) and analyzed with the GeneMapper PLoS ONE | www.plosone.org January 2010 | Volume 5 | Issue 1 | e8689 7 PITX3 Mutation in Sheep Found
at:
doi:10.1371/journal.pone.0008689.s002
(0.53
MB
PDF) Supporting Information (A) Alignment of human PITX3 (NP_005020) and
human PITX2 isoform a, b, and c (NP_700476; NP_700475;
NP_000316) protein sequences. The conserved domains (homeo-
domain and the C-terminal OAR domain) are indicated in bold
face type. The mutated arginine at PITX3 position 113 is shown in
red. (B) Structure of the PITX2 homeodomain-DNA complex
(Code 1YZ8 taken from PDB [54]). The tertiary structure of the
PITX2 homeodomain (96% identical to sheep PITX3 homeodo-
main) is composed of three alpha helices (shown in light grey). The
wildtype PITX3 Arg113 equivalent PITX2 Arg90 (shown with
carbon atoms in pink) is located in the third alpha helix and binds
with its positively charged capped guanidinium group (shown with
blue nitrogen atoms) via two hydrogen bonds (indicated in black)
to the DNA backbone (oxygens are shown in red, nitrogen atoms
in blue, and carbon atoms in green and light blue). Table S4
Primer sequences for the amplification of ovine PITX3
and PAX2 cDNA. Found
at:
doi:10.1371/journal.pone.0008689.s006
(0.01
MB
PDF) Author Contributions Conceived and designed the experiments: CD. Performed the experiments:
DB JT AB DB. Analyzed the data: DB JT AB JK CD. Contributed
reagents/materials/analysis tools: MG JK. Wrote the paper: DB JT CD. Conceived and designed the experiments: CD. Performed the experiments:
DB JT AB DB. Analyzed the data: DB JT AB JK CD. Contributed
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Optimizing photo-Fenton like process for the removal of diesel fuel from the aqueous phase
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Abstract Background: In recent years, pollution of soil and groundwater caused by fuel leakage from old underground
storage tanks, oil extraction process, refineries, fuel distribution terminals, improper disposal and also spills during
transferring has been reported. Diesel fuel has created many problems for water resources. The main objectives of
this research were focused on assessing the feasibility of using photo-Fenton like method using nano zero-valent
iron (nZVI/UV/H2O2) in removing total petroleum hydrocarbons (TPH) and determining the optimal conditions using
Taguchi method. Results: The influence of different parameters including the initial concentration of TPH (0.1-1 mg/L), H2O2
concentration (5-20 mmole/L), nZVI concentration (10-100 mg/L), pH (3-9), and reaction time (15-120 min) on TPH
reduction rate in diesel fuel were investigated. The variance analysis suggests that the optimal conditions for TPH
reduction rate from diesel fuel in the aqueous phase are as follows: the initial TPH concentration equals to 0.7
mg/L, nZVI concentration 20 mg/L, H2O2 concentration equals to 5 mmol/L, pH 3, and the reaction time of 60 min
and degree of significance for the study parameters are 7.643, 9.33, 13.318, 15.185 and 6.588%, respectively. The
predicted removal rate in the optimal conditions was 95.8% and confirmed by data obtained in this study which
was between 95-100%. Conclusion: In conclusion, photo-Fenton like process using nZVI process may enhance the rate of diesel
degradation in polluted water and could be used as a pretreatment step for the biological removal of TPH from
diesel fuel in the aqueous phase. Keywords: Photo-Fenton like process, nZVI, Taguchi method, TPH, Diesel fuel Keywords: Photo-Fenton like process, nZVI, Taguchi method, TPH, Diesel fuel lesser amount of aromatic compounds and includes mol-
ecules containing sulfur, nitrogen and metal oxides [2]. The toxicity of diesel fuel is mostly due to BTEX aro-
matic hydrocarbons. The carcinogen property of diesel
fuel is due to C10 and C20 alkenes and alkylated benzene. Therefore, developing an efficient method for the re-
moval of diesel fuel from contaminated water resources
is very crucial [4]. Different physical, chemical, and bio-
logical techniques have been used to degrade the contami-
nated soils and water [6-10]. The aromatic hydrocarbons
with high toxic nature cannot be degraded simply by
conventional treatment methods [11]. Most conventional
techniques such as evaporation, oil phase separation,
filtration, dissolved air flotation, coagulation, flocculation,
absorption, and ultrafiltration only transfer pollutions
from one media to another [12,13]. RESEARCH ARTICLE Open Access * Correspondence: mahdifarzadkia@gmail.com
2Department of Environmental Health Engineering, Iran University of Medical
Sciences, Tehran, Iran
Full list of author information is available at the end of the article Abstract Therefore, another
method should be developed to remove hydrocarbon pol-
lutions [14]. In other words, these methods do not remove Optimizing photo-Fenton like process for the
removal of diesel fuel from the aqueous phase Mansooreh Dehghani1, Esmaeel Shahsavani1, Mahdi Farzadkia2* and Mohammad Reza Samaei © 2014 Dehghani 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 credited. JOURNAL OF
ENVIRONMENTAL HEALTH
SCIENCE & ENGINEERING JOURNAL OF
ENVIRONMENTAL HEALTH
SCIENCE & ENGINEERING Dehghani et al. Journal of Environmental Health Science & Engineering 2014, 12:87
http://www.ijehse.com/content/12/1/87 Dehghani et al. Journal of Environmental Health Science & Engineering 2014, 12:87
http://www.ijehse.com/content/12/1/87 JOURNAL OF
ENVIRONMENTAL HEALTH
SCIENCE & ENGINEERING Chemicals and analytical method In this research, regular diesel fuel from a gas station in
Shiraz was used as a pollutant model. Water-oil emulsion
was prepared using emulsifier SDS. Nano zero-valent iron
(nZVI) was supplied by Iran Oil Industry Research Center. The rest of the chemicals were purchased from Merck
(Germany). UV lamp, 125 Kw, 247.3 nm wave length,
(ARDA, Netherland) was used as the radiation source. In this study, photo-Fenton like process which is based
on electrochemical system using nano zero-valent iron
(nZVI) and peroxide hydrogen as reductive and oxidative
reagents, respectively, was used. In photo-Fenton process,
the formation of ferrous ion reduces the process effective-
ness and eventually it will halt the reaction. In photo-
Fenton like process (Fe°/UV/H2O2), ferrous (Fe+2) and
ferric (Fe+3) are formed, respectively. These ions practic-
ally enhance the efficiency of the process [19]: The standard method No. 8015B of Environment Pro-
tection Agency at the United States (EPA) was used to
detect petroleum hydrocarbons especially gasoline range
organics (GROs, C6-C10) with the boiling point of 60-
170°C, and diesel fuel range organics (DROs, C10-C28)
with the boiling point of 170-430°C [20]. DRO was
prepared using liquid-liquid extraction method with n-
hexane as solvent [21]. The recovery of the sample from
the aqueous phase with this method of extraction was
98%. The method provides the chromatographic condi-
tions for detecting non-halogen volatile organic com-
pounds. Diesel standard was supplied by Merck (Germany,
analytical grade GC). For TPH detection in diesel fuel in
the aqueous phase a Varian Model gas chromatography
(Australia) system with fused silica capillary columns (CP-
SIL 5 CB column model, 30 m*0.25 mm, 25 um) was
calibrated and tested prior to injection of the samples. A
Flame Ionization Detector (FID) was used to detect TPH
in the samples. The injector and detector temperatures
were set at 200°C and 340°C, respectively. The column
temperature was maintained at 45°C for 3 min, and then
increased to 275°C (at a rate of 12°C/min), where it was
kept for 12 min. Introduction In recent years, pollution of soil and groundwater caused
by fuel leakage from old underground storage tank, re-
fineries, fuel distribution terminals, improper disposal
and also spills during transferring, has been reported
[1-3]. The accidental spills of more than 2 million tons
of refined oil products per year into the environment are
a worldwide problem [4]. Large amounts of benzene,
toluene, ethyl benzene, and xylenes (BTEX) have been
detected in polluted water resources [5]. The leaching of
petroleum hydrocarbons such as diesel fuel into water
resources causes many serious environmental problems
[6]. Diesel fuel consists of a complex compound includ-
ing paraffin, olefins, aliphatic hydrocarbons, as well as a * Correspondence: mahdifarzadkia@gmail.com
2Department of Environmental Health Engineering, Iran University of Medical
Sciences, Tehran, Iran
Full list of author information is available at the end of the article Page 2 of 7 Dehghani et al. Journal of Environmental Health Science & Engineering 2014, 12:87
http://www.ijehse.com/content/12/1/87 the pollutants, but generally produce highly concentrated
wastes in lower volume. In addition, these methods have
lower efficiency in removing smaller oil droplets and
emulsions [15]. Nowadays, biological and bioremediation
techniques are used to treat polluted soils and sewages,
but these methods need improvement for enhancing the
enzymatic activity in microbial population [16]. Therefore,
it is very important to use advanced methods in order to
remove oily compounds from groundwater resources. Advanced oxidation processes (AOPs) is an efficient
environment-friendly method in which hydroxyl radi-
cals (OH°) are used to oxidize recalcitrant organic pol-
lutants and convert them to harmless end-products
such as H2O and CO2 [17,18]. to (i) evaluate the feasibility of using photo-Fenton like
method (nZVI/UV/H2O2) in removing total petroleum
hydrocarbons (TPH) and (ii) determine the optimal condi-
tions using Taguchi method so that the standard limit can
be achieved by further complementary treatment. Chemicals and analytical method The flow rate of helium carrier gas and Fe∘þ H2O2 →Fe2þ þ OH∘þ OH−
ð1Þ
Fe2þ þ H2O2 →Fe3þ þ OH∘þ OH−
ð2Þ
Organicmaterial þ OH∘→Oxidizedcompound þ H2O
ð3Þ Fe∘þ H2O2 →Fe2þ þ OH∘þ OH−
ð1Þ
Fe2þ þ H2O2 →Fe3þ þ OH∘þ OH−
ð2Þ
Organicmaterial þ OH∘→Oxidizedcompound þ H2O
ð3Þ ð1Þ ð2Þ Organicmaterial þ OH∘→Oxidizedcompound þ H2O
ð3Þ ð3Þ Previous studies in Iran have reported soil and water
pollution caused by petroleum compounds near Shiraz,
Esfahan and Tehran refineries [6]. Since Fars (in Southern
part of Iran) enjoys a top rank in oil refinery, storage, and
distribution of oil products in the country in recent years,
there is a concern regarding the effect of petroleum hy-
drocarbons in water resources on people's health and the
environment. Therefore, the objectives of the study were 5
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Figure 1 GC chromatograms for the initial sample of diesel fuel. Figure 1 GC chromatograms for the initial sample of diesel fuel. 5
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Figure 2 GC chromatograms for the treated sample of diesel fuel. Dehghani et al. Journal of Environmental Health Science & Engineering 2014, 12:87
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Figure 2 GC chromatograms for the treated sample of diesel fuel. Figure 2 GC chromatograms for the treated sample of diesel fuel. nitrogen make up gas, were set at 5-7 mL/min and 30 mL/
min, respectively. The detection limit for the sample was
0.01 mg/L. GC chromatograms for the initial and treated
samples of diesel fuel are presented in Figures 1 and 2. TPH reduction rate was analyzed using QT4 software. The most effective parameters for removing diesel fuel
from the aqueous phase, the rate of efficiency, and the level
of precision, and optimal conditions were determined. The effects of nZVI particles concentration According to Figure 4, the optimal nZVI concentration
and the reduction rate of TPH were 20 mg/L and 77.7%,
respectively. The TPH reduction rate in diesel fuel in-
creased from 65.9 to 77.7% as the nZVI concentration
increased from 10 to 20 mg/L. However, an increase in
the concentration from 20 to 100 mg/L, caused a decrease
in the reduction rate of diesel degradation (58.3%). In the
current study (nZVI/UV/H2O2), increasing nZVI increased
the production rate of hydroxyl radical. Due to the fact that
by increasing nZVI the metal active surface was increased
to make better contact with peroxide hydrogen and UV ra-
diation. The reduction rate was decreased by increasing
the nZVI concentration above the optimal concentration;
in this condition the process is in favor of producing more
ferrous ions rather than producing hydroxyl radical [15]. Therefore, TPH reduction rate increased with nZVI con-
centration up to a specific level (10-20 mg/L) and then
began to decrease (20-100 mg/L). The reduction of the
pollutant is basically proportional to the formation of hy-
droxyl radicals on the surface of the catalyst. Due to high
turbidity of the solution at higher concentrations of nZVI
(20-100 mg/L), UV radiation cannot penetrate into the Sample preparation Diesel fuel (commercial grade) at the concentration of
100 mL was added gradually to 1 liter of distilled water
using 2.5 mL of 0.1 g/L SDS as emulsifier. The water-
diesel emulsion was mixed using Jar test at the speed of
200 rpm for 24 h. In order to separate non-dispersed
diesel in oil-water emulsion, the solution was kept at rest
for 1 h. Then, the emulsion was transferred to a separ-
ator funnel and the supernatant was removed and the
rest was collected and passed through Whatman filter
paper (20 μm in diameter). The prepared emulsion was
used as stock solution and different concentrations 0.1-
1 mg/L were prepared by diluting with distilled water. The specification of photochemical reactor is shown in
Figure 3. One liter samples were used in 2 liter volume
reactor. Test was performed in a closed reactor with ad-
justable mixer. The UV radiation source, 1020 μw/Cm2,
was embedded at the bottom of reactor. The UV lamp
was protected by Quartz tube. A thermometer was placed
inside the photochemical cell to record the temperature. The temperature inside the reactor was kept at the range
of 24-26°C using cooling water recirculation system. Experimental setup Five parameters including the initial concentration of
TPH, H2O2 concentration, nZVI concentration, pH, and
reaction time were selected in 4 levels to analyze the re-
moval efficiency of TPH in diesel fuel in the aqueous
phase (Table 1). Taguchi’s statistical method and Qualitek-4 (QT4)
software were used for the experimental design. Using
this software, 16 tests were designed randomly to reduce
the errors. Two replications were done for each sample. Table 1 Parameters and the selected levels of photo-Fenton
like treatment process (nZVI/UV/H2O2) for the reduction
of TPH from diesel fuel in the aqueous phase Table 1 Parameters and the selected levels of photo-Fenton
like treatment process (nZVI/UV/H2O2) for the reduction
of TPH from diesel fuel in the aqueous phase
Variables level
Level 1
Level 2
Level 3
Level 4
Concentration TPH (mg/L)
0.1
0.4
0.7
1
nZVI Concentration (mg/L)
10
20
40
100
H2O2 Concentration (mmole/L)
5
10
15
20
pH
3
5
7
9
Reaction time (min)
15
60
90
120 Table 1 Parameters and the selected levels of photo-Fenton
like treatment process (nZVI/UV/H2O2) for the reduction
of TPH from diesel fuel in the aqueous phase Dehghani et al. Journal of Environmental Health Science & Engineering 2014, 12:87
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http://www.ijehse.com/content/12/1/87 Figure 3 Photochemical reactor. and biological processes especially advanced oxidation
efficiency. In addition, pH has a considerable effect on
the solubility of petroleum hydrocarbon, catalyst surface
charge, as well as the mechanism of hydroxyl radical
production [11]. The Fenton and photo-Fenton reactions
depend on the pH. The feasibility of hydroxyl radical pro-
duction and oxidation efficiency also depend on pH [23]. The reduction rate of TPH reduced in higher pH, because
of the formation of ferric hydroxide which in turn reduced
the intensity of UV radiation and the potential of hydroxyl
radical production as well [24]. Additionally, high pH
values intensify the formation of HO2
−2 ions and de-
struction of hydroxyl radicals by carbonate and bicar-
bonate ions (Figure 5). solution and mostly adsorbed by the particles. In addition,
the rate of H2O2 photolysis depends on the intensity of UV
radiation. Therefore, lower UV radiation results in redu-
cing the hydroxyl radical production as well as the deg-
radation rate [20]. The additional Fe ions react with the
hydroxyl radical and therefore reduce the efficiency of
the process [22]. pH effect
D Data regarding the effect of pH shows that as pH in-
creased from 3.0 to 9, the rate of TPH reduction de-
creased. Based on the data obtained in the present study,
pH of 3 is optimal for TPH degradation. The reduction
rate was more than 83% in this case. Generally, pH is
one of the most important factors affecting chemical The effect of H2O2 concentration The effect of H2O2 concentration mostly tend toward a semi-Fenton process and producing
more hydroxyl radicals [26]. Many advanced processes
such as ultrasonic and photochemical reaction were used
to remove organic pollutants (e.g. diesel) from aqueous
solution [27-30]. 2
2
The effect of H2O2 concentration on TPH reduction was
shown in Figure 7. Based on the data obtained in the
present study, H2O2 concentration of 5 mmol/L is opti-
mal for diesel fuel degradation and the reduction rate
was 81.7%. As H2O2 concentration increased from 5 to
20 mmol/L, TPH reduction rate decreased to 42.5%. Add-
ing extra H2O2 concentration (more than 5 mmol/L) will
act as the scavenger for hydroxyl radical and form HO2°
which has lower oxidative ability and longer lifetime
comparing to OH° [15]. The decomposition of hydrogen
peroxide into oxygen and water occurred at H2O2 concen-
tration of more than optimal. Therefore, it can be
concluded that high concentrations of H2O2 act as an in-
hibitor for the formation of hydroxyl radicals' formation
and consequently reduced the efficiency of the process
[22]. Adding hydrogen peroxide to nZVI considerably re-
duces production of free electrons and the reaction will The effects of reaction time
h
ff
f The effect of reaction time on TPH reduction rate from
diesel fuel in the aqueous phase was studied at four
levels 15, 60, 90, and 120 min (Figure 6). Data regarding
the effect of reaction time shows that as the time in-
creased from 15 to 60 min, the rate of reduction in-
creased by 75%. However, from 60 to 120 min there was
a reduction rate in TPH degradation (63.5%). Based on
our findings, 60 min reaction time is optimal for TPH
degradation (Figure 6). The optimization of reaction time
is one of the most important parameters in studying the
removal processes. Basically, an optimal contact time is a
very important parameter for any chemical reactions. At
equilibrium, TPH degradation reached a plateau. If the re-
action time exceeds equilibrium, the process will be no
longer economical [25]. Coelho et al. showed that the op-
timal reaction time for the removal of TPH from petrol-
eum wastewater was equal to one hour [26]. Figure 4 The effect of nZVI on the reduction rate of TPH
from diesel fuel in the aqueous phase using photo- Fenton
like process. Figure 4 The effect of nZVI on the reduction rate of TPH
from diesel fuel in the aqueous phase using photo- Fenton
like process. Figure 4 The effect of nZVI on the reduction rate of TPH
from diesel fuel in the aqueous phase using photo- Fenton
like process. Dehghani et al. Journal of Environmental Health Science & Engineering 2014, 12:87
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http://www.ijehse.com/content/12/1/87 Page 5 of 7 Figure 5 The effect of pH on the reduction rate of TPH from
diesel fuel in the aqueous phase using photo- Fenton
like process. Figure 7 The effect of H2O2 on the reduction rate of TPH from
diesel fuel in the aqueous phase using photo- Fenton
like process. Figure 7 The effect of H2O2 on the reduction rate of TPH from
diesel fuel in the aqueous phase using photo- Fenton
like process. Figure 7 The effect of H2O2 on the reduction rate of TPH from
diesel fuel in the aqueous phase using photo- Fenton
like process. The effects of reaction time
h
ff
f Figure 7 The effect of H2O2 on the reduction rate of TPH from
diesel fuel in the aqueous phase using photo- Fenton
like process. Figure 5 The effect of pH on the reduction rate of TPH from
diesel fuel in the aqueous phase using photo- Fenton
like process. Figure 7 The effect of H2O2 on the reduction rate of TPH from
diesel fuel in the aqueous phase using photo- Fenton
like process. The effects of initial TPH concentration Wang S: A comparative study of Fenton and Fenton-like reaction kinetics
in decolorization of wastewater. Dyes Pigm 2008, 76:714–720. 6. Wang S: A comparative study of Fenton and Fenton-like reaction kinetics
in decolorization of wastewater. Dyes Pigm 2008, 76:714–720. 7. Dehghani M, Naseri S, Zamanian Z: Biodegradation of alachlor in liquid
and soil cultures under variable carbon and nitrogen sources by
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and soil cultures under variable carbon and nitrogen sources by
bacterial consortium isolated from corn field soil. J Environ Health Sci Eng
2013, 10(1):21. doi:10.1186/1735-2746-10-21. Conclusion Our results suggest that the photo-Fenton like process
(nZVI/UV/H2O2) can be used as a pre-treatment step for
the biological removal of TPH from diesel fuel in the
aqueous phase. In the conventional photo-Fenton like
methods, high concentrations of ferrous salts were used. Therefore, the large amount of ferrous sludge was formed. The current study revealed that a lower amount of Fe is
needed and the nZVI particles can be reused in a mag-
netic field. The variance analysis suggests that the optimal
conditions for TPH reduction rate from diesel fuel in the
aqueous phase using photo-Fenton like method (nZVI/
UV/H2O2) are as follows: the initial TPH concentration
equals to 0.7 mg/L, nZVI concentration 20 mg/L, H2O2
concentration equals to 5 mmol/L, pH 3, and the reaction
time of 60 min and degree of significance for the study pa-
rameters are 7.643, 9.33, 13.318, 15.185 and 6.588 percent,
respectively. The predicted removal rate in optimal condi-
tions was 95.8%, confirmed by the results of our study
which was between 95-100%. 8. Dehghani M, Naseri S, Karamimanesh M: Removal of
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tion rate of diesel fuel in the aqueous phase have been
shown in Figure 8. As initial TPH concentration of
diesel fuel increased from 0.1 to 0.7 mg/L, TPH reduc-
tion rate increased from 57.8 to 75.8% (Figure 8). How-
ever, as the initial TPH concentration increased from 0.7
to 1.0 mg/L, a decrease in the reduction rate in diesel
fuel degradation (72.7%) was seen. The decrease in the
TPH reduction rate at more than 0.7 mg/L was possibly
the result of increasing the turbidity of the solution and
consequently decreasing the UV radiation permeability. Figure 6 The effect of reaction time on the reduction rate of
TPH from diesel fuel in the aqueous phase using photo- Fenton
like process. Figure 8 The effect of initial TPH concentration on the
reduction rate of TPH from diesel fuel in the aqueous phase
using photo- Fenton like process. Figure 8 The effect of initial TPH concentration on the
reduction rate of TPH from diesel fuel in the aqueous phase
using photo- Fenton like process. Figure 6 The effect of reaction time on the reduction rate of
TPH from diesel fuel in the aqueous phase using photo- Fenton
like process. Figure 6 The effect of reaction time on the reduction rate of
TPH from diesel fuel in the aqueous phase using photo- Fenton
like process. Figure 8 The effect of initial TPH concentration on the
reduction rate of TPH from diesel fuel in the aqueous phase
using photo- Fenton like process. Figure 8 The effect of initial TPH concentration on the
reduction rate of TPH from diesel fuel in the aqueous phase
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http://www.ijehse.com/content/12/1/87 Besides, H2O2 photolysis depends on UV radiation in-
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the potential formation of hydroxyl radical and diesel
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Chiavone-Filho O: Application of the photo-Fenton process to the
treatment of wastewaters contaminated with diesel. Sci Total Environ
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Chiavone-Filho O: Application of the photo-Fenton process to the
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process for the removal of diesel fuel from the aqueous phase. Journal
of Environmental Health Science & Engineering 2014 12:87. 28. Mahvi AH, Maleki A, Rezaee R, Safari M: Reduction of humic substances in
water by application of ultrasound waves and ultraviolet irradiation. Iranian J Environ Health Sci En 2009, 6(4):233–240. 29. Dobaradran S, Nabizadeh R, Mahvi AH, Mesdaghinia AR, Naddafi K, Yunesian M,
Rastkari N, Nazmara S: Survey on degradation rates of trichloroethylene in
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7(4):307–312. 30. Mahvi AH: Application of ultrasonic technology for water and
wastewater treatment. Iranian J Environ Health Sci En 2009, 38(2):1–17. doi:10.1186/2052-336X-12-87
Cite this article as: Dehghani et al.: Optimizing photo-Fenton like
process for the removal of diesel fuel from the aqueous phase. Journal
of Environmental Health Science & Engineering 2014 12:87. 28. Mahvi AH, Maleki A, Rezaee R, Safari M: Reduction of humic substances in
water by application of ultrasound waves and ultraviolet irradiation. Iranian J Environ Health Sci En 2009, 6(4):233–240. 29. Dobaradran S, Nabizadeh R, Mahvi AH, Mesdaghinia AR, Naddafi K, Yunesian M,
Rastkari N, Nazmara S: Survey on degradation rates of trichloroethylene in
aqueous solutions by ultrasound. Iranian J Environ Health Sci En 2010,
7(4):307–312. 30. Mahvi AH: Application of ultrasonic technology for water and
wastewater treatment. Iranian J Environ Health Sci En 2009, 38(2):1–17. doi:10.1186/2052-336X-12-87
Cite this article as: Dehghani et al.: Optimizing photo-Fenton like
process for the removal of diesel fuel from the aqueous phase. Journal
of Environmental Health Science & Engineering 2014 12:87. 28. Mahvi AH, Maleki A, Rezaee R, Safari M: Reduction of humic substances in
water by application of ultrasound waves and ultraviolet irradiation. Iranian J Environ Health Sci En 2009, 6(4):233–240. 29. Dobaradran S, Nabizadeh R, Mahvi AH, Mesdaghinia AR, Naddafi K, Yunesian M,
Rastkari N, Nazmara S: Survey on degradation rates of trichloroethylene in
aqueous solutions by ultrasound. Iranian J Environ Health Sci En 2010,
7(4):307–312. doi:10.1186/2052-336X-12-87
Cite this article as: Dehghani et al.: Optimizing photo-Fenton like
process for the removal of diesel fuel from the aqueous phase. Journal
of Environmental Health Science & Engineering 2014 12:87. Submit your next manuscript to BioMed Central
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Biological control of weeds in the 22 Pacific island countries and territories: current status and future prospects
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NeoBiota
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Abstract Biological control of introduced weeds in the 22 Pacific island countries and territories (PICTs) began in
1911, with the lantana seed-feeding fly introduced into Fiji and New Caledonia from Hawaii. To date, a to
tal of 62 agents have been deliberately introduced into the PICTs to control 21 weed species in 17 countries. A further two agents have spread naturally into the region. The general impact of the 36 biocontrol agents
now established in the PICTs ranges from none to complete control of their target weed(s). Fiji has been
most active in weed biocontrol, releasing 30 agents against 11 weed species. Papua New Guinea, Guam,
and the Federated States of Micronesia have also been very active in weed biocontrol. For some weeds such
as Lantana camara, agents have been released widely, and can now be found in 15 of the 21 PICTs in which
the weed occurs. However, agents for other commonly found weeds, such as Sida acuta, have been released
in only a few countries in which the weed is present. There are many safe and effective biocontrol agents
already in the Pacific that could be utilised more widely, and highly effective agents that have been released
elsewhere in the world that could be introduced following some additional host specificity testing. This pa
per discusses the current status of biological control efforts against introduced weeds in the 22 PICTs and re
views options that could be considered by countries wishing to initiate weed biological control programmes. Biological control of weeds in the 22 Pacific island
countries and territories: current status
and future prospects Michael D. Day1, Rachel L. Winston2 1 Department of Agriculture and Fisheries, Ecosciences Precinct, GPO Box 267, Brisbane, Qld 4001 Australia
2 MIA Consulting, 316 N. Hansen Ave., Shelley, ID 83274 USA Corresponding author: Michael D. Day (michael.day@daf.qld.gov.au) Academic editor: C. Daehler | Received 6 November 2015 | Accepted 28 March 2016 | Published 23 June 2016 Academic editor: C. Daehler | Received 6 November 2015 | Accepted 28 March 2016 | Published 23 June 2016 Citation: Day MD, Winston RL (2016) Biological control of weeds in the 22 Pacific island countries and territories:
current status and future prospects. In: Daehler CC, van Kleunen M, Pyšek P, Richardson DM (Eds) Proceedings of 13th
International EMAPi conference, Waikoloa, Hawaii. NeoBiota 30: 167–192. doi: 10.3897/neobiota.30.7113 Biological co
NeoBiota 30: 167–192 (2016)
doi: 10.3897/neobiota.30.7113
http://neobiota.pensoft.net Biological co
NeoBiota 30: 167–192 (2016)
doi: 10.3897/neobiota.30.7113
http://neobiota.pensoft.net Biological co
NeoBiota 30: 167–192 (2016)
doi: 10.3897/neobiota.30.7113
http://neobiota.pensoft.net n the 22 Pacific isla
REVIEW ARTICLE Copyright Michael D. Day, Rachel L. Winston. 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. Introduction Introduced invasive weeds are of increasing concern and importance in the Pacific
region, which is reflected by the growing number of publications and websites docu
menting their distribution and impacts (e.g. Swarbrick 1997, Waterhouse 1997, Mey
er 2000, Shine et al. 2003, PIER 2013). Weeds decrease food security and income by
smothering crops, infesting plantations, and overgrowing grazing lands (Waterhouse
and Norris 1987, Orapa 2001, Day et al. 2012). Weeds also affect ecosystem processes
through impacts such as degrading soil and reducing water quality and quantity, and
are second only to land clearing as a major threat to biodiversity (Meyer 2000, Sherley
and Lowe 2000, Dovey et al. 2004). Since 1985, at least six workshops have been held
in the Pacific region to prioritise weeds for improved management (e.g. Waterhouse
and Norris 1987, Sherley 2000, Shine et al. 2003, Dodd and Hayes 2009, Day 2013). Biological control is a long-term, self-sustaining and feasible option for managing
many weeds (Dovey et al. 2004, Julien et al. 2007). Biocontrol of weeds is particu
larly beneficial and applicable to many Pacific island countries and territories (PICTs)
where the capacity to tackle major weed problems is often restricted due to limited
infrastructure, resources, and skills (Dovey et al. 2004). The earliest case of the delib
erate introduction of biocontrol agents from their native range to control a weed was
in 1902 when 23 insect species were imported into Hawaii from Mexico to control
Lantana camara (Swezey 1923). One agent, the seed-feeding fly Ophiomyia lantanae,
which successfully established in Hawaii, was subsequently introduced into Fiji and
New Caledonia in 1911 (Guiterrez and Forno 1989), becoming the first weed biocon
trol agent released in the PICTs. Introduced invasive weeds are of increasing concern and importance in the Pacific
region, which is reflected by the growing number of publications and websites docu
menting their distribution and impacts (e.g. Swarbrick 1997, Waterhouse 1997, Mey
er 2000, Shine et al. 2003, PIER 2013). Weeds decrease food security and income by
smothering crops, infesting plantations, and overgrowing grazing lands (Waterhouse
and Norris 1987, Orapa 2001, Day et al. 2012). Weeds also affect ecosystem processes
through impacts such as degrading soil and reducing water quality and quantity, and
are second only to land clearing as a major threat to biodiversity (Meyer 2000, Sherley
and Lowe 2000, Dovey et al. 2004). Introduction Since 1985, at least six workshops have been held
in the Pacific region to prioritise weeds for improved management (e.g. Waterhouse
and Norris 1987, Sherley 2000, Shine et al. 2003, Dodd and Hayes 2009, Day 2013). y
y
y
Biological control is a long-term, self-sustaining and feasible option for managing
many weeds (Dovey et al. 2004, Julien et al. 2007). Biocontrol of weeds is particu
larly beneficial and applicable to many Pacific island countries and territories (PICTs)
where the capacity to tackle major weed problems is often restricted due to limited
infrastructure, resources, and skills (Dovey et al. 2004). The earliest case of the delib
erate introduction of biocontrol agents from their native range to control a weed was
in 1902 when 23 insect species were imported into Hawaii from Mexico to control
Lantana camara (Swezey 1923). One agent, the seed-feeding fly Ophiomyia lantanae,
which successfully established in Hawaii, was subsequently introduced into Fiji and
New Caledonia in 1911 (Guiterrez and Forno 1989), becoming the first weed biocon
trol agent released in the PICTs. Over 60 weed biocontrol agents have since been introduced deliberately into 17
of the 22 PICTs, not including Australia, New Zealand, or Hawaii (Winston et al. 2014). However, for most biocontrol agents, the number of PICTs in which they have
been introduced or naturally spread is only a fraction of the number of PICTs where
the target weeds occur. Consequently, there is great potential for further introductions
within the PICTs. In addition, there are many more weeds present for which biocon
trol has not been attempted in the PICTs. Effective biocontrol agents for some of these
are available elsewhere and could be introduced. One of the limiting factors for weed biocontrol in many PICTs is the knowledge
of what agents are available and effective. Numerous workshops involving the PICTs
have been conducted, with the last being held in Auckland in 2009 (Dodd and Hayes
2009) where potential biocontrol agents were discussed. These workshops have often
resulted in new biocontrol programs being implemented, with new or existing agents
being introduced into one or more countries (Winston et al. 2014).hf This paper reviews the current status of biocontrol efforts against introduced weeds
in the PICTs and identifies existing biocontrol agents that could be moved around the
Pacific as well as additional effective biocontrol agents that could be introduced into
the region. Keywordsi Host specificity, establishment, biocontrol agents Copyright Michael D. Day, Rachel L. Winston. 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. 168 Michael D. Day & Rachel L. Winston / NeoBiota 30: 167–192 (2016) Materials and methods The number of weed biocontrol agents introduced into the 22 PICTs, their estab
lishment status, and their current impact were extracted from Winston et al. (2014)
and supplemented by recent publications and personal communications with local
researchers to provide an updated account through to 2015. The assessment did not
include Australia, Norfolk Island (a territory of Australia), New Zealand, Hawaii, and
Easter Island (a territory of Chile).f From the compiled dataset, we determined the weed biocontrol effort of each
country, including the number of weeds targeted and the number of agents deliber
ately introduced. We also analysed the dataset by target weed to determine how many
biocontrol agents have been introduced into the region, how many have established,
and their overall level of impact against their target weeds. The level of impact was
obtained from Winston et al. (2014) or from the perception of local researchers and
took into consideration varying habitats and climates, with the understanding that a
weed may not be under the same level of control in all areas where it exists. The two
analyses allowed us to ascertain which weeds were most amenable to biocontrol, and
which biocontrol agents were the most widespread, damaging, and effective against
their target weed. g
Numerous sources were utilized to determine the distribution of weeds in the Pa
cific, including workshop reports, websites, and personal communications with local
land managers (Swarbrick 1997, Waterhouse 1997, Meyer 2000, Shine et al. 2003,
Dodd and Hayes 2009, PIER 2013, Endemia 2015). Some of the weed biocontrol
prioritisation workshops utilized herein asked participants to list the top 10 weeds in
their country. In these circumstances, not all weeds present in a country were captured. The weed lists were then collated into a comprehensive compilation of weeds occurring
in each country and cross-checked against weed species that have already been targeted
for biocontrol worldwide (Winston et al. 2014), as well as against weed species being
evaluated as potential new candidates now or in the near future (Q. Paynter, Landcare
Research pers. comm. 2015, T. Johnson, US Department of Agriculture, pers. comm. 2015). Weed species not targeted for weed biocontrol were deleted from the dataset. After combining the two datasets, we determined which biocontrol agents could
be introduced into particular countries where the target weed occurs but no biocon
trol agents have established to date. Introduction This information provides a platform for PICTs to identify the best and
most appropriate weed biocontrol opportunities to pursue, and should be considered Biological control of weeds in the 22 Pacific island countries and territories... 169 against other factors such as weed importance and available resources in each country. Australia, Norfolk Island (a territory of Australia), New Zealand, and Hawaii are not
included in this paper as they already have well-established biocontrol programmes,
and extensive reviews on their programmes have already been conducted (Conant et
al. 2013, Fowler et al. 2000, 2010, Funasaki 1988, Julien et al. 2012, Smith 2002,
Trujillo 2005). Materials and methods In doing so, we only considered those biocontrol
agents that had been deliberately released into at least one country. This excluded spe Michael D. Day & Rachel L. Winston / NeoBiota 30: 167–192 (2016) 170 cies that had found their way into countries naturally but had never been deliberately
introduced into any country. The rationale behind excluding these species is that they
are not bona fide biocontrol agents, nor have they been subjected to detailed host
specificity testing; consequently, there is a risk of non-target impacts if introduced into
a new region. There are no native species in the Pacific region that have been used as
weed biocontrol agents. Results were separated into three lists based on whether 1) the agent is already es
tablished in at least one of the PICTs and is having at least a medium impact (weed is
partially or fully controlled in most areas) on the target weed, 2) the agent is not yet in
any PICTs but has at least a medium impact on the target weed elsewhere, and 3) the
agent has only a slight impact (may cause damage but does not reduce weed popula
tions) on the target weed either in any of the PICTs or elsewhere. A fourth list docu
ments the agents that have been recently released and are still being evaluated, and any
new target weeds for which agent exploration or host specificity testing of new agents
are currently being conducted. As much of the data on weed presence or importance
by country is not well defined, no attempt was made to suggest specific actions. ii
Our analysis excluded agents that did not establish in any country in which they
were introduced, agents that had established in at least one country but were consid
ered to have no impact against the target weed, and agents that have caused significant
impacts to non-target species. We determined that these agents were unlikely to suc
ceed in terms of achieving establishment and causing a significant impact to the target
weed and/or had great potential to damage non-target species in a new country (Julien
et al. 2007, Paynter et al. 2015). Results Seventeen of the 22 PICTs have deliberately introduced at least one biocontrol agent
(Table 1). Fiji (30 biocontrol agents introduced against 11 weed species) and Papua
New Guinea (19 agents released against 12 weed species) have been the most active. Guam (16 agents against 4 weed species), Federated States of Micronesia (13 agents
against 3 weed species), and Palau (11 agents against 4 weed species) have also been
actively involved in weed biocontrol. Five countries, namely Kiribati, Pitcairn Islands,
Tokelau, Tuvalu, and Wallis and Futuna, have not deliberately introduced any weed
biocontrol agents to date. These countries mainly consist of small, low-lying atolls, and
weeds may not be at sufficient densities to warrant biocontrol. fi
Since 1911, there has been a steady stream of biocontrol agents introduced into
the PICTs (Fig. 1). A total of 62 biocontrol agents targeting 21 weed species have been
deliberately released into at least one country in the PICTs (Table 2). Of these, 32
agents have established on 17 weed species. Two biocontrol agents, Neogalea sunia and
Epiblema strenuana, did not establish when deliberately introduced into the region,
but were later found to have spread into some PICTs of their own accord (Table
2). In addition, Acalitus adoratus and Maravalia cryptostegiae also self-introduced into Biological control of weeds in the 22 Pacific island countries and territories... 171 Table 1. The number of weed species targeted for biocontrol and the number of biocontrol agents that
have been deliberately introduced (intentional) and agents that were not deliberately introduced but have
been found (unintentional) in the PICTs. Table 1. The number of weed species targeted for biocontrol and the number of biocontrol agents that
have been deliberately introduced (intentional) and agents that were not deliberately introduced but have
been found (unintentional) in the PICTs. Intentional introductions
Unintentional
introductions
Combined
introductions
Country
No. of
weed
species
No. of
agents
released
No. of
agents
establ. No. of
weed
species
No. of
agents
establ. No. of
weed
species
No. of
agents
establ. Results American Samoa
2
2
2
0
0
2
2
Cook Islands
4
11
2
0
0
4
2
Federated States of
Micronesia
3
13
10
2
2
3
12
Fiji
11
30
17
0
0
11
17
French Polynesia
2
3
3
0
0
2
3
Guam
4
16
9
2
4
4
13
Marshall Islands
1
1
1
0
0
1
1
Nauru
1
1
0
0
0
1
0
New Caledonia
4
7
6
3
4
5
10
Niue
2
4
3
1
1
3
4
Northern Mariana
Islands
4
8
7
2
5
4
12
Palau
4
11
6
2
4
4
10
Papua New Guinea
12
19
12
3
6
13
18
Samoa
4
5
3
1
1
4
4
Solomon Islands
5
7
4
2
2
5
6
Tonga
3
6
5
2
2
4
7
Vanuatu
8
9
8
3
6
9
14
Figure 1. Cumulative number of deliberate biocontrol agent introductions in the PICTs since 1911. The
values include those introductions where the agent failed to establish in any country. Intentional introductions
Unintentional
introductions
Combined
introductions
Country
No. of
weed
species
No. of
agents
released
No. of
agents
establ. No. of
weed
species
No. of
agents
establ. No. of
weed
species
No. of
agents
establ. American Samoa
2
2
2
0
0
2
2
Cook Islands
4
11
2
0
0
4
2
Federated States of
Micronesia
3
13
10
2
2
3
12
Fiji
11
30
17
0
0
11
17
French Polynesia
2
3
3
0
0
2
3
Guam
4
16
9
2
4
4
13
Marshall Islands
1
1
1
0
0
1
1
Nauru
1
1
0
0
0
1
0
New Caledonia
4
7
6
3
4
5
10
Niue
2
4
3
1
1
3
4
Northern Mariana
Islands
4
8
7
2
5
4
12
Palau
4
11
6
2
4
4
10
Papua New Guinea
12
19
12
3
6
13
18
Samoa
4
5
3
1
1
4
4
Solomon Islands
5
7
4
2
2
5
6
Tonga
3
6
5
2
2
4
7
Vanuatu
8
9
8
3
6
9
14 Figure 1. Cumulative number of deliberate biocontrol agent introductions in the PICTs since 1911. The
values include those introductions where the agent failed to establish in any country. Michael D. Day & Rachel L. Results Winston / NeoBiota 30: 167–192 (2016) 172 Table 2. Status of weed biocontrol agents deliberately released (intentional) and/or spread of their own accord (unintentional) into the 22 PICTs and the potential
countries in which they could be introduced. Countries: AS=American Samoa, CI=Cook Islands, FSM=Federated States of Micronesia, Fi=Fiji, FP=French Poly
nesia, Gu=Guam, Ki=Kiribati, MI=Marshall Islands, Na=Nauru, NC=New Caledonia, Ni=Niue, NMI=Northern Mariana Islands, Pa=Palau, PNG=Papua New
Guinea, PI=Pitcairn Islands, Sa=Samoa, SI=Solomon Islands, Tk=Tokelau, To=Tonga, Tu=Tuvalu, Va=Vanuatu, WF=Wallis & Futuna. Status: I=intentionally intro
duced, U=unintentionally introduced, E=established, F=failed to establish. Impact: H=high, M=moderate, N=none, S=slight, V=variable, ?=unknown. * Potential
countries where agents could be introduced (based on weed occurrence in each country, not weed density). Weed family
Weed species
Biocontrol
agent family
Biocontrol agent species
AS
CI
FSM
Fi
FP
Gu
Ki MI Na NC
Ni
NMI
Pa
PNG PI
Sa
SI
Tk
To
Tu
Va
WF
Apocynaceae
Cryptostegia
grandiflora
R. Br. Chaconiaceae
Maravalia cryptostegiae
(Cummins) Ono
UE? Araceae
Pistia stratiotes
L. Curculionidae
Neohydronomus affinis
Hustache
*
*
*
*
*
*
*
IEV
*
IEV
Asteraceae
Chromolaena
odorata (L.) R. M. King & H. Rob. Eriophyidae
Acalitus adoratus Keifer
UES
UES
UES
UES
UES
Brentidae
Apion brunneonigrum
Béguin-Billecocq
IF
Agromyzidae
Calycomyza eupatorivora
Spencer
IF
Tephritidae
Cecidochares connexa
Macquart
IEH
IEM
*
*
IEH
IES
IEV
Erebidae
Pareuchaetes
pseudoinsulata Rego
Barros
IEV
IEM
IEV
IF
IEV
Pyralidae
Phestinia costella
Hampson
IF
Elephantopus
mollis Kunth
Tephritidae
Tetraeuaresta
obscuriventris (Loew)
*
*
IEM
*
*
*
UE? *
*
*
*
UEN
UEN
*
Mikania
micrantha
Kunth
Phlaeothripidae
Liothrips mikaniae
(Priesner)
IF
Pucciniaceae
Puccinia spegazzinii De
Toni
*
*
IE? *
*
*
*
*
*
*
*
IE? *
*
UE? *
*
*
IE? *
Parthenium
hysterophorus L. Tortricidae
Epiblema strenuana
(Walker)
*
*
UE? Chrysomelidae
Zygogramma bicolorata
Pallister
I? Xanthium
strumarium L. Tortricidae
Epiblema strenuana
(Walker)
*
*
*
*
*
IF*
*
Tephritidae
Euaresta aequalis Loew
IF of weed biocontrol agents deliberately released (intentional) and/or spread of their own accord (unintentional) into the 22 PICTs and the potential
h they could be introduced. Countries: AS=American Samoa, CI=Cook Islands, FSM=Federated States of Micronesia, Fi=Fiji, FP=French Poly
, Ki=Kiribati, MI=Marshall Islands, Na=Nauru, NC=New Caledonia, Ni=Niue, NMI=Northern Mariana Islands, Pa=Palau, PNG=Papua New
irn Islands, Sa=Samoa, SI=Solomon Islands, Tk=Tokelau, To=Tonga, Tu=Tuvalu, Va=Vanuatu, WF=Wallis & Futuna. Status: I=intentionally intro
ntionally introduced, E=established, F=failed to establish. Impact: H=high, M=moderate, N=none, S=slight, V=variable, ?=unknown. Results * Potential
gents could be introduced (based on weed occurrence in each country, not weed density). Table 2. Status of weed biocontrol agents deliberately released (intentional) and/or spread of their own accord (unintentional) into the 22 PICTs and the potential
countries in which they could be introduced. Countries: AS=American Samoa, CI=Cook Islands, FSM=Federated States of Micronesia, Fi=Fiji, FP=French Poly
nesia, Gu=Guam, Ki=Kiribati, MI=Marshall Islands, Na=Nauru, NC=New Caledonia, Ni=Niue, NMI=Northern Mariana Islands, Pa=Palau, PNG=Papua New
Guinea, PI=Pitcairn Islands, Sa=Samoa, SI=Solomon Islands, Tk=Tokelau, To=Tonga, Tu=Tuvalu, Va=Vanuatu, WF=Wallis & Futuna. Status: I=intentionally intro
duced, U=unintentionally introduced, E=established, F=failed to establish. Impact: H=high, M=moderate, N=none, S=slight, V=variable, ?=unknown. * Potential
countries where agents could be introduced (based on weed occurrence in each country, not weed density). Biological control of weeds in the 22 Pacific island countries and territories... 173 Weed family
Weed species
Biocontrol
agent family
Biocontrol agent species
AS
CI
FSM
Fi
FP
Gu
Ki MI Na NC
Ni
NMI
Pa
PNG PI
Sa
SI
Tk
To
Tu
Va
WF
Cerambycidae
Nupserha vexator (Pascoe)
IF
Pucciniaceae
Puccinia xanthii
Schweinitz
I? Cactaceae
Acanthocereus
tetragonus (L.)
Hummelinck
Pseudococcidae
Hypogeococcus festerianus
(Lizer y Trelles)
IF
Opuntia spp. Dactylopiidae
Dactylopius sp. nr
confusus (Cockerell)
IEH
Opuntia ficus-
indica (L.) Mill. Pyralidae
Cactoblastis cactorum
(Berg)
*
*
Opuntia
monacantha
(Willd.) Haw. Pyralidae
Cactoblastis cactorum
(Berg)
*
*
*
*
*
*
*
*
*
*
*
*
*
Opuntia stricta
(Haw.) Haw. Pyralidae
Cactoblastis cactorum
(Berg)
IEH
*
*
Cucurbitaceae
Coccinia grandis
(L.) Voigt
Curculionidae
Acythopeus burkhartorum
O’Brien & Pakaluk
IF
IF
Curculionidae
Acythopeus cocciniae
O’Brien & Pakaluk
*
*
IEH
*
IEM
*
*
*
*
*
*
Sesiidae
Melittia oedipus
Oberthür
*
*
IEH
*
IE? *
*
*
*
*
*
Cyperaceae
Cyperus
rotundus L. Curculionidae
Athesapeuta cyperi
Marshall
IF
IF
IEN
Tortricidae
Bactra minima Meyrick
IF
IF
IEN
Tortricidae
Bactra venosana (Zeller)
IF
IEN
UE? Fabaceae
Mimosa
diplotricha C. Wright
Psyllidae
Heteropsylla spinulosa
Muddiman, Hodkinson
& Hollis
IEH IEH IEH
IEH
*
IE? *
IEH
IE? IEH
IEV
IEH IEH
IEH
IES
*
Saturniidae
Psigida walkeri (Grote)
IF
Coreidae
Scamurius sp. IF
Mimosa pigra L. Chrysomelidae
Acanthoscelides puniceus
Johnson
I? Chrysomelidae
Acanthoscelides
quadridentatus
(Schaeffer)
I? Malvaceae
Sida acuta
Burm. f. Chrysomelidae
Calligrapha pantherina
Stål
*
*
*
IEH
*
*
*
*
*
UE? *
*
*
IEH
I? *
*
IEH
Sida rhombifolia
L. Results Chrysomelidae
Calligrapha pantherina
Stål
*
*
*
IEH
*
*
*
*
*
*
*
*
IEH
*
I? *
*
*
*
IEH
* Michael D. Day & Rachel L. Winston / NeoBiota 30: 167–192 (2016) 174 Weed family
Weed species
Biocontrol
agent family
Biocontrol agent species
AS
CI
FSM
Fi
FP
Gu
Ki MI Na NC
Ni
NMI
Pa
PNG PI
Sa
SI
Tk
To
Tu
Va
WF
Melastomataceae
Clidemia hirta
(L.) D. Don
Crambidae
Ategumia matutinalis
(Guenée)
IF
Phlaeothripidae
Liothrips urichi Karny
IEM
*
IEH
IEV
*
*
IF*
*
Miconia
calvescens DC. Glomerellaceae
Colletotrichum
gloeosporioides (Penz.)
Penz. & Sacc. f. sp. miconiae Killgore & L. Sugiyama
IEV
*
*
Pontederiaceae
Eichhornia
crassipes (Mart.)
Solms
Erirhinidae
Neochetina bruchi
Hustache
*
*
*
*
*
*
*
*
*
*
*
IEH
*
*
IE? Erirhinidae
Neochetina eichhorniae
Warner
*
*
*
IEH
*
*
*
I? *
*
*
IEH
*
IES
IEH
Crambidae
Niphograpta albiguttalis
(Warren)
IF
Crambidae
Xubida infusella (Walker)
I? Salviniaceae
Salvinia molesta
D.S. Mitch. Erirhinidae
Cyrtobagous salviniae
Calder & Sands
*
IEH
*
*
*
*
IEH
Erirhinidae
Cyrtobagous singularis
Hustache
IEN
Pauliniidae
Paulinia acuminata (De
Geer)
IEN
Crambidae
Samea multiplicalis
(Guenée)
IEN
Verbenaceae
Lantana camara
L. sens. lat. Agromyzidae
Calycomyza lantanae
(Frick)
UEM
IE? UE? UE? UES
UES
UES
Chrysomelidae
Charidotis pygmaea Klug
IF
Tortricidae
Crocidosema lantana
Busck
IEM
UEM
IE? UEM UEM
UES
Noctuidae
Diastema tigris Guenée
IF
IF
Erebidae
Hypena laceratalis Walker
IEN
IES
IEN
UES
UEN
UES
UES
Pterophoridae
Lantanophaga
pusillidactyla (Walker)
IEM
UEM
UEM
IES
UES
Tingidae
Leptobyrsa decora Drake
I? IF
IF
IF
I? Noctuidae
Neogalea sunia (Guenée)
IF
UES
Chrysomelidae Octotoma championi Baly
IF
Chrysomelidae
Octotoma scabripennis
Guérin-Méneville
*
IF*
*
IF*
*
IF*
*
*
*
IES
IF*
*
*
*
*
*
I? *
*
*
* 175 Biological control of weeds in the 22 Pacific island countries and territories... Weed family
Weed species
Biocontrol
agent family
Biocontrol agent species
AS
CI
FSM
Fi
FP
Gu
Ki MI Na NC
Ni
NMI
Pa
PNG PI
Sa
SI
Tk
To
Tu
Va
WF
Agromyzidae
Ophiomyia lantanae
(Froggatt)
I? IE? IES
IE? IEM
IEM
UEM UEM
UE? UE? UE? UE? Cerambycidae
Plagiohammus spinipennis
(Thomson)
IF
IF
Crambidae
Pseudopyrausta
santatalis (Barnes &
McDunnough)
IF
IF
Crambidae
Salbia haemorrhoidalis
Guenée
IES
IES
IF
IF
Lycaenidae
Strymon bazochii
(Godart)
I? Results Tingidae
Teleonemia elata Drake
IF
Tingidae
Teleonemia scrupulosa Stål
*
*
IEV
IEV
IE? IEV
*
*
*
IEV IEH
IEV
IEV
IEH
*
IEH IEH
IES
*
IES
*
Lycaenidae
Tmolus echion (L.)
IF
Chrysomelidae
Uroplata fulvopustulata
Baly
IF
Chrysomelidae
Uroplata girardi Pic
*
IEH IEM
IEV
*
IEM
*
*
*
IEM IEM
IEV
IEM
IES
*
IEM IEH
IEM
*
IES
*
gophyllaceae
Tribulus cistoides
L. Curculionidae
Microlarinus lareynii
(Jacquelin du Val)
IF
Microlarinus lypriformis
(Wollaston)
*
*
*
*
*
*
*
IEH
Tribulus
terrestris L. Curculionidae
Microlarinus lypriformis
(Wollaston)
*
* Michael D. Day & Rachel L. Winston / NeoBiota 30: 167–192 (2016) 176 some PICTs. In total, 36 weed biocontrol agents are now confirmed as present in the
PICTs, attacking 19 weed species. The overall impact of these biocontrol agents ranges
from no damage to high impact on the target weed, depending on country and region
(Tables 2, 3). Of the weed species on which at least one biocontrol agent has established, seven are
deemed to be under complete control overall, due to the high impact of the agent(s) (Ta
ble 3). A further six weed species are deemed to be under partial to full control. The im
pacts of biocontrol agents on two weed species have been variable. For four weed species
where biocontrol agents have only recently established, the establishment and impacts of
biocontrol agents are still being evaluated. There are three weed species for which agents
have either not established, or there is little, no, or unknown impact of biocontrol agents.h The most widespread and damaging biocontrol agent in the PICTs is the psyl
lid Heteropsylla spinulosa, which was introduced and has established in 13 of the 16
countries where its target weed Mimosa diplotricha occurs. In most areas within most
countries, M. diplotricha is under control (Tables 2, 3). However, in high rainfall areas,
control is not always achieved because heavy rain can wash the psyllids from plants. Sida acuta and S. rhombifolia are deemed under control in three of the four coun
tries where the leaf-feeding beetle Calligrapha pantherina was intentionally introduced
and established. The establishment of C. pantherina in the fourth country, Samoa,
is not known. Calligrapha pantherina has recently been reported in New Caledonia,
although its mode of entry and impact on the Sida spp. are unknown. Results Other weeds
considered under control by biocontrol agents in the PICTs include Salvinia molesta,
Tribulus cistoides, Opuntia stricta, and unspecified Opuntia spp. (Tables 2, 3). i
Eichhornia crassipes and Pistia stratiotes are generally under a high degree of control
in each of the countries where their respective biocontrol agents have been released and
established (Tables 2, 3). Control of E. crassipes is generally higher if both Neochetina
eichhorniae and N. bruchi are present. Control of both aquatic weeds appears to be
incomplete in shaded locations. Cecidochares connexa has established and is aiding the control of Chromolaena odo
rata in all five countries in which it has been introduced (Tables 2, 3). However, C. connexa appears to be less effective at altitudes greater than 1000 m above sea level or
in areas where rainfall is high, such as West New Britain, Papua New Guinea. Of the two agents introduced to control Clidemia hirta, only Liothrips urichi estab
lished. This agent appears to be effective at controlling C. hirta in only sunny areas of
the three countries in which it has established (Tables 2, 3); there is little impact where
C. hirta is growing in shaded areas. Three agents have been released against Coccinia grandis, but only two have estab
lished. Melittia oedipus has been released in Guam and the Northern Mariana Islands,
and is having a high degree of impact in both countries. Acythopeus cocciniae is having
a high degree of impact in Guam, while its establishment in the Northern Mariana
Islands has not been confirmed (Tables 2, 3). i
Twenty biocontrol agents have been intentionally introduced against L. camara
in the PICTs. Of these, nine agents have established in at least one country (Table 2). Biological control of weeds in the 22 Pacific island countries and territories... 177 Table 3. Summary of the biocontrol effort against each target weed species, including the number of
PICTs where biocontrol agents have established without being deliberately released. For weeds where
multiple agents have been released, numbers have been pooled. Weed family
Weed species
No. countries
weed occurs
No. agents
established in
the Pacific
No. Results countries
all agents
established
Overall impact
on weed**
Apocynaceae
Cryptostegia grandiflora
8
1
1
unknown
Araceae
Pistia stratiotes
9
1
2
medium to
high
Asteraceae
Chromolaena odorata
7
3
5
medium to
high
Elephantopus mollis
14
1
4
variable
Mikania micrantha
20
1
4
still evaluating
Parthenium hysterophorus
3
1
1
still evaluating
Xanthium strumarium
7
0*
0
still evaluating
Cactaceae
Acanthocereus tetragonus
1
0
0
none
Opuntia spp. 1
1
1
high
Opuntia stricta
3
1
1
high
Cucurbitaceae
Coccinia grandis
11
2
2
medium to
high
Cyperaceae
Cyperus rotundus
21
3
2
none
Fabaceae
Mimosa diplotricha
16
1
13
high
Mimosa pigra
1
0*
0
still evaluating
Malvaceae
Sida acuta
18
1
4
high
Sida rhombifolia
22
1
3
high
Melastomataceae
Clidemia hirta
9
1
3
low to high
Miconia calvescens
3
1
1
variable
Pontederiaceae
Eichhornia crassipes
15
2
4
medium to
high
Salviniaceae
Salvinia molesta
7
4
2
high
Verbenaceae
Lantana camara
21
10
15
slight to high
Zygophyllaceae
Tribulus cistoides
8
1
1
high
* Biocontrol agents have recently been released, but establishment is not confirmed
** Rating is based on the overall level of control as per Winston et al. (2014) * Biocontrol agents have recently been released, but establishment is not confirmed
** Rating is based on the overall level of control as per Winston et al. (2014) Uroplata girardi and Teleonemia scrupulosa have been released and have established in
13 countries; both reportedly have a moderate to high overall impact in most coun
tries where they have established. Crocidosema lantana, Lantanophaga pusillidactyla,
and Ophyiomyia lantanae have a moderate impact in some countries but only a slight
impact in other countries. The remaining agents have little or no impact on L. camara. ph
g g
p
Of the biocontrol agents that have established in the PICTs and are having a me
dium to high impact on the target weed, many have not been released in all PICTs
where their respective target weed has been recorded. For example, C. pantherina has
proven very effective against S. acuta and S. rhombifolia in three countries, and could
potentially be introduced into 14 and 18 additional countries, respectively. Likewise, 178 Michael D. Day & Rachel L. Winston / NeoBiota 30: 167–192 (2016) N. bruchi and N. eichhorniae could potentially be introduced against E. crassipes in 13
additional countries, while the biocontrol agents for C. Results grandis could be introduced
into nine countries. Cactoblastis cactorum was introduced into New Caledonia to control O. stricta. However, the agent also attacks Opuntia monacantha, and so could be released in the
13 countries in which this weed occurs. Similarly, Microlarinus lypriformis was released
against Tribulus cistoides, but could also be used against Tribulus terrestris in Fiji and
Papua New Guinea. The countries in which established and effective agents within
the PICTs could potentially be redistributed are listed in Table 2. Because biocontrol
agents can spread naturally between islands, it is recommended that countries conduct
surveys to determine what biocontrol agents are present prior to any introductions.hf There are also opportunities to introduce biocontrol agents that have proven effec
tive outside the PICTs (Table 4), provided target weed densities are sufficiently high to
warrant this. Additional agents attacking L. camara, O. stricta, and Parthenium hystero
phorus could be introduced in the PICTs to supplement the biocontrol agents already
established against these species. There are also effective agents for weeds that have not
been targeted for biocontrol in the PICTs to date. These weed species include Arundo
donax (present in 12 countries), Dolichandra unguis-cati (7 countries), and Melaleuca
quinquenervia (7 countries) (Table 4). Because biocontrol agents may do poorly in one region and have spectacular suc
cess elsewhere, agents having slight or variable impacts on their target weed(s) in at
least one country within or outside the Pacific region are listed in Table 5. i
Numerous weed species occurring in the PICTs are currently weed biocontrol tar
gets elsewhere, but the agents have either been only recently released and not yet evalu
ated or not yet released (Table 6). In addition, there are several previously targeted
weeds (e.g. C. odorata, E. crassipes, and L. camara) for which new agents were recently
released and are currently being evaluated for establishment and/or impact (Table 6). Should any of these agents prove to be specific and effective against their target weeds,
they could also be considered for introduction in the PICTs in the future. Discussion Biological control of weeds has been practiced in the PICTs for over 100 years, with
over 20 weed species targeted. In that time, 17 countries have deliberately introduced
at least one biocontrol agent (Winston et al. 2014). In addition to agents deliberately
released into the PICTs, four biocontrol agents have found their way into the Pacific
region either through natural means or unintentionally on imported goods. For over
half the weed species targeted, biocontrol agents are having a medium to high impact. Consequently, weed biocontrol to date has been very cost-effective and has provided
relief to farmers and land managers trying to control those weeds, and has resulted in
increased production and income (e.g. Julien and Orapa 2001, Day et al. 2013a, Day
and Bule this edition). Biological control of weeds in the 22 Pacific island countries and territories... 179 Table 4. Weed biocontrol agents that have medium to high impacts in at least one country outside the PICTs and could be introduced into the region. Prior to
introduction, additional host specificity testing may be needed. Countries: AS=American Samoa, CI=Cook Islands, FSM=Federated States of Micronesia, Fi=Fiji,
FP=French Polynesia, Gu=Guam, Ki=Kiribati, MI=Marshall Islands, Na=Nauru, NC=New Caledonia, Ni=Niue, NMI=Northern Mariana Islands, Pa=Palau,
PNG=Papua New Guinea, PI=Pitcairn Islands, Sa=Samoa, SI=Solomon Islands, Tk=Tokelau, To=Tonga, Tu=Tuvalu, Va=Vanuatu, WF=Wallis & Futuna. Weed family
Weed species
Biocontrol agent family
Biocontrol agent species
Possible countries for
introduction#
Asteraceae
Ageratina adenophora (Spreng.) R. M. King
& H. Rob. Mycosphaerellaceae
Passalora ageratinae Crous & A.R. Wood
FP
Parthenium hysterophorus
Chrysomelidae
Zygogramma bicolorata*
FP, NC, Va
Curculionidae
Listronotus setosipennis (Hustache)
FP, NC, Va
Xanthium strumarium
Pucciniaceae
Puccinia xanthii Schweinitz*
CI, Fi, FP, Gu, NC, PNG, To
Azollaceae
Azolla filiculoides Lam. Erirhinidae
Stenopelmus rufinasus Gyllenhal
CI
Basellaceae
Anredera cordifolia (Ten.) Steenis
Chrysomelidae
Plectonycha correntina Lacordaire
CI, Fi, FP, NC, Ni, PI
Bignoniaceae
Dolichandra unguis-cati (L.) L. G. Lohmann
Buprestidae
Hedwigiella jureceki (Obenberger)
CI, FSM, FP, Gu, NC, Ni, Va
Tingidae
Carvalhotingis visenda Drake
CI, FSM, FP, Gu, NC, Ni, Va
Cactaceae
Opuntia ficus-indica
Dactylopiidae
Dactylopius opuntiae (Cockerell)
FP, NC
Opuntia monacantha
Dactylopiidae
Dactylopius ceylonicus (Green)
AS, CI, FSM, Fi, Gu, Na, NC,
Ni, NMI, Pa, Sa, SI, To
Opuntia stricta
Dactylopiidae
Dactylopius opuntiae (Cockerell)
NC, Sa, SI
Pereskia aculeata Mill. Chrysomelidae
Phenrica guerini Bechyné
FP, NC, Pa
Fabaceae
Acacia dealbata Link
Curculionidae
Melanterius maculatus Lea
FP
Acacia mearnsii De Wild. Discussion Curculionidae
Anthonomus santacruzi Hustache
CI, Fi, FP, NC, SI, To
Tingidae
Gargaphia decoris Drake
CI, Fi, FP, NC, SI, To
Verbenaceae
Lantana camara
Agromyzidae
Ophiomyia camarae Spencer
AS, CI, FSM, Fi, FP, Gu, Ki,
MI, Na, NC, Ni, NMI, Pa,
PNG, PI, Sa, SI, To, Tu, Va, WF
Eriophyidae
Aceria lantanae (Cook)
AS, CI, FSM, Fi, FP, Gu, Ki,
MI, Na, NC, Ni, NMI, Pa,
PNG, PI, Sa, SI, To, Tu, Va, WF
Miridae
Falconia intermedia (Distant)
AS, CI, FSM, Fi, FP, Gu, Ki,
MI, Na, NC, Ni, NMI, Pa,
PNG, PI, Sa, SI, To, Tu, Va, WF
Zygophyllaceae
Tribulus cistoides
Curculionidae
Microlarinus lareynii*
CI, Fi, FP, Gu, Ki, MI, NC,
PNG
Tribulus terrestris
Curculionidae
Microlarinus lareynii
Fi, PNG
* Introduced previously but failed to establish
# Based on weed occurrence in each country, not weed density Biological control of weeds in the 22 Pacific island countries and territories... 181 Table 5. Weed biocontrol agents that have slight, variable, or unknown impacts in at least one country within or outside the PICTs that could be investigated
further to assess their suitability for introduction/redistribution in the region. Prior to introduction, additional host specificity testing may be needed. Coun
tries: AS=American Samoa, CI=Cook Islands, FSM=Federated States of Micronesia, Fi=Fiji, FP=French Polynesia, Gu=Guam, Ki=Kiribati, MI=Marshall Islands,
Na=Nauru, NC=New Caledonia, Ni=Niue, NMI=Northern Mariana Islands, Pa=Palau, PNG=Papua New Guinea, PI=Pitcairn Islands, Sa=Samoa, SI=Solomon
Islands, Tk=Tokelau, To=Tonga, Tu=Tuvalu, Va=Vanuatu, WF=Wallis & Futuna. Weed family
Weed species
Biocontrol agent
family
Biocontrol agent species
No. of countries
in Pacific agent
established
Possible countries for
introduction#
Apocynaceae
Cryptostegia grandiflora
Crambidae
Euclasta whalleyi Popescu-Gorj &
Constantinescu
Fi, FP, Gu, MI, NC, NMI, PNG, SI
Chaconiaceae
Maravalia cryptostegiae
1
Fi, FP, Gu, MI, NC, NMI, SI
Asteraceae
Ageratina adenophora
Pterophoridae
Oidaematophorus beneficus Yano & Heppner
FP
Tephritidae
Procecidochares utilis Stone
FP
Chromolaena odorata
Agromyzidae
Calycomyza eupatorivora
FSM, Gu, MI, NC, NMI, Pa, PNG
Erebidae
Pareuchaetes insulata (Walker)
FSM, Gu, MI, NC, NMI, Pa, PNG
Erebidae
Pareuchaetes pseudoinsulata
5
MI, NC
Cirsium vulgare (Savi) Ten. Curculionidae
Larinus carlinae (Olivier)
NC
Curculionidae
Rhinocyllus conicus (Frölich)
NC
Curculionidae
Trichosirocalus horridus (Panzer)
NC
Syrphidae
Cheilosia grossa (Fallén)
NC
Tephritidae
Urophora stylata (Fabricius)
NC
Parthenium hysterophorus
Bucculatricidae
Bucculatrix parthenica Bradley
FP, NC, Va
Curculionidae
Conotrachelus albocinereus Fiedler
FP, NC, Va
Curculionidae
Smicronyx lutulentus Dietz
FP, NC, Va
Delphacidae
Stobaera concinna (Stål)
FP, NC, Va
Pucciniaceae
Puccinia abrupta Dietel & Holw. var. partheniicola (H.S. Discussion Cecidomyiidae
Dasineura rubiformis Kolesik
CI
Curculionidae
Melanterius maculatus Lea
CI
Acacia melanoxylon R. Br. Curculionidae
Melanterius acaciae Lea
CI
Acacia pycnantha Benth. Curculionidae
Melanterius maculatus Lea
Gu
Pteromalidae
Trichilogaster signiventris (Girault)
Gu
Mimosa pigra
Chrysomelidae
Acanthoscelides spp. PNG
Chrysomelidae
Malacorhinus irregularis Jacoby
PNG
Curculionidae
Chalcodermus serripes Fåhraeus
PNG
Geometridae
Macaria pallidata (Warren)
PNG
Gracillariidae
Neurostrota gunniella (Busck)
PNG
Sesiidae
Carmenta mimosa Eichlin & Passoa
PNG Michael D. Day & Rachel L. Winston / NeoBiota 30: 167–192 (2016) 180 Weed family
Weed species
Biocontrol agent family
Biocontrol agent species
Possible countries for
introduction#
Paraserianthes lophantha (Willd.) Nielsen
Curculionidae
Melanterius servulus Pascoe
CI
Ulex europaeus L. Tetranychidae
Tetranychus lintearius Dufour
PNG
Vachellia nilotica subsp. indica (Benth.) Kyal. & Boatwr
Geometridae
Chiasmia assimilis (Warren)
FP, NC, SI, WF
Hydrocharitaceae
Hydrilla verticillata (L. f.) Royle
Ephydridae
Hydrellia pakistanae Deonier
Fi, Gu, NC, PNG
Lamiaceae
Marrubium vulgare L. Pterophoridae
Wheeleria spilodactylus (Curtis)
NC
Sesiidae
Chamaesphecia mysiniformis Rambur
NC
Myrtaceae
Melaleuca quinquenervia (Cav.) S. T. Blake
Cecidomyiidae
Lophodiplosis trifida Gagné
FSM, Fi, FP, Gu, NC, Pa, PNG
Curculionidae
Oxyops vitiosa Pascoe
FSM, Fi, FP, Gu, NC, Pa, PNG
Psyllidae
Boreioglycaspis melaleucae Moore
FSM, Fi, FP, Gu, NC, Pa, PNG
Pucciniaceae
Puccinia psidii G. Winter
FSM, Fi, FP, Gu, NC, Pa, PNG
Passifloraceae
Passiflora tarminiana Coppens & V. E. Barney
Mycosphaerellaceae
Septoria passiflorae Pallister
Gu
Poaceae
Arundo donax L. Eurytomidae
Tetramesa romana Walker
CI, FSM, Fi, FP, Gu, Na, NC,
Pa, PNG, Sa, To, WF
Polygonaceae
Rumex crispus L. Sesiidae
Pyropteron doryliformis (Ochsenheimer)
Fi, FP, NC, PNG
Solanaceae
Solanum mauritianum Scop. Discussion Jacks.) Parmelee
FP, NC, Va
Pucciniaceae
Puccinia xanthii Schwein. var. parthenii-
hysterophorae Seier, H.C. Evans & Á. Romero
FP, NC, Va
Sesiidae
Carmenta sp. nr ithacae (Beutenmüller)
FP, NC, Va
Tortricideae
Platphalonidia mystica (Razowski & Becker)
FP, NC, Va Michael D. Day & Rachel L. Winston / NeoBiota 30: 167–192 (2016) 182 Weed family
Weed species
Biocontrol agent
family
Biocontrol agent species
No. of countries
in Pacific agent
established
Possible countries for
introduction#
Pluchea carolinensis (Jacq.)
G. Don
Tephritidae
Acinia picturata (Snow)
CI, FP, Gu, Ki, MI, Na, NC, NMI,
Pa, To, Va, WF
Xanthium strumarium
Cerambycidae
Nupserha vexator
CI, Fi, FP, Gu, NC, PNG, To
Bignoniaceae
Dolichandra unguis-cati
Chrysomelidae
Charidotis auroguttata Boheman
CI, FSM, FP, Gu, NC, Ni, Va
Tingidae
Carvalhotingis hollandi Drake
CI, FSM, FP, Gu, NC, Ni, Va
Cactaceae
Opuntia ficus-indica
Cerambycidae
Lagocheirus funestus Thomson
FP, NC
Dryophthoridae
Metamasius spinolae (Gyllenhal)
FP, NC
Nectriaceae
Fusarium oxysporum Schlecktendahl
FP, NC
Opuntia monacantha
Dactylopiidae
Dactylopius opuntiae
AS, CI, FSM, Fi, Gu, Na, NC, Ni,
NMI, Pa, Sa, SI, To
Opuntia stricta
Cerambycidae
Moneilema blapsides (Newman) subsp. ulkei
Horn
NC, Sa, SI
Convolvulaceae
Convolvulus arvensis L. Eriophyidae
Aceria malherbae Nuzzaci
Pa
Noctuidae
Tyta luctuosa (Denis & Schiffermüller)
Pa
Fabaceae
Acacia podalyriifolia A. Cunn. ex G. Don
Curculionidae
Melanterius maculatus
NC
Caesalpinia decapetala
(Roth) Alston
Chrysomelidae
Sulcobruchus subsuturalis (Pic)
Fi, FP, NC
Leucaena leucocephala
(Lam.) de Wit
Chrysomelidae
Acanthoscelides macrophthalmus (Schaeffer)
AS, CI, FSM, Fi, FP, Gu, Ki, MI,
Na, NC, Ni, NMI, Pa, PNG, PI, Sa,
SI, To, Tu, Va, WF
Mimosa pigra
Brentidae
Coelocephalapion pigrae Kissinger
PNG
Cerambycidae
Rhytiphora piperitia Hope
PNG
Chrysomelidae
Chlamisus mimosae Karren
PNG
Geometridae
Leuciris fimbriaria (Stoll)
PNG
Parkinsonia aculeata
Chrysomelidae
Penthobruchus germaini (Pic)
FSM, FP, Gu, NC, SI
Prosopis juliflora (Sw.) DC. Chrysomelidae
Algarobius prosopis (Le Conte)
FP, PNG
Ulex europaeus
Brentidae
Exapion ulicis (Forster)
PNG
Oecophoridae
Agonopterix umbellana (Fabricius)
PNG Biological control of weeds in the 22 Pacific island countries and territories... 183 Weed family
Weed species
Biocontrol agent
family
Biocontrol agent species
No. of countries
in Pacific agent
established
Possible countries for
introduction#
Pyralidae
Pempelia genistella (Duponchel)
PNG
Tetranychidae
Tetranychus linterarius Dufour
PNG
Thripidae
Sericothrips staphylinus Haliday
PNG
Tortricidae
Cydia succedana (Denis & Schiffermüller)
PNG
Vachellia nilotica subsp. Discussion indica
Chrysomelidae
Bruchidius sahlbergi Schilsky
FP, NC, SI, WF
Melastomataceae
Clidemia hirta
Buprestidae
Lius poseidon Napp
AS, FSM, Fi, Pa, PNG, Sa, SI, Va,
WF
Crambidae
Ategumia matutinalis (Guenée)
AS, FSM, Fi, Pa, PNG, Sa, SI, Va,
WF
Erebidae
Antiblemma acclinalis Hübner
AS, FSM, Fi, Pa, PNG, Sa, SI, Va,
WF
Glomerellaceae
Colletotrichum clidemiae B. Weir & P.R. Johnst. AS, FSM, Fi, Pa, PNG, Sa, SI, Va,
WF
Momphidae
Mompha trithalama Meyrick
AS, FSM, Fi, Pa, PNG, Sa, SI, Va,
WF
Poaceae
Arundo donax
Diaspididae
Rhizaspidiotus donacis Leonardi
CI, FSM, Fi, FP, Gu, Na, NC, Pa,
PNG, Sa, To, WF
Polygonaceae
Emex australis
Brentidae
Perapion antiquum (Gyllenhal)
NC
Pontederiaceae
Eichhornia crassipes
Crambidae
Niphograpta albiguttalis
AS, CI, FSM, Fi, FP, Gu, MI, Na,
NC, NMI, Pa, PNG, Sa, SI, Va
Crambidae
Xubida infusella
AS, CI, FSM, Fi, FP, Gu, MI, Na,
NC, NMI, Pa, PNG, Sa, SI, Va
Galumnidae
Orthogalumna terebrantis Wallwork
AS, CI, FSM, Fi, FP, Gu, MI, Na,
NC, NMI, Pa, PNG, Sa, SI, Va
Miridae
Eccritotarsus catarinensis (Carvalho)
AS, CI, FSM, Fi, FP, Gu, MI, Na,
NC, NMI, Pa, PNG, Sa, SI, Va
Salviniaceae
Salvinia molesta
Crambidae
Samea multiplicalis
CI, Fi, FP, Gu, NC, NMI, PNG
Pauliniidae
Paulinia acuminata
CI, Fi, FP, Gu, NC, NMI, PNG Michael D. Day & Rachel L. Winston / NeoBiota 30: 167–192 (2016) 184 Weed family
Weed species
Biocontrol agent
family
Biocontrol agent species
No. of countries
in Pacific agent
established
Possible countries for
introduction#
Scrophulariaceae
Buddleja davidii Franch. Curculionidae
Cleopus japonicus Wingelmüller
Fi, NC, PNG
Verbenaceae
Lantana camara
Agromyzidae
Calycomyza lantanae
7
AS, CI, FP, Ki, MI, Na, NC, Ni,
NMI, PI, Sa, To, Tu, WF
Agromyzidae
Ophiomyia lantanae
11
AS, CI, Ki, MI, Na, Ni, PI, SI, Tu,
WF
Brentidae
Coelocephalapion camarae Kissinger
AS, CI, FSM, Fi, FP, Gu, Ki, MI,
Na, NC, Ni, NMI, Pa, PNG, PI, Sa,
SI, To, Tu, Va, WF
Mycosphaerellaceae
Passalora lantanae (Chupp) U. Braun & Crous
var. lantanae
AS, CI, FSM, Fi, FP, Gu, Ki, MI,
Na, NC, Ni, NMI, Pa, PNG, PI, Sa,
SI, To, Tu, Va, WF
Mycosphaerellaceae
Septoria sp. Discussion AS, CI, FSM, Fi, FP, Gu, Ki, MI,
Na, NC, Ni, NMI, Pa, PNG, PI, Sa,
SI, To, Tu, Va, WF
Cerambycidae
Plagiohammus spinipennis
AS, CI, FSM, Fi, FP, Gu, Ki, MI,
Na, NC, Ni, NMI, Pa, PNG, PI, Sa,
SI, To, Tu, Va, WF
Chrysomelidae
Octotoma championi
AS, CI, FSM, Fi, FP, Gu, Ki, MI,
Na, NC, Ni, NMI, Pa, PNG, PI, Sa,
SI, To, Tu, Va, WF
Chrysomelidae
Uroplata fulvopustulata
AS, CI, FSM, Fi, FP, Gu, Ki, MI,
Na, NC, Ni, NMI, Pa, PNG, PI, Sa,
SI, To, Tu, Va, WF
Crambidae
Salbia haemorrhoidalis
2
AS, CI, FP, Gu, Ki, MI, Na, NC, Ni,
NMI, Pa, PNG, PI, Sa, SI, To, Tu,
Va, WF
Erebidae
Hypena laceratalis
7
AS, CI, FP, Ki, MI, Na, Ni, Pa, PI,
Sa, SI, To, Tu, WF
Gracillariidae
Cremastobombycia lantanella Busck
AS, CI, FSM, Fi, FP, Gu, Ki, MI,
Na, NC, Ni, NMI, Pa, PNG, PI, Sa,
SI, To, Tu, Va, WF 185 Biological control of weeds in the 22 Pacific island countries and territories... Weed family
Weed species
Biocontrol agent
family
Biocontrol agent species
No. of countries
in Pacific agent
established
Possible countries for
introduction#
Noctuidae
Neogalea sunia
1
AS, CI, FSM, Fi, FP, Gu, Ki, MI,
Na, Ni, NMI, Pa, PNG, PI, Sa, SI,
To, Tu, Va, WF
Pterophoridae
Lantanophaga pusillidactyla
5
AS, CI, Fi, FP, Ki, MI, Na, NC, Ni,
PI, Sa, SI, To, Tu, Va, WF
Uropyxidaceae
Prospodium tuberculatum (Spegazzini) Arthur
AS, CI, FSM, Fi, FP, Gu, Ki, MI,
Na, NC, Ni, NMI, Pa, PNG, PI, Sa,
SI, To, Tu, Va, WF
Tephritidae
Eutreta xanthochaeta Aldrich
AS, CI, FSM, Fi, FP, Gu, Ki, MI,
Na, NC, Ni, NMI, Pa, PNG, PI, Sa,
SI, To, Tu, Va, WF
Tingidae
Leptobyrsa decora
AS, CI, FSM, Fi, FP, Gu, Ki, MI,
Na, NC, Ni, NMI, Pa, PNG, PI, Sa,
SI, To, Tu, Va, WF
Tortricideae
Crocidosema lantana
6
AS, CI, Fi, FP, Ki, Na, NC, Ni,
PNG, PI, Sa, SI, To, Tu, WF
Lantana montevidensis
(Spreng.) Briq. Agromyzidae
Calycomyza lantanae
7
FP, NC, WF
Erebidae
Hypena laceratalis
7
FP, SI, WF
Pterophoridae
Lantanophaga pusillidactyla
5
Fi, FP, NC, SI, WF
Based on weed occurrence in each country, not weed density Weed family Michael D. Day & Rachel L. Winston / NeoBiota 30: 167–192 (2016) 186 Table 6. Weed species currently under evaluation outside the PICTs. Discussion Crambidae
Neomusotima conspurcatalis (Warren)
FSM, Fi, Gu, NMI, Pa, PNG, SI
Eriophyidae
Floracarus perrepae Knihinicki & Boczek
FSM, Fi, Gu, NMI, Pa, PNG, SI
Myrtaceae
Psidium cattleianum Sabine
Eriococcidae
Tectococcus ovatus Hempel
CI, FSM, Fi, FP, NC, Pa, PNG, PI, Sa, SI
Passifloraceae
Passiflora rubra L.*
AS, CI
Pontederiaceae
Eichhornia crassipes
Acrididae
Cornops aquaticum (Brüner)
AS, CI, FSM, Fi, FP, Gu, MI, Na, NC, NMI, Pa,
PNG, Sa, SI, Va
Delphacidae
Megamelus scutellaris Berg
AS, CI, FSM, Fi, FP, Gu, MI, Na, NC, NMI, Pa,
PNG, Sa, SI, Va
Sapindaceae
Cardiospermum grandiflorum Sw. Curculionidae
Cissoanthonomus tuberculipennis Hustache
CI, FP
Verbenaceae
Lantana camara
Chrysomelidae
Longitarsus bethae Savini & Escalona
AS, CI, FSM, Fi, FP, Gu, Ki, MI, Na, NC, Ni,
NMI, Pa, PNG, PI, Sa, SI, To, Tu, Va, WF
Zingiberaceae
Hedychium gardnerianum Sheppard ex
Ker Gawl.*
CI, FSM, Fi, FP, NC
# Based on weed occurrence in each country, not weed density
* Field exploration and host specificity being conducted Weed family
Weed species
Biocontrol agent
family
Biocontrol agent species
Possible countries for introduction#
Lygodiaceae
Lygodium microphyllum (Cav.) R. Br. Crambidae
Neomusotima conspurcatalis (Warren)
FSM, Fi, Gu, NMI, Pa, PNG, SI
Eriophyidae
Floracarus perrepae Knihinicki & Boczek
FSM, Fi, Gu, NMI, Pa, PNG, SI
Myrtaceae
Psidium cattleianum Sabine
Eriococcidae
Tectococcus ovatus Hempel
CI, FSM, Fi, FP, NC, Pa, PNG, PI, Sa, SI
Passifloraceae
Passiflora rubra L.*
AS, CI
Pontederiaceae
Eichhornia crassipes
Acrididae
Cornops aquaticum (Brüner)
AS, CI, FSM, Fi, FP, Gu, MI, Na, NC, NMI, Pa,
PNG, Sa, SI, Va
Delphacidae
Megamelus scutellaris Berg
AS, CI, FSM, Fi, FP, Gu, MI, Na, NC, NMI, Pa,
PNG, Sa, SI, Va
Sapindaceae
Cardiospermum grandiflorum Sw. Curculionidae
Cissoanthonomus tuberculipennis Hustache
CI, FP
Verbenaceae
Lantana camara
Chrysomelidae
Longitarsus bethae Savini & Escalona
AS, CI, FSM, Fi, FP, Gu, Ki, MI, Na, NC, Ni,
NMI, Pa, PNG, PI, Sa, SI, To, Tu, Va, WF
Zingiberaceae
Hedychium gardnerianum Sheppard ex
Ker Gawl.*
CI, FSM, Fi, FP, NC
# Based on weed occurrence in each country, not weed density
* Field exploration and host specificity being conducted Weed family
Weed species
Biocontrol agent
family
Biocontrol agent species
Possible countries for introduction#
Lygodiaceae
Lygodium microphyllum (Cav.) R. Br. Discussion Agents have either not been released to date, or have been released and not yet evaluated. Bio
control agents could potentially be introduced against these weeds in the PICTs in the future. Countries: AS=American Samoa, CI=Cook Islands, FSM=Federated
States of Micronesia, Fi=Fiji, FP=French Polynesia, Gu=Guam, Ki=Kiribati, MI=Marshall Islands, Na=Nauru, NC=New Caledonia, Ni=Niue, NMI=Northern
Mariana Islands, Pa=Palau, PNG=Papua New Guinea, PI=Pitcairn Islands, Sa=Samoa, SI=Solomon Islands, Tk=Tokelau, To=Tonga, Tu=Tuvalu, Va=Vanuatu,
WF=Wallis & Futuna. Weed family
Weed species
Biocontrol agent
family
Biocontrol agent species
Possible countries for introduction#
Asteraceae
Ageratina adenophora
Pucciniosiraceae
Baeodromus eupatorii (Arthur) Arthur
FP
Chromolaena odorata
Curculionidae
Lixus aemulus Petri
FSM, Gu, MI, NC, NMI, Pa, PNG
Tortricidae
Dichrorampha odorata Brown &
Zachariades
FSM, Gu, MI, NC, NMI, Pa, PNG
Bignoniaceae
Spathodea campanulata P. Beauv.*
AS, CI, FSM, Fi, FP, Gu, Ki, MI, Na, NC, Ni,
NMI, Pa, PNG, PI, Sa, SI, To, Va, WF
Tecoma stans (L.) Juss. ex Kunth var. stans
Coccinellidae
Mada polluta (Mulsant)
AS, CI, FSM, Fi, FP, Gu, Ki, MI, Na, NC, Ni,
NMI, Pa, PNG, Sa, SI, To, WF
Dolichandra unguis-cati
Pyralidae
Hypocosmia pyrochroma Jones
CI, FSM, FP, Gu, NC, Ni, Va
Cactaceae
Pereskia aculeata
Coreidae
Catorhintha schaffneri Brailovsky & Garcia
FP, NC, Pa
Commelinaceae
Tradescantia fluminensis Vell. Chrysomelidae
Lema basicostata Monros
FP, Na
Chrysomelidae
Neolema abbreviata Lacordaire
FP, Na
Chrysomelidae
Neolema ogloblini (Monros)
FP, Na
Dioscoreaceae
Dioscorea bulbifera L. Chrysomelidae
Lilioceris cheni Gressitt & Kimoto
AS, FSM, Fi, FP, Gu, MI, Ni, NMI, Pa, PNG, Sa,
SI, To, Va, WF
Fabaceae
Falcataria moluccana (Miq.) Barneby &
J.W. Grimes*
AS, CI, FSM, Fi, FP, Gu, NC, Ni, Pa, PNG, Sa,
SI, To, WF
Mimosa pigra
Chrysomelidae
Nesaecrepida infuscata (Schaeffer)
PNG
Raveneliaceae
Diabole cubensis (Arthur & J.R. Johnst.)
Arthur
PNG
Parkinsonia aculeata L. Geometridae
Eueupithecia cisplatensis Prout
FSM, FP, Gu, NC, SI
Lamiaceae
Clerodendrum chinensis (Osbeck) Mabb. Chrysomelidae
Phyllocharis undulata (L.)
AS, CI, FSM, Fi, FP, Gu, Ni, NMI, PNG, Sa, SI,
To, Va 187 Biological control of weeds in the 22 Pacific island countries and territories... Weed family
Weed species
Biocontrol agent
family
Biocontrol agent species
Possible countries for introduction#
Lygodiaceae
Lygodium microphyllum (Cav.) R. Br. Discussion Crambidae
Neomusotima conspurcatalis (Warren)
FSM, Fi, Gu, NMI, Pa, PNG, SI
Eriophyidae
Floracarus perrepae Knihinicki & Boczek
FSM, Fi, Gu, NMI, Pa, PNG, SI
Myrtaceae
Psidium cattleianum Sabine
Eriococcidae
Tectococcus ovatus Hempel
CI, FSM, Fi, FP, NC, Pa, PNG, PI, Sa, SI
Passifloraceae
Passiflora rubra L.*
AS, CI
Pontederiaceae
Eichhornia crassipes
Acrididae
Cornops aquaticum (Brüner)
AS, CI, FSM, Fi, FP, Gu, MI, Na, NC, NMI, Pa,
PNG, Sa, SI, Va
Delphacidae
Megamelus scutellaris Berg
AS, CI, FSM, Fi, FP, Gu, MI, Na, NC, NMI, Pa,
PNG, Sa, SI, Va
Sapindaceae
Cardiospermum grandiflorum Sw. Curculionidae
Cissoanthonomus tuberculipennis Hustache
CI, FP
Verbenaceae
Lantana camara
Chrysomelidae
Longitarsus bethae Savini & Escalona
AS, CI, FSM, Fi, FP, Gu, Ki, MI, Na, NC, Ni,
NMI, Pa, PNG, PI, Sa, SI, To, Tu, Va, WF
Zingiberaceae
Hedychium gardnerianum Sheppard ex
Ker Gawl.*
CI, FSM, Fi, FP, NC
# Based on weed occurrence in each country not weed density 188 Michael D. Day & Rachel L. Winston / NeoBiota 30: 167–192 (2016) However, many biocontrol agents that have established in the PICTS are only
found in a fraction of the countries in which their respective target weed occurs. This
could be because weed densities in countries where agents are not present are not
high enough to warrant biocontrol, or because human population base, infrastructure,
expertise, experience and funding to implement biocontrol programmes are limited
(Dovey et al. 2004). Both the Secretariat of the Pacific Community (SPC) and the South Pacific Re
gional Environmental Program (SPREP) have a responsibility in helping member
countries in agricultural and environmental issues respectively, and could therefore
assist in coordination of biocontrol programmes, while Australia, the USA and New
Zealand could help in a technical capacity, especially regarding the additional testing
of biocontrol agents (Dovey et al. 2004). g
Another constraint to successfully implementing biocontrol in the PICTs is due to
the nature of the Pacific. The Pacific region covers 30 million km2, of which only 2%
is landmass and is spread over 7,500 islands (Shine et al. 2003). Therefore, releasing
biocontrol agents into all countries and on all islands where target weeds occur can
be challenging and expensive (Dovey et al. 2004, Day et al. 2013a, c). This contrasts
greatly with Asia or Africa where biocontrol agents have readily spread within and to
other countries, as weed populations are often contiguous (Winston et al. 2014). Discussion To
help overcome these logistical difficulties, many biocontrol programs in the Pacific
region have been funded by donor organisations from Australia, Europe, the USA and
New Zealand and/or have involved the assistance of the SPC. Within these programs, substantial funds are frequently allocated to conducting
weed and biocontrol agent distribution surveys in order to identify locations where
a target weed is present but no agents have established. Such surveys have been con
ducted recently in Papua New Guinea and Vanuatu, with funding from the Australian
Government. Program funds are also frequently spent on increasing capacity, such as
improving infrastructure and training staff, as well as releasing biocontrol agents.f f
A cost-effective solution to weed biocontrol research in the PICTs is to redistribute
effective agents already established in the region (Dovey et al. 2004, Julien et al. 2007,
Paynter et al. 2015). In general, redistribution of agents within the Pacific requires
little to no extra host specificity testing because plant assemblages are often similar be
tween countries, and many agents have been established long enough to both identify
the most highly effective agents and to detect any non-target impacts. Utilising tried
and proven agents overcomes the considerable cost of host specificity testing of new
agents, and reduces the likelihood of agents not establishing or having minimal impact
on the target weeds (Julien et al. 2007, Paynter et al. 2015).i Countries wishing to introduce any biocontrol agent from within the Pacific re
gion should conduct surveys to determine what agents are already present in their
country. There are many examples of agents previously not reported, being found in
countries following the conduct of dedicated or even opportunistic surveys (Winston
et al. 2014). Regardless of the mode of entry into a country, once established within
the region, biocontrol agents can spread naturally to new islands and/or countries. Cal Biological control of weeds in the 22 Pacific island countries and territories... 189 ligrapha pantherina was released onto only 14 islands in Vanuatu and is now present
on 21 islands (Day and Bule this edition). Within the PICTs, Calycomyza lantanae was
deliberately released into only Fiji for the control of L. camara, but it is now found in
seven countries in the PICTs. Incidentally, although C. lantanae has only ever been
deliberately released into three countries (Australia, Fiji and South Africa), it is now
found in 28 countries worldwide (Day et al. Discussion 2003, Winston et al. 2014). In addition to redistributing agents already established within the PICTS, there
are many more biocontrol agents released outside the PICTs that cause medium to
high impacts on their target weed(s) and could be considered for introduction into the
PICTs (Winston et al. 2014). However, such agents may not have the same efficacy
in the PICTs, so climate-matching and other suitability studies may need to be con
ducted prior to their consideration. More importantly, because host specificity testing
of these agents may have occurred in regions with very different plant assemblages,
PICTs wishing to import particular agents from outside the region should determine
if additional host specificity testing is required prior to the agents’ importation. pi
g
q
p
g
p
Under an Australian Government funded programme, Puccinia spegazzinii was
tested against an additional 17 local plant species by CABI prior to its introduction
into PNG and Fiji. This was despite the agent being tested against 170 species on
behalf of India and China prior to its introduction into those countries (Day et al. 2013b). Conversely, both Neochetina spp. and C. pantherina were introduced into the
PICTs without any additional testing following their testing and subsequent release in
Australia (Julien et al. 2007). J
Biocontrol is seen as the most cost-effective, environmentally friendly, and sustain
able option to manage many weeds in the Pacific and elsewhere. Utilising tried and
proven agents that are both host specific and effective against the target weed species in
other countries maximises the chance of success in new countries while minimising the
risks of non-target impacts (Dovey et al. 2004, Julien et al. 2007, Paynter et al. 2015). With over 60 agents already deliberately released against more than 20 weed species,
biocontrol of weeds in the PICTs is not a new concept. However, as many of these
agents are found in only a few countries, there is great potential to manage the target
weeds in other countries in the Pacific through their redistribution. In addition, highly
damaging and host specific agents established outside the Pacific could be introduced
to control those weed species not yet targeted.h g
Through coordinated responses, possibly involving the SPC and the SPREP, as
well as Australia, the USA and New Zealand, the impacts of weeds in the Pacific region
can be reduced through biocontrol, and food security for its inhabitants increased. Acknowledgements The authors wish to thank researchers within National Agricultural Research Institute
and National Agricultural Quarantine and Inspection Authority, Papua New Guinea,
Biosecurity Vanuatu, and the Secretariat of the Pacific Community for their input into Michael D. Day & Rachel L. Winston / NeoBiota 30: 167–192 (2016) 190 projects over the past few decades. The USDA Forest Service assisted with funding the
production of the weed biocontrol catalogue, while Landcare Research New Zealand
Ltd funded attendance at the EMAPI Conference held in Hawaii. The authors are
grateful to Lynley Hayes and Drs Anthony Pople, Curt Daehler and Clifford Smith for
providing helpful comments on the manuscript. References Conant P, Garcia JN, Johnson MT, Nagamine WT, Hirayama CK, Markin GP, Hill RL
(2013) Releases of natural enemies in Hawaii since 1980 for classical biological control of
weeds. In: Wu Y, Johnson T, Sing S, Raghu S, Wheeler G, Pratt P, Warner K, Center T,
Goolsby J, Reardo R (Eds) Proceedings of the XIII International Symposium on Biological
Control of Weeds, Waikoloa (Hawaii USA), September 2011. Forest Health Technology
Enterprise Team, Morgantown WV, 230–246. Day MD (2013) Evaluating prospects for biological control of invasive weeds in Melanesia. ACIAR Report, Canberra, 1–4. Day MD, Bule S (2016) The status of weed biological control in Vanuatu. In: Daehler CC,
van Kleunen M, Pyšek P, Richardson DM (Eds) Proceedings of 13th International EMAPi
conference, Waikoloa, Hawaii. NeoBiota 30: 151–166. doi: 10.3897/neobiota.30.7049 Day MD, Bofeng I, Nabo I (2013a) Successful biological control of Chromolaena odorata (Aster
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Wu Y, Johnson T, Sing S, Raghu S, Wheeler G, Pratt P, Warner K, Center T, Goolsby J,
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Weeds, Waikoloa (Hawaii USA), September 2011. Forest Health Technology Enterprise
Team, Morgantown WV, 400–408. Day MD, Kawi AP, Ellison CA (2013b) Assessing the potential of the rust fungus Puccinia
spegazzinii as a classical biological control agent for the invasive weed Mikania micran
tha in Papua New Guinea. Biological Control 67: 253–261. doi: 10.1016/j.biocon
trol.2013.08.007 Day MD, Kawi AP, Fidelis J, Tunabuna A, Orapa W, Swamy B, Ratutini J, Saul-Maora J,
Dewhurst CF (2013c) Biology, field release and monitoring of the rust fungus Puccinia
spegazzinii (Pucciniales: Pucciniaceae), a biological control agent of Mikania micrantha
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Wheeler G, Pratt P, Warner K, Center T, Goolsby J, Reardo R (Eds) Proceedings of the
XIII International Symposium on Biological Control of Weeds, Waikoloa (Hawaii USA),
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Orapa W (2012) Distribution and socio-economic impacts of Mikania micrantha in Papua
New Guinea and Fiji and prospects for its biocontrol. Pakistan Journal of Weed Science
Research 18: 169–179. Day MD, Wiley CJ, Playford J, Zalucki MP (2003) Lantana: Current Management Status and
Future Prospects. References Australian Centre for International Agriculture Research, Canberra, 128 pp. Biological control of weeds in the 22 Pacific island countries and territories... 191 Dodd S, Hayes L (2009) Report on Pacific Biocontrol Strategy Workshop. Landcare Research
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(Australia), May 2003. CSIRO Entomology Australia, Canberra, 36–41.i Endemia (2015) Calligrapha pantherina. http://www.endemia.nc/faune/fiche6800.html [accessed:
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Zealand: an overview of nearly 85 years. In: Zydenbos SM (Ed.) Proceedings of the 17th
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ronmental weeds in New Zealand. Austral Ecology 25: 553–562. doi: 10.1046/j.1442-
9993.2000.01075.x Funasaki GY, Lai P-Y, Nakahara LM, Beardsley JW, Ota AK (1988) A review of biological
control introductions in Hawaii: 1890–1985. Proceedings of the Hawaiian Entomological
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of lantana: Octotoma scabripennis and Uroplata girardi (Coleoptera: Chrysomelidae). Acta
Œcologica 10: 19–29. Julien M, McFadyen R, Cullen J (Eds) (2012) Biological Control of Weeds in Australia. CSIRO Publishing Melbourne, 620 pp. Julien MH, Orapa W (2001) Insects used for the control of the aquatic weed, water hyacinth
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in New Zealand. Biological Control 80: 119–127. doi: 10.1016/j.biocontrol.2014.10.008 PIER - Pacific Island Ecosystems at Risk (2013) Plant threats to Pacific ecosystems. http://
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duced weeds in Hawaii. Biological Control 33(1): 113–122. doi: 10.1016/j.biocon
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FHTET-2014-04: 1–838.
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Prevalence and Risk Factors for Positive Nasal Methicillin-Resistant Staphylococcus aureus Carriage among Orthopedic Patients in Korea
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Journal of clinical medicine
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cc-by
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Received: 14 April 2019; Accepted: 7 May 2019; Published: 8 May 2019 Abstract: Methicillin-resistant Staphylococcus aureus (MRSA) causes purulent skin and soft tissue
infections as well as other life-threatening diseases. Recent guidelines recommend screening for MRSA
at the time of admission. However, few studies have been conducted to determine the prevalence
and risk factors for MRSA colonization. A prospective data collection and retrospective analysis was
performed. MRSA screening tests were performed using nasal swabs in patients enrolled between
January 2017 and July 2018. Demographic data, socio-economic data, medical comorbidities, and
other risk factors for MRSA carriage were evaluated among 1577 patients enrolled in the study. The
prevalence of MRSA nasal carriage was 7.2%. Univariate regression analysis showed that colonization
with MRSA at the time of hospital admission was significantly related to patient age, body mass
index, smoking, alcohol, trauma, recent antibiotic use, and route of hospital admission. Multiple
logistic regression analysis for the risk factors for positive MRSA nasal carriage showed that being
under- or overweight, trauma diagnosis, antibiotic use one month prior to admission, and admission
through an emergency department were related to MRSA colonization. This study highlights the
importance of a preoperative screening test for patients scheduled to undergo surgery involving
implant insertion, particularly those at risk for MRSA. Keywords:
Methicillin-resistant Staphylococcus aureus (MRSA); nasal screening test; infection;
prevalence; risk factors Sung-Woo Choi 1,†,*, Jae Chul Lee 1
, Jahyung Kim 1, Ji Eun Kim 2, Min Jung Baek 3,†,
Se Yoon Park 4, Suyeon Park 5 and Byung-Joon Shin 1,* 1
Department of Orthopedic Surgery, Soonchunhyang University Hospital, College of Medicine,
Seoul 04401, Korea; jlee@schmc.ac.kr (J.C.L.); t0152@schmc.ac.kr (J.K.) 1
Department of Orthopedic Surgery, Soonchunhyang University Hospital, College of Medicine,
Seoul 04401, Korea; jlee@schmc.ac.kr (J.C.L.); t0152@schmc.ac.kr (J.K.) Department of Laboratory Medicine, Soonchunhyang University College of Medicine, Seoul 04401, Korea;
jkim@schmc.ac.kr 2
Department of Laboratory Medicine, Soonchunhyang University College of Medicine, Seoul 04401, Kore
jkim@schmc.ac.kr 3
Department of Obstetrics and Gynecology, Bundang CHA Hospital, Seongnam 13496, Korea;
goodgood75@naver.com Department of Obstetrics and Gynecology, Bundang CHA Hospital, Seongnam 13496, Korea;
goodgood75@naver.com 4
Division of Infectious Diseases, Department of Internal Medicine, Soonchunhyang University Hospital
College of Medicine, Seoul 04401, Korea; sypark@schmc.ac.kr g
yp
5
Department of Biostatistics, Soonchunhyang University College of Medicine, Seoul 04401, Korea;
sue3517@schmc.ac.kr *
Correspondence: swchoi@schmc.ac.kr (S.-W.C.); schsbj@schmc.ac.kr (B.-J.S.); Tel.: +82-2-709-9259 (S.-W.C.)
+82-2-709-9250 (B.-J.S.) †
These authors contributed equally to this paper. Received: 14 April 2019; Accepted: 7 May 2019; Published: 8 May 2019 Journal of
Clinical Medicine Journal of
Clinical Medicine Journal of
Clinical Medicine Journal of
Clinical Medicine Prevalence and Risk Factors for Positive Nasal
Methicillin-Resistant Staphylococcus aureus Carriage
among Orthopedic Patients in Korea Sung-Woo Choi 1,†,*, Jae Chul Lee 1
, Jahyung Kim 1, Ji Eun Kim 2, Min Jung Baek 3,†,
Se Yoon Park 4, Suyeon Park 5 and Byung-Joon Shin 1,* 1. Introduction The emergence of methicillin-resistant Staphylococcus aureus (MRSA) since the early 1960s and its
spread throughout hospitals and communities decades later have complicated antibiotic therapy [1–5]. The rate of methicillin resistance is higher in the orthopedics department, where artificial implantation
is more common than in other medical specialties [6]. If the infection is caused by MRSA, either due to J. Clin. Med. 2019, 8, 631; doi:10.3390/jcm8050631 www.mdpi.com/journal/jcm www.mdpi.com/journal/jcm J. Clin. Med. 2019, 8, 631 2 of 11 antibiotic resistance or biofilm formation, the treatment of an orthopedic infection becomes difficult
and may lead to a higher economic burden [7]. Therefore, early detection and prevention of MRSA are
important, particularly among orthopedic patients. The recent guidelines published by the Society for Health Care Epidemiology for America
recommend surveillance of cultures at the time of hospital admission for patients scheduled to undergo
high-risk surgeries, including some orthopedic and cardiothoracic procedures [8,9]. The Korea Center
for Disease Control and Prevention suggests a screening test only for patients who are admitted to
high-risk departments such as the intensive care unit, the hemato-oncology department, and the organ
transplantation department; patients with a prior diagnosis of MRSA; and patients transferred from a
nursing facility [10]. The recommended screening method for MRSA is nasal sampling because the
anterior nasal cavity is one of the preferred carrier sites of this bacterium and because the frequency of
skin colonization depends on nasal carriage [11,12]. In addition, Yano et al. [13] reported that patients
with positive preoperative nasal cultures for MRSA had a higher occurrence of surgical site infection
with MRSA after orthopedic surgery. However, a limited number of studies have been conducted to determine the prevalence and risk
factors for colonization at the time of admission in orthopedic patients [14–18], especially in Korea. The prevalence of MRSA is extremely high in Korea. The Asian Network for Surveillance of Resistant
Pathogens (ANSORP) study, which included seven hospitals in Korea, showed an average MRSA
prevalence of 77.6% for nosocomial S. aureus isolates [19]. The recent report of the Regional Resistance
Surveillance (RRS) program showed that 73% of the clinical S. aureus isolates from two hospitals in
Korea were MRSA [20]. Korea has the highest MRSA prevalence among the 12 surveillance countries
in the RRS program [20,21]. The present study primarily aimed to assess the prevalence of MRSA carriage in the orthopedic
department. 1. Introduction The secondary objective was to identify potential risk factors for MRSA colonization with
respect to the demographic and medical data. 2.2. Sampling The patients, who were admitted to the hospital during the study period and satisfied the inclusion
criteria, had nasal swabs collected from them. All sampling was performed by well-trained orthopedic
nurses. Nasal swabs were obtained within a day of admission. A sterile transport swab (COPAN,
Brescia, Italy) was rotated in the anterior nasal cavity of the patients by the nurses. The swab was
transported at room temperature and each swab was processed as described here within four hours of
collection. Direct culture was performed onto chromID MRSA agar (bioMérieux, Nürtingen, Germany)
and was examined at 24 and 48 hours after incubation. A positive culture was defined as growth
with morphological features comparable to MRSA. This was confirmed by the coagulase test using a
commercial latex agglutination kit (Pastorex Staph Plus, Bio Rad Laboratories, Hemel Hempstead, UK). 2.1. Inclusion and Exclusion Criteria The patients admitted to the orthopedic surgery department of Soonchunhyang University
Hospital, Korea were enrolled in the study based on the following inclusion criteria: patients aged 18
to 90 years were included in the study and patients with a current infection were excluded. 2. Material and Methods The study involved prospective data collection and retrospective data analysis and was conducted
between January 2017 and July 2018 at the orthopedic surgery department of Soonchunhyang University
Hospital and approved by the Institutional Review Board (IRB No.: SCHUH 2018-11-021). 2.3. Data Collection Data collection was coordinated by an orthopedic surgeon who completed standardized forms
for each included patient. Overall data was recorded for the following variables: demographic 3 of 11 J. Clin. Med. 2019, 8, 631 characteristics, socio-economic characteristics, medical comorbidities, and other risk factors for MRSA
carriage. Sex, age, body mass index (BMI), history of smoking and alcohol consumption, diagnosis of
trauma or disease, and body part involved were analyzed as demographic data. BMI was divided into
four groups (underweight (<18.5 kg/m2), normal weight (18.5–25 kg/m2), overweight (25–30 kg/m2),
and obese (>30 kg/m2)) [22]. Smoking history was classified into three groups (never-smoker, ex-smoker,
and current-smoker). An ex-smoker was defined as someone who had smoked more than 100 cigarettes
in their lifetime but had not smoked in the last 28 days [23]. A current-smoker was defined as
an adult who had smoked more than 100 cigarettes in his or her lifetime and was still smoking. History of alcohol consumption was divided into three groups (never-drinker, former-drinker, and
current-drinker). A current-drinker was a person who consumed up to 12 drinks per year, while a
former-drinker was a patient who had stopped drinking for >1 year [24]. The body part involved
was divided into five categories (spine, knee and shoulder, hand and elbow, hip, and foot). Using
the ICD-10-CM (International Classification of Diseases, Tenth Revision, Clinical Modification) codes,
patients were divided into two groups (coded as trauma (S) and disease (M)). Education status was
collected as socio-economic data [25–27]. Medical comorbidities included hypertension, diabetes
mellitus, cardiovascular diseases, hepatic diseases, and dialysis. Other risk factors for MRSA carriage
were recent hospitalization, recent antibiotics use, presence of a ureteral catheter, and type of hospital
admission (i.e., whether emergency or outpatient department) (Table 1). Recent hospitalization and
recent antibiotic use were defined as stationary hospitalization within 12 months prior to admission
and antimicrobial therapy within one month before the screening test, respectively [28]. Table 1. Overview of collected patient data. Table 1. Overview of collected patient data. 2.3. Data Collection Patient Data
Type of Acquired Information
Demographic
Gender
Age
Body mass index (BMI)
Smoking (current-smoker/ex-smoker/never-smoker)
Regular consumption of alcohol (current-drinker/former-drinker/never-drinker)
Socio-economic
Status of education (under high school/high school graduate/denied or
unanswered)
Medical comorbidities
Hypertension, diabetes mellitus, cardiovascular diseases, hepatic disease, dialysis
Other risk factors
Stationary hospitalization in last 12 months
Antimicrobial therapy in the past month
Presence of urethral catheter
Route of admission (ER/OPD)
Trauma/disease
Body parts involved (spine/knee and shoulder/hand and elbow/hip/foot)
ER = Emergency Room, OPD = Outpatient department. Table 1. Overview of collected patient data. Table 1. Overview of collected patient data. Patient Data
Type of Acquired Information
Demographic
Gender
Age
Body mass index (BMI)
Smoking (current-smoker/ex-smoker/never-smoker)
Regular consumption of alcohol (current-drinker/former-drinker/never-drinker)
Socio-economic
Status of education (under high school/high school graduate/denied or
unanswered)
Medical comorbidities
Hypertension, diabetes mellitus, cardiovascular diseases, hepatic disease, dialysis
Other risk factors
Stationary hospitalization in last 12 months
Antimicrobial therapy in the past month
Presence of urethral catheter
Route of admission (ER/OPD)
Trauma/disease
Body parts involved (spine/knee and shoulder/hand and elbow/hip/foot)
ER = Emergency Room, OPD = Outpatient department. 2.4. Statistical Analysis Statistical analysis was performed using parametric or nonparametric tests, where appropriate. Logistic regression analysis was used to identify independent predictors of colonization with MRSA at
the time of admission to the hospital as well as clinical conditions associated with the development of
symptomatic MRSA infection. The association between the risk factors and MRSA colonization was
analyzed using the chi-square test or Fisher’s exact test, as appropriate. Data were analyzed using
SPSS Statistics (Statistical Package for the Social Sciences, version 25.0; 2017, IBM Corp. Armonk, NY,
USA) and two-tailed p-values of ≤0.05 were considered as statistically significant. 4 of 11 J. Clin. Med. 2019, 8, 631 3. Results 3.1. Overall Result 3.1. Overall Result A total of 1577 patients were enrolled during the study period. Of these, 617 (39.1%) were women
and 960 (60.9%) were men. The average patient age was 59.2 years; particularly, 288 patients (18.3%)
were aged under 40 years, 776 (49.2%) were aged between 40 and 70 years, and 513 (32.5%) were
aged older than 70 years. A total of 1332 patients (84.5%) were tested in the ward after admission,
while 245 (15.5%) were tested in the outpatient department. Ultimately, 114 patients were found to be
colonized with MRSA; thus, the prevalence of nasal MRSA carriage was 7.2%. Of the 114 colonized
samples, 99 (86.8%) samples were collected after admission and 15 (13.2%) samples were collected in
the outpatient department. 3.2.1. Univariate Logistic Regression Analysis Selected variables of interest were subjected to analysis (Tables 2 and 3). The results of the
univariate logistic regression analysis showed that the colonization with MRSA at the time of hospital
admission was significantly related to patient age, BMI, smoking, alcohol consumption, body part
involved, diagnosis of trauma, type of hospital admission, and recent antibiotic use (Table 4). Patients
aged 40 to 70 years and those older than 70 years had 0.237 (95% Confidence Interval (CI): 0.151–0.374;
p < 0.001) and 0.292 (95% CI: 0.180–0.474; p < 0.001) times the risk of being a carrier of MRSA as
compared to those aged under 40 years, respectively. Additionally, patients whose BMI was under
18.5 kg/m2, 25 to 30 kg/m2, and higher than 30 kg/m2 had 3.432 (95% CI: 1.308–9.000; p = 0.012), 0.290
(95% CI: 0.153–0.550; p < 0.01), 0.637 (95% CI: 0.238–1.702; p = 0.369) times the risk of being an MRSA
carrier than those with a normal BMI. Current-smokers had an odds ratio of 4.204 (95% CI: 2.801–6.309;
p < 0.01) and current-drinkers had an odds ratio of 4.204 (95% CI: 3.250–7.223; p < 0.001). Patients
diagnosed with trauma had 3.401 (95% CI: 1.593–7.261; p < 0.01) times the risk of being an MRSA
carrier than those diagnosed with disease. Furthermore, those who were recently treated with
antibiotics tended to have an increased chance of being an MRSA carrier, with an odds ratio of 4.199
(95% CI: 2.831–6.228; p < 0.01). Lastly, patients admitted through the emergency department had
6.915 (95% CI: 2.863–16.702; p < 0.01) times the risk of being an MRSA carrier than those admitted
through the outpatient department. Meanwhile, there was no strong association between MRSA
carriage and other medical comorbidities, recent hospitalization, the presence of a urinary catheter,
and socio-economic background. Table 2. Descriptive logistic regression analysis from data obtained at admission (demographics,
socio-economic status). Number of
Patients
MRSA (−)
(1463 Patients)
MRSA (+)
(114 Patients)
p-Value a
N = 1577 (%)
N (%)
N (%)
Gender
0.359
Male
617 (39.1%)
577 (93.5%)
40 (6.5%)
Female
960 (60.9%)
886 (92.3%)
74 (7.7%)
Age group
<0.01
<40
288 (18.3%)
239 (82.9%)
49 (17.1%)
40–70
776 (49.2%)
740 (95.4%)
36 (4.6%)
≥70
513 (32.7%)
484 (94.3%)
29 (5.7%)
BMI group
<0.01
Underweight (<18.5)
159 (10.1%)
116 (73.0%)
43 (27.0%) Table 2. Descriptive logistic regression analysis from data obtained at admission (demographics,
socio-economic status). 5 of 11 J. 3.2.1. Univariate Logistic Regression Analysis Clin. Med. 2019, 8, 631 Table 2. Cont. Number of
Patients
MRSA (−)
(1463 Patients)
MRSA (+)
(114 Patients)
p-Value a
N = 1577 (%)
N (%)
N (%)
Normal (18.5–25)
735 (46.6%)
686 (93.3%)
49 (6.7%)
Overweight (25–30)
566 (35.9%)
549 (97.0%)
17 (3.0%)
Obese (≥30)
117 (7.4%)
112 (95.7%)
5 (4.3%)
Smoking
<0.01
Never-smoker
1294 (82.1%)
1226 (94.7%)
68 (5.3%)
Current-smoker
238 (15.1%)
193 (81.1%)
45 (18.9%)
Ex-smoker
45 (2.9%)
44 (97.8%)
1 (2.2%)
Alcohol
<0.01
Never-drinker
1292 (81.9%)
1228 (95.0%)
64 (5.0%)
Current-drinker
248 (15.7%)
198 (79.8%)
50 (20.2%)
Former-drinker
37 (2.3%)
37 (100.0%)
0 (0.0%)
Status of education
0.372
Under high school
89 (5.6%)
82 (92.1%)
7 (7.9%)
High school graduate
420 (26.6%)
374 (89.0%)
46 (11.0%)
Denied, Unanswered
1068 (67.7%)
1007 (94.3%)
61 (5.7%)
x% = Column percentage; N (xx%) = Number of patients (row percentage). OR = Odds ratio; CI = Confidence
Interval; a = chi-square test. MRSA = Methicillin-resistant Staphylococcus aureus. = Column percentage; N (xx%) = Number of patients (row percentage). OR = Odds ratio; CI = Confidence
erval; a = chi-square test. MRSA = Methicillin-resistant Staphylococcus aureus. Table 3. Descriptive logistic regression analysis from data obtained at admission (medical comorbidities,
other risk factors for MRSA). able 3. Descriptive logistic regression analysis from data obtained at admission (medical comorbiditie
ther risk factors for MRSA). Number of
Patients
MRSA (−)
(1463 Patient)
MRSA (+)
(114 Patient)
p-Value a
N = 1577 (%)
N (%)
N (%)
Hypertension
0.059
No
919 (58.3%)
843 (91.7%)
76 (8.3%)
Yes
658 (41.7%)
620 (94.2%)
38 (5.8%)
Cardiovascular
disease
0.510
No
1486 (94.2%)
1377 (92.7%)
109 (7.3%)
Yes
91 (5.8%)
86 (94.5%)
5 (5.5%)
Diabetes Mellitus
0.697
No
1236 (78.4%)
1145 (92.6%)
91 (7.4%)
Yes
341 (21.6%)
318 (93.3%)
23 (6.7%)
Hepatic disease
0.516
No
1502 (95.2%)
1392 (92.7%)
110 (7.3%)
Yes
75 (4.8%)
71 (94.7%)
4 (5.3%)
Dialysis
0.405 *
No
1565 (99.2%)
1451 (92.7%)
114 (7.3%)
Yes
12 (0.8%)
12 (100.0%)
0 (0.0%)
Recent
hospitalization
0.951
No
1172 (74.3%)
1087 (92.7%)
85 (7.3%)
Yes
405 (25.7%)
376 (92.8%)
29 (7.2%)
Recent antibiotics
<0.01
No
1105 (70.1%)
1061 (96.0%)
44 (4.0%)
Yes
472 (29.9%)
402 (85.2%)
70 (14.8%) 6 of 11 J. Clin. Med. 2019, 8, 631 Table 3. Cont. 4. Discussion According to this study, the overall prevalence of nasal MRSA carriage is 7.2%. There is
a huge difference in the epidemiology of MRSA worldwide. For example, the prevalence of
community-associated MRSA infection in Japan, Germany, Turkey, Taiwan, and Malta was found
to be 0.94% [29], 1.2% [17], 1.2% [30], 3.8% [31], and 8.81% [6] respectively. These differences can be
attributed to variations in microbiological methods (sampling technique, culture sites, and method of
MRSA identification), local infection control standards, and the local prevalence of MRSA. Gi et al. [32]
reported that the methicillin resistance rate of staphylococcal isolates in Korea is slightly higher
than that in other countries. Another study showed that the prevalence of MRSA is 73% among
the nosocomial S. aureus isolates in Korea, which is higher than that in other Asian countries [21]. Therefore, we considered it worthwhile to study the prevalence and risk factors for MRSA colonization
in the orthopedic field, where aseptic surgery is essential. Several studies have evaluated the risk factors associated with MRSA carriage [33–38]. Of the
many known risk factors, the present study identified being underweight, having a diagnosis of
trauma, recent antibiotic use, and admission through the emergency department as significant risk
factors for MRSA carriage. Contrary to some prior studies, the present study did not reveal age as a
significant risk factor [35,36]. The majority of studies have reported that the elderly patients have a
higher tendency of being an MRSA carrier. An increased burden of infection in the elderly is linked to
age-related decline in immune function, malnutrition, and anatomical and physiological changes [35]. However, some studies have reported that MRSA colonization is not influenced by age [39], and others
found a higher carriage rate in younger patients [36]. Therefore, the association between age and
MRSA colonization remains controversial. Our results showed that being underweight is a risk factor for MRSA colonization and being
overweight decreases the colonization rate. However, obesity did not turn out to be a significant
risk factor for MRSA colonization in this study. Neidhart et al. [17] reported a reduced risk for
S. aureus carriage in obese (BMI ≥30.0 kg/m2) compared to overweight patients (BMI of 25.0 to
30 kg/m2). However, other studies reported the opposite result. Olsen et al. [40] found a significant
positive correlation between BMI and MRSA carriage only in women, particularly among those aged
30–43 years. Campbell et al. 3.2.2. Multiple Logistic Regression Analysis Table 4 shows the results of the multiple logistic regression analysis for the risk factors found
statistically significant in the univariate logistic regression analysis. Being underweight, having trauma,
recent antibiotic use, and admission through the emergency room were found to be risk factors. Patients
with a BMI lower than 18.5 kg/m2 had a 2.026 times higher risk of nasal MRSA carriage than patients
with normal BMI. Patients diagnosed with trauma had a 1.795 times higher rate of nasal MRSA carriage
than those diagnosed with disease. In addition, patients with recent antibiotics use had a 1.946 times
higher risk of MRSA carriage than patients without antibiotic use. Furthermore, patients admitted
through the emergency department had a 3.998 times higher MRSA carriage rate than those admitted
through the outpatient department. 3.2.1. Univariate Logistic Regression Analysis Number of
Patients
MRSA (−)
(1463 Patient)
MRSA (+)
(114 Patient)
p-Value a
N = 1577 (%)
N (%)
N (%)
Urinary catheter
0.985
No
1329 (84.3%)
1233 (92.8%)
96 (7.2%)
Yes
248 (15.7%)
230 (92.7%)
18 (7.3%)
Route of admission
<0.01
ER
227 (14.4%)
174 (76.7%)
53 (23.3%)
OPD
1350 (85.6%)
1289 (95.5%)
61 (4.5%)
Body part involved
0.343
Spine
567 (36.0%)
543 (95.8%)
24 (4.2%)
Knee, shoulder
531 (33.7%)
506 (95.3%)
25 (4.7%)
Hand, elbow
64 (4.1%)
64 (100.0%)
0 (0.0%)
Hip
127 (8.1%)
119 (93.7%)
8 (6.3%)
Foot
288 (18.1%)
231 (80.2%)
57 (19.8%)
Disease/Trauma
<0.01
Trauma
560 (35.5%)
500 (89.3%)
60 (10.7%)
Disease
1017 (64.5%)
963 (94.7%)
54 (5.3%)
x% = Column percentage; N (xx%) = Number of patients (row percentage). OR = Odds ratio; CI = Confidence
Interval. a = chi-square test or * Fisher’s exact test. x% = Column percentage; N (xx%) = Number of patients (row percentage). OR = Odds ratio; CI = Confidence
Interval. a = chi-square test or * Fisher’s exact test. Table 4. Univariable and multivariable logistic regression analysis of the risk factors associated with
MRSA colonization. Table 4. Univariable and multivariable logistic regression analysis of the risk factors associated with
MRSA colonization. Variables
Univariable Analysis
OR (95% CI)
p-Value
Multivariable Analysis
Adjusted OR (95% CI)
p-Value
Age
<40
1
40–70
0.237 (0.151–0.374)
<0.01 **
>70
0.292 (0.180–0.474)
<0.01 **
BMI
Normal weight
1
1
Underweight
3.432 (1.308–9.000)
0.012 **
2.026 (1.115–3.680)
0.020**
Overweight
0.290 (0.153–0.550)
<0.01 **
0.389 (0.219–0.689)
<0.01 **
Obese
0.637 (0.238–1.702)
0.369
0.637 (0.246–1.650)
0.354
Smoking
Never-smoker
1
Current-smoker
4.204 (2.801–6.309)
<0.01 **
Ex-smoker
0.410 (0.056–3.019)
0.381
Alcohol
Never-drinker
1
Current-drinker
4.204 (3.250–7.223)
<0.01 **
Former-drinker
N/A
Hypertension
0.680 (0.454–1.017)
0.061
Code
Disease
1
1
Trauma
3.401 (1.593–7.261)
<0.01 **
1.795 (0.984–3.275)
0.048 **
Recent antibiotics use
4.119 (2.831–6.228)
<0.01 **
1.946 (1.178–3.217)
<0.01 **
Route of admission
OPD
1
1
ER
6.915 (2.863–16.702)
<0.01 **
3.998 (2.003–7.979)
0.047 **
**: p < 0.05. J. Clin. Med. 2019, 8, 631 7 of 11 7 of 11 4. Discussion [41] reported an increased risk of S. aureus colonization in patients with
both obesity and asthma. The analysis of skin and soft tissue staphylococcal infections showed that
obesity is related to the presence of methicillin resistance. Therefore, further studies are needed to
prove a definite relation between BMI and MRSA colonization. In this study, smoking was not found to be a statistically significant risk factor for MRSA carriage. The influence of smoking on the colonization of MRSA is still controversial. Some studies proposed
that smokers have higher rates of MRSA colonization than nonsmokers, thus increasing their risk of
serious and difficult-to-treat infections [42,43]. Ellisa et al. reported that cigarette smoke increases
MRSA hydrophobicity, thus increasing MRSA adherence and invasion [43]. They also stated that
cigarette smoking increases the MRSA expression of genes linked to cell surface changes. In contrast,
the smoke itself seems to influence the S. aureus load as indicated by the reduced identification rate J. Clin. Med. 2019, 8, 631 8 of 11 in the upper respiratory tract [44]. A possible explanation for this finding is the inherent toxicity of
smoke. The capability of smoke to inhibit the growth capacity of Gram-positive bacteria, particularly
S. aureus, has already been demonstrated [45]. However, whether smoking affects MRSA colonization
remains unclear. In our study, patients admitted through the emergency department and those diagnosed with
trauma had 3.998 and 1.795 times higher risk of MRSA colonization, respectively. Most trauma patients
tend to visit the emergency department, and these two factors were considered by us to be similar. Quach et al. [46] reported that a visit to the emergency department is associated with more than
threefold increased risk of acute infection. Patients with morbid conditions are more likely to visit the
emergency department than patients with less morbid conditions. In addition, admission through
the emergency department may increase the chance of contact with these patients, which may be
expected to increase the risk of acquiring an infection. Also, patterns of injury differ between different
mechanisms of trauma [47] which may cause patients diagnosed with trauma to be more susceptible to
MRSA. For example, certain mechanisms of trauma may result in skin injuries, which may serve as a
portal for entry. Furthermore, overcrowding of the emergency department creates more opportunities
for cross-transmission [48], and overworked medical staffmay not follow preventive procedures and
take inadequate precautions [49,50]. 4. Discussion Our findings on the correlation between trauma, admission
through the emergency department, and MRSA detection may be due to these factors. This study proves the relationship between antibiotic use prior to admission and MRSA
colonization. Muller et al. [28] reported individual exposure to fluoroquinolones and collective
exposure to penicillin to be associated with MRSA isolation after adjustment for colonization pressure
and other potential confounders. Tacconelli et al. [51] performed a meta-analysis of over 76 studies
included 24,230 patients and found that the development of MRSA is significantly related to the length
of antibiotic exposure. According to the Society for Healthcare Epidemiology of America guidelines
for preventing nosocomial transmission of MRSA and vancomycin-resistant enterococci, the use of
antimicrobial agents in US hospitals is commonly excessive or unnecessary [9]. This widespread use of
antibiotics may create conditions in which resistant bacteria experience a competitive advantage. There are some limitations to our study. First, only nasal swabs were performed by us as the
screening test considering cost- and time-effectiveness. Some studies suggest that collecting samples
from additional sites (throat, groin, and thorax) may contribute to a higher detection rate than using
a single site [17]. The prevalence of MRSA colonization might have been higher if samples from
additional sites were collected. However, the anterior nasal cavity is known to be the most common
carrier site of MRSA [11,12]. Second, we focused mainly on the screening test but whether the result of
the screening test actually contributed to the development of post-operative infection has not been
investigated. Third, this is a single-institute and single-department study. Additional multi-center
studies must be performed to determine the actual frequency and risk factors for MRSA in Korea. However, the current study is valuable in that it is among the first to focus on the prevalence and
risk factors for MRSA in the orthopedic patients in Korea. The importance of the nasal MRSA screening
test will be further validated by analyzing the clinical outcomes of the patients with a positive swab. References 1. Su, C.-H.; Chang, S.-C.; Yan, J.-J.; Tseng, S.-H.; Chien, L.-J.; Fang, C.-T. Excess Mortality and Long-Term
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for Disease Control & Prevention 2012. 5. Conclusions The present study focused on the prevalence of MRSA colonization and associated risk factors
among patients admitted to the orthopedic surgery department. Being underweight, having a
trauma diagnosis, antibiotic use one month prior to admission, and admission through the emergency
department are risk factors for MRSA infection. The relatively high prevalence of MRSA in this study
highlights the importance of pre-operative screening tests for patients scheduled for surgery involving
implant insertion, particularly those at risk for MRSA. Among high risk patients, elective surgery
could be delayed until the confirmation of the MRSA screening test. Prophylactic treatment prior to
surgery is recommended for patients with positive MRSA colonization. J. Clin. Med. 2019, 8, 631 9 of 11 Author Contributions: Conceptualization, S.-W.C.; methodology, S.-W.C.; validation, S.-W.C.; formal analysis, J.K.;
investigation, J.K. and S.-W.C.; resources, S.-W.C.; data curation, J.K. and S.P.; writing—original draft preparation,
J.K.; writing—review and editing, J.C.L. and J.E.K. and M.J.B. and S.Y.P.; visualization, J.C.L.; supervision, B.-J.S.;
funding acquisition, S.-W.C. Author Contributions: Conceptualization, S.-W.C.; methodology, S.-W.C.; validation, S.-W.C.; formal analysis, J.K.;
investigation, J.K. and S.-W.C.; resources, S.-W.C.; data curation, J.K. and S.P.; writing—original draft preparation,
J.K.; writing—review and editing, J.C.L. and J.E.K. and M.J.B. and S.Y.P.; visualization, J.C.L.; supervision, B.-J.S.;
funding acquisition, S.-W.C. Funding: This study was supported by the Soonchunhyang University research foundation and National Research
Foundation of Korea (NRF) grant funded by the Korea government (MIST). (2019R1A2B5B01005924). Funding: This study was supported by the Soonchunhyang University research foundation and National Research
Foundation of Korea (NRF) grant funded by the Korea government (MIST). (2019R1A2B5B01005924). Acknowledgments: The authors sincerely thank Soon-hyo Kwon and Jae Heon Kim for their assistance with
patient data analysis and consolidation. Acknowledgments: The authors sincerely thank Soon-hyo Kwon and Jae Heon Kim for their assistance with
patient data analysis and consolidation. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
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of methicillin-resistant Staphylococcus aureus (MRSA) isolation? A systematic review and meta-analysis. J. Antimicrob. Chemother. 2008, 61, 26–38. [CrossRef] © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/). © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
|
https://openalex.org/W4293661304
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https://journal2.unusa.ac.id/index.php/MTPHJ/article/download/3187/1749
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Indonesian
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The Relationship between Snack Consumption Patterns and Nutritional Status in Adolescents in Malang City Indonesia
|
Medical technology and public health journal
| 2,022
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cc-by-sa
| 2,785
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ABSTRACT Background : The consumption of snacks is often done between meals. About 20% of the
daily intake of adolescents comes from snacks. Snacks contribute ‘empty’ energy but provide
an excess intake of substances that are not beneficial to the teenager’s body. Objectives; this
study aims to determine the pattern of snack consumption and nutritional status of
adolescents in Malang City. Material and Method; This study used a cross-sectional
approach to determine the relationship between snack consumption patterns and nutritional
status in adolescent in Malang City. The population of this study consisted of 128 high school
students (aged 15-18 years) who were selected using the multistage random sampling method
( a combination of the cluster method and simple random sampling) from July-September
2015. Results : Respondents who experiences poor nutritional status were 40 pople (8,6%)
and overweight as many as 88 people (19%). Respondents who consumed snacks as much as
3-4x/week showed less nutritional status 14people (35%) and overweight 35 people (39,8%). Conclusion; This study concludeds that the frequency of snack consumption affects the
nutritional status of underweight and overweight in adolescents aged 15-18 years in Malang
City Keywords: consumption pattern, snacks, nutrition status, adolescents Ira Dwijayanti1*, Jane C-J Chao2 Ira Dwijayanti1*, Jane C-J Chao2 1 Program Studi S1 Gizi, Fakultas Kesehatan, Universitas Nahdlatul Ulama Surabaya,
Surabaya, Indonesia
2 School of Nutrition and Health Sciences, College of Nutrition, Taipei Medical University,
Taipei, Taiwan
*iradwijayanti@unusa.ac.id ABSTRAK Pendahuluan : Konsumsi makanan ringan atau camilan sering dilakukan di antara waktu
makan utama. Sekitar 20% dari kebutuhan asupan energi harian remaja berasal dari camilan. Camilan menyumbang energi ‘kosong’ tetapi memberikan asupan berlebih zat yang tidak
bermanfaat pada tubuh remaja. Penelitian ini bertujuan untuk mengetahui pola konsumsi
camilan dan status gizi pada remaja di Kota Malang. Bahan dan Metode : Penelitian ini
menggunakan pendekatan cross-sectional untuk mengetahui hubungan pola konsumsi
camilan dan status gizi pada remaja di Kota Malang, Indonesia. Populasi penelitian ini terdiri
dari 128 siswa SMA (usia 15-18 tahun) yang dipilih menggunakan metode multistage random
sampling (gabungan dari metode cluster dan simple random sampling) dari Bulan Juli-
September 2015. Hasil : Responden yang mengalami status gizi kurang sebanyak 40 orang
(8,6%) dan status gizi lebih sebanyak 88 orang (19%). Reponden yang mengonsumsi camilan
sebanyak 3-4x/minggu menunjukkan status gizi kurang 14 orang (35%) dan status gizi lebih
35 orang (39,8%). Ada perbedaan signifikan antara frekuensi konsumsi camilan dan status gizi remaja (p=0.00). Kesimpulan : Penelitian ini menyimpulkan bahwa frekuensi konsumsi
camilan berpengaruh terhadap status gizi kurang dan lebih pada remaja usia 15-18 tahun di
Kota Malang. Kata Kunci : Pola konsumsi, camilan, status gizi, remaja PENDAHULUAN telah
meningkat
secara
signifikan
bersamaan dengan tingkat obesitas pada
remaja. Sekitar 20% dari kebutuhan asupan
energi harian remaja berasal dari camilan
(5). Camilan menyumbang energi ‘kosong’
tetapi memberikan asupan berlebih zat
yang tidak bermanfaat pada tubuh remaja
(6). Berat badan lebih dan obesitas
merupakan permasalahan gizi remaja di
Indonesia. Prevalensi remaja usia 16-18
tahun yang mengalami kelebihan berat
badan
dan
obesitas
adalah
13,5%. Prevalensi remaja gemuk di Indonesia
mengalami peningkatan sebanyak 2,2%
dalam waktu 5 tahun. Provinsi Jawa Timur
termasuk dalam daerah yang memiliki
prevalensi
remaja
gemuk
melebihi
prevalensi nasional yaitu 11,3% (1,2)
Remaja dengan kelebihan berat badan atau
obesitas memiliki risiko yang lebih tinggi
untuk mengalami penyakit kronis seprti
hipertensi, diabetes tipe 2 dan dislipidemia
serta konsekuensi psikososial seperti stres
dan depresi. Selain itu, remaja obesitas
memiliki risiko tetap mengalami obesitas
saat dewasa (3). Sebagian besar remaja di Indonesia
menggunakan waktu luang untuk kegiatan
tidak aktif, sepertiga remaja makanan
camilan buatan pabrik atau makanan
olahan, dan sepertiga lainnya sering
mengonsumsi kue basah, roti basah,
gorengan dan kerupuk (7). Perubahan gaya
hidup saat ini membuat remaja semakin
terhubung degan akses internet sehingga
remaja lebih sering membuat keputusan
mandiri dalam memilih makanan. Pilihan
makanan yang dibuat seringkali kurang
tepat sehingga dapat menyebakan masalah
gizi (5)(8). Keadaan
status
gizi
remaja
di
Indonesia harus diperbaiki. Berdasarkan
penelitian sebelumnya, ditemukan adanya
perubahan pola makanan dan aktivitas fisik
pada remaja (4). Konsumsi makanan
ringan atau camilan sering dilakukan di
antara waktu makan utama merupakan
salah satu pola makan yang banyak
ditemui pada remaja. Konsumsi camilan Pendekatan
pola
makan
untuk
mengurangi
konsumsi
camilan
sering
ditargetkan untuk mencapai pengurangan
kalori dalam program pengurangan berat
badan remaja. Memahami hubungan antara
pola konsumsi camilan dan status gizi pada
remaja ini sangat penting. Penelitian sebelumnya hanya fokus terhadap asupan
makanan
remaja
dari
laporan
atau
wawancara orang tua. Potensi bias dalam
laporan asupan makan remaja yang tidak
dilakukan secara langsung akan membatasi
kemampuan peneliti untuk memperkirakan
hubungan yang sebenarnya (2). di Kota Malang. Beberapa kriteria inklusi
penelitian ini adalah (1) Kriteria umur
subjek penelitian adalah 15-18 tahun; (2)
Siswa berdomisili di Malang; (3) Siswa
wajib mengisi persetujuan orang tua atau
wali pada formulir perijinan penelitian;
dan (4) Subjek penelitian harus mengisi
dan
melengkapi
kuesioner
penelitian
secara mandiri. Enam puluh enam siswa
tidak mengisi kuesioner secara lengkap
sehingga dikeluarkan dari penelitian ini. Penelitian ini telah disetujui oleh Komisi
Etika
Penelitian
Kesehatan
Fakultas
Kedokteran Universitas Brawijaya (No. 465/EC/KEPK/088/2015). PENDAHULUAN Upaya perbaikan gizi pada remaja
yang dilakukan oleh sektor kesehatan tidak
akan tercapai maksimal tanpa adanya
intervensi sensitif yang sesuai dengan
keadaan
masyarakat. Perlu
dilakukan
penelitian untuk mendapatkan gambaran
data dasar pola makan dan status gizi
remaja. Oleh karena itu, penelitian ini
bertujuan untuk mengetahui pola konsumsi
camilan dan status gizi pada remaja di
Kota Malang. Data
tinggi
dan
berat
badan
responden
diukur
untuk
menentukan
Indeks Massa Tubuh (IMT)/Umur (U). IMT/U dikategorikan berdasarkan nilai
persentil sesuai dengan grafik World
Health Organization (WHO) yaitu berat
badan kurang (IMT <5 th persentile),
normal ≥ 5 th - <85 th dan gemuk/obesitas
≥85th persentile (10). Data pola makan
dikumpulkan
dengan
menggunakan
kuesioner
Semi
Quantitative-Food
Frequency
Questionnaire
(SQ-FFQ). Semua bahan makanan diadopsi dari
pedoman makan Indonesia yang disajikan
dalam tumpeng gizi seimbang. Tumpeng
gizi seimbang atau piramida makanan
merupakan pedoman pola makan yang
digambarkan melalui 4 lapisan piramida
dengan tujuan untuk menyeimbangkan gizi METODE Penelitian
ini
menggunakan
pendekatan
cross-sectional
untuk
mengetahui
hubungan
pola
konsumsi
camilan dan status gizi pada remaja di
Kota Malang, Indonesia. Penelitian cross-
sectional memberikan keuntungan peneliti
untuk mendapatkan berbagai hasil dengan
eksposur secara bersamaan dalam satu
waktu (9). Populasi penelitian ini terdiri
dari 128 siswa SMA (usia 15-18 tahun)
yang
dipilih
menggunakan
metode
multistage random sampling (gabungan
dari metode cluster dan simple random
sampling) dari Bulan Juli-September 2015 ini. Semua analiss statistik dilakukan
dengan menggunakan SPSS versi 22
(Armonk, NY:IBM Corp). dan porsi makan harian. Data food recall
24 hours juga dikumpulkan dari 50 orang
siswa yang dipilih secara acak untuk
menambah daftar makanan lain dan
menentukan ukuran porsi rata-rata dalam
kuesioner. Frekuensi jenis makanan diukur
dengan sebelas kategori yaitu tidak pernah,
kurang dari 1 kali sebulan, 1 sampai 3 kali
sebulan, 1 kali seminggu, 2 kali seminggu,
3 kali seminggu, 4 kali seminggu, 5 kali
seminggu, 6 kali seminggu, 7 kali
seminggu dan lebih dari 1 kali sehari. Analisis
pola
makan
menggunakan
Principal Component Analysis (PCA). Total 84 jenis makanan yang terdapat di
kuesioner dikurangi menjadi 23 jenis
makanan berdasarkan faktor loading ≥±
0,30. Empat
faktor
diidentifikasi
berdasarkan nilai eigen lebih besar dari
>0,10, bentuk plot scree dan interpretasi
pola makan yang bermakna. Interpretasi
komponen makanan menggunakan rotasi
varimax. Pola makan camilan terdiri dari
jenis makanan kue ketan, kue mangkok,
cenil, dan kue pukis (11). Analisis
data
menggunakan HASIL Hubungan antara konsumsi camilan dan status gizi remaja usia 15-18 tahun di Kota
Malang (n=128)
Variabel
Kurus
n=40
Gemuk
n=88
pa
Konsumsi Camilan (n,%)
Tidak pernah
0 (0.0)
4 (4.5)
0.00
1-2x/ minggu
6 (15.0)
27 (30.7)
3-4x/ minggu
14 (35.0)
35 (39.8)
5-6x/ minggu
6 (15.0)
10 (11.4)
7x/ minggu
14 (35.0)
12 (13.6)
aData analisis menggunakan uji korelatif spearman . Hubungan antara konsumsi camilan dan status gizi remaja usia 15-18 tahun di Kota
Malang (n=128) remaja
yang
cenderung
mengonsumsi
makanan ringan yang mengandung tinggi
lemak
dan
tinggi
kalori
dapat
mengakibatkan gizi lebih (15). Kebiasaan
konsumsi camilan juga dapat menjadikan
seseorang melewatkan waktu makan utama
sehingga
menyumbang
asupan
energi
kosong
dan
menyebabkan
kurangnya
asupan zat penting untuk tubuh (16). HASIL Data karakteristik dasar responden
terdiri dari 189 laki-lai (40,1%) dan 278
perempuan (59,9%) yang berusia 15
sampai 18 tahun. Pendidikan ayah dan ibu
menunjukkan
46,8%
dan
47,8%
merupakan lulusan sarjana. Ayah bekerja
penuh waktu (±≥ 8 jam per hari) sebanyak
67% dan ibu tidak bekerja sebanyak
43,8%. Penghasilan keluarga menunjukkan
sebanyak Rp 3.000.000 rupiah sebanyak
55,3%. Tidak
ada
perbedaan
yang
signifikan dalam data demografi antara
jenis kelamin. Konsumsi camilan dan pengukuran
antropometri sesuai dengan IMT menurut
usia disajikan dalam tabel 1. Responden
melaporkan
paling
banyak
konsumsi
camilan 3-4x seminggu (35,8%) dan 7x
seminggu (22,2%). Tabel 2 menunjukkan
bahwa
8,6%
dan
19%
responden
dikasifikasikan
sebagai
kurus
(IMT
menurut usia <5persentile) dan kelebihan
berat badan/obesitas (IMT menurut usia
>85persentile). Analisis
data
menggunakan
pengukuran
non-parametrik
karena
distribusi data tidak normal dan data
merupakan data nominal. Proporsi dan
perbandingan antar variabel diukur dengan
tes chi square. Korelasi Spearman dan
regresi logistik digunakan untuk mengukur
korelasi antar variable. Nilai p kurang dari
0,05 dianggap signifikan dalam penelitian Hubungan antara konsumsi camilan
dan status gizi remaja usia 15-18 tahun di
Kota Malang ditunjukkan pada tabel 2. Responden yang mengalami status gizi
kurang (8,6%) dan lebih (19%) sebanyak
128 orang. Reponden yang mengonsumsi signifikan
antara
frekuensi
konsumsi
camilan dan status gizi remaja. 3-4x/minggu sebanyak camilan menunjukkan status gizi kurang (35%) dan
status gizi lebih (39,8%). Ada perbedaan Tabel 1. Data konsumsi camilan dan status gizi remaja usia 15-18 tahun di Kota Malang
(n=128)
Variabel
Laki-laki
Perempuan
Total
IMT/U (n, %)
Kurus (≤5th presentile)
19 (10.2)
21 (7.6)
40 (8.6)
Gemuk (≥85th presentile)
45 (24.2)
43 (15.5)
88 (19.0)
Tabel 2 Hubungan antara konsumsi camilan dan status gizi remaja usia 15-18 tahun di Kota Tabel 1. Data konsumsi camilan dan status gizi remaja usia 15-18 tahun di Kota Malang
(n=128)
Variabel
Laki-laki
Perempuan
Total
IMT/U (n, %)
Kurus (≤5th presentile)
19 (10.2)
21 (7.6)
40 (8.6)
Gemuk (≥85th presentile)
45 (24.2)
43 (15.5)
88 (19.0)
Tabel 2. Hubungan antara konsumsi camilan dan status gizi remaja usia 15-18 tahun di Kota
Malang (n=128)
Variabel
Kurus
n=40
Gemuk
n=88
pa
Konsumsi Camilan (n,%)
Tidak pernah
0 (0.0)
4 (4.5)
0.00
1-2x/ minggu
6 (15.0)
27 (30.7)
3-4x/ minggu
14 (35.0)
35 (39.8)
5-6x/ minggu
6 (15.0)
10 (11.4)
7x/ minggu
14 (35.0)
12 (13.6)
aData analisis menggunakan uji korelatif spearman Tabel 2. PEMBAHASAN Konsumsi makanan ringan atau
camilan
dalam
frekuensi
sering
menyebabkan asupan makanan berlebih dan
memiliki potensi untuk meningkatkan berat
badan
(12). Jumlah
camilan
yang
dikonsumsi
cenderung
lebih
sedikit
dibandingkan dengan jumlah menu pada
makanan utama, namun frekuensi camilan
yang sering itu menjadikan asupan kalori
yang setara atau berlebih (13). Kebiasaan
mengonsumsi energi melebihi kebutuhan
tubuh maka kelebihan energi akan disimpan
dalam bentuk lemak sebagai cadangan
energi (14). Perubahan gaya hidup pada Pemilihan
jenis
makanan
dan
frekuensi konsumsi camilan menjadi sangat
berpengaruh terhadap tingkat kecukupan
asupan gizi. Responden menyebutkan jenis
makanan camilan kue ketan, kue mangkok,
cenil, dan kue pukis. Jenis makanan yang mengandung tepung berkontribusi terhadap
peningkatan berat badan (17). Seseorang
yang
mengonsumsi
camilan
dengan
frekuensi yang sering sedangkan tetap
mengonsumsi
makanan
utama
maka
cenderung akan meningkatkan berat badan. Sebaliknya, jika frekuensi camilan sering
namun melewatkan waktu makan utama
dan asupan zat gizi tidak terpenuhi makan
seseorang cenderung menjadi kurus (18). mineral(21). mineral(21). KESIMPULAN 6. Zuhdy N, Ani LS, Utami WAU. Aktivitas Fisik , Pola Makan dan Status
Gizi Pelajar Putri SMA di Denpasar
Utara
(Physical
Activity
,
Food
Consumption and Nutritional Status
among Female High School Students in
North Denpasar ). Public Heal Prev
Med Arch [Internet]. 2015;3(1):78–83. Tersedia
pada:
https://www.researchgate.net/publicatio
n/332348005 Penelitian ini menyimpulkan bahwa
frekuensi konsumsi camilan berpengaruh
terhadap status gizi kurang dan lebih pada
remaja usia 15-18 tahun di Kota Malang. Penelitian ini diharapkan dapat dilanjutkan
menggunakan metode prospektif untuk
mengetahui efek dalam jangka panjang
camilan terhadap status gizi di masa depan. 7. Irdiana W, Nindya TS. Hubungan
Kebiasaan Sarapan dan Asupan Zat
Gizi dengan Status Gizi Siswi SMAN 3
Surabaya. Amerta Nutr. 2017;1(3):227. mineral(21). Penelitian
menunjukkan
bahwa
konsumsi camilan berfungsi mengganjal
rasa
lapar
di
antara
waktu
makan. Masyarakat
Indonesia
rata-rata
mengonsumsi 3x makanan ringan per hari,
melebihi jumlah rata-rata global. Selain itu,
tersedianya waktu luang dan rendahnya
aktivitas fisik mengarahkan remaja untuk
mekan berlebihan terutama camilan setelah
mengonsumsi makanan utama atau sekedar
pengganti waktu makan. Remaja memiliki
risiko yang besar untuk mandapatkan
asupan gizi yang tidak seimbang (17). Perbaikan pola makan remaja yang bersifat
spesifik dan sensitif gizi perlu diupayakan
dan memiliki integrasi yang baik untuk
mencapai status gizi remaja yang optimal. Pendidikan
gizi
untuk
meningkatkan
pengetahuan, sikap dan efikasi diri remaja
terkait makanan sehat harus dilakukan
secara kontinyu(22). Penelitian ini menunjukkan bahwa
responden yang memiliki status gizi kurang
dan lebih didapatkan mengonsumsi camilan
dengan frekuensi 3-4x/minggu. Penelitian
lain menunjukkan bahwa hasil serupa
dimana
responden
yang
terbiasa
mengonsumsi camilan berhubungan dengan
peningkatan asupan energi dan signifikan
terhadap status gizi (19). Penelitian lain
menunjukan bahwa ada hubungan antara
konsumsi
camilan
dan
status
gizi. Responden
yang
sering
mengonsumsi
camilan dilaporkan mengalami defisiensi
mikronutrien seperti kalsium dan vitamin A
sehingga menghambat pertumbuhan. Status
gizi diperburuk dengan konsumsi camilan
dalam jumlah besar dengan kandungan
mikronutrien rendah. Oleh karena itu
konsumsi camilan harus dikurangi dan
diganti dengan makanan yang mengandung
padat energi (20). Penelitian lain juga
menunjukkan bahwa remaja lebih menyukai
makanan dengan kandungan natrium dan
lemak yang tinggi tetapi rendah vitamin dan Keterbatasan dalam penelitian kami
adalah penelitian dilakukan di sekolah
menengah atas di Kota malang sehingga
membatasi untuk generalisasi hasil di kota
lain. Jumlah sampel juga mungkin dapat
membatasi untuk menemukan hasil yang
serupa pada populasi umum. Penyebab
langsung antara konsumsi camilan pada
remaja usia 15-18 tahun di Kota Malang
tidak dapat diukur dari waktu ke watu
karena menggunakan desain penelitian
cross-sectional. DAFTAR PUSTAKA 1. Setiawati
FS,
Mahmudiono
T,
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Di SMA Negeri 6 Surabaya Tahun
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Public health application of predictive modeling: an example from farm vehicle crashes
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Injury epidemiology.
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© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Many variables are needed to predict a future event
with good accuracy, with an implicit assumption that the
predicted event has consistent explanatory factors. For
example, to predict tomorrow’s weather in a given city, we
will need information on today’s weather in that city,
today’s weather in all places surrounding that city, the
wind speed, and the season and annual averages based on
past weather trends. In the current highly digitized world
with Big Data, large sets of variables with sophisticated
predictive algorithms may allow forecasting a future event
with increasing fidelity. Ranapurwala et al. Injury Epidemiology (2019) 6:31
https://doi.org/10.1186/s40621-019-0208-9 Public health application of predictive
modeling: an example from farm vehicle
crashes Shabbar I. Ranapurwala1,2*
, Joseph E. Cavanaugh3,4, Tracy Young3, Hongqian Wu4, Corinne Peek-Asa3 and
Marizen R. Ramirez3,5 Abstract Background: The goal of predictive modelling is to identify the likelihood of future events, such as the predictive
modelling used in climate science to forecast weather patterns and significant weather occurrences. In public
health, increasingly sophisticated predictive models are used to predict health events in patients and to screen high
risk individuals, such as for cardiovascular disease and breast cancer. Although causal modelling is frequently used
in epidemiology to identify risk factors, predictive modelling provides highly useful information for individual risk
prediction and for informing courses of treatment. Such predictive knowledge is often of great utility to physicians,
counsellors, health education specialists, policymakers or other professionals, who may then advice course correction
or interventions to prevent adverse health outcomes from occurring. In this manuscript, we use an example dataset
that documents farm vehicle crashes and conventional statistical methods to forecast the risk of an injury or death in a
farm vehicle crash for a specific individual or a scenario. Results: Using data from 7094 farm crashes that occurred between 2005 and 2010 in nine mid-western states, we
demonstrate and discuss predictive model fitting approaches, model validation techniques using external datasets, and
the calculation and interpretation of predicted probabilities. We then developed two automated risk prediction tools
using readily available software packages. We discuss best practices and common limitations associated with predictive
models built from observational datasets. Conclusions: Predictive analysis offers tools that could aid the decision making of policymakers, physicians, and
environmental health practitioners to improve public health. Keywords: Forecasting, Predictions, Decision support techniques, Motor vehicles Yahoo, travel websites, or elsewhere. The information
needed for such prediction may, for example, come from
an individual’s browsing history or grocery shopping
habits. Many variables are needed to predict a future event
with good accuracy, with an implicit assumption that the
predicted event has consistent explanatory factors. For
example, to predict tomorrow’s weather in a given city, we
will need information on today’s weather in that city,
today’s weather in all places surrounding that city, the
wind speed, and the season and annual averages based on
past weather trends. In the current highly digitized world
with Big Data, large sets of variables with sophisticated
predictive algorithms may allow forecasting a future event
with increasing fidelity. Yahoo, travel websites, or elsewhere. The information
needed for such prediction may, for example, come from
an individual’s browsing history or grocery shopping
habits. * Correspondence: sirana@email.unc.edu
1Injury Prevention Research and Department of Epidemiology, University of
North Carolina at Chapel Hill, 137 E Franklin St, Suite 500, CB# 7505, Chapel
Hill, NC 27599, USA
2Department of Epidemiology, College of Public Health, University of Iowa,
Iowa City, IA, USA
Full list of author information is available at the end of the article Introduction Predictive models or algorithms are routinely utilized to
predict many everyday events or decisions. The list of
such outcomes include daily or inclement weather events
(Saha et al. 2014); department store purchases that com-
panies use to provide coupons at grocery stores (Wu and
Bryniolfsson 2013; Schoen et al. 2013); or targeted adver-
tisement and news stories that we receive on Facebook, Page 2 of 11 Page 2 of 11 Ranapurwala et al. Injury Epidemiology (2019) 6:31 Ranapurwala et al. Injury Epidemiology (2019) 6:31 In healthcare settings, medical tests, (e.g., blood pres-
sure or cholesterol measurements) are one form of
predictive data that allow physicians to assess the pro-
bability that the patient may (or may not) have an
adverse event in the (near or distant) future. We have
utilized many such medical tests to predict good or bad
health outcomes, for example, to predict breast cancer
(Gail et al. 2007; Quante et al. 2012) and 5-year or
10-year risks of cardiovascular diseases (Keys et al. 1972;
Kleinbaum et al. 1971; Wilson et al. 1998). The latter
work (Keys et al. 1972; Kleinbaum et al. 1971; Wilson et
al. 1998) led to the development of a web-based tool to
forecast the 10-year risk of cardiovascular disease, pre-
dicted by age, sex, smoking status, blood pressure (in-
cluding
medications),
and
cholesterol
levels
(AHA
2017). Similarly, predictive algorithms are also now
being used to estimate the probability that an individual
may suffer from an opioid overdose in a defined future
period (Glanz et al. 2018). Such predictive algorithms
are helpful for a physician, counselor, health education
specialist, social worker, police or parole officer, policy
maker or other professionals to identify individuals at an
increased risk of having certain adverse outcome within
a given timeframe. Predictive models can also be used to
help educate target populations so that individuals can
understand their personal risks given their unique set of
demographics, behavioral characteristics or environmen-
tal / social conditions. may discover that the risk of a hypothetical adverse event
may be twice as high among the exposed than the un-
exposed, i.e., a risk ratio of 2. While on a population or
sample level this may hold true, the risk of actually ex-
periencing the adverse event for each individual in the
exposed and unexposed subsets differ. Introduction Indeed, some indi-
viduals in the exposed subset will never experience the ad-
verse event, and some individuals will experience the
adverse event even when they were not exposed. A pre-
dictive algorithm allows us to identify or measure
subject-specific probabilities of experiencing an adverse
events, thereby aiding decision making at an individual or
situational level. Predictive models can be developed using the same
traditional regression analyses methods used for causal
modelling (e.g. linear, log binomial, logistic, Poisson, Cox
regression). In this manuscript, we illustrate the de-
velopment of a simplistic predictive algorithm for an
injury-related outcome that incorporates our prior under-
standing of the substantive area and utilizes a large
database to predict the probability of an adverse event. Predictive models have widespread applicability in the
area of injury and violence prevention, especially as the
field continues to develop and implement interventions
that target individual behaviors. For example, a physician
could use predictive tools based on a patient’s diagnosis,
mental and physical health history and previous medi-
cation prescriptions to titrate the supply of opioid pain
medication for that patient so that the pain is adequately
controlled while also reducing the risk of an opioid use
disorder. Using similar patient-history driven predictive
tools, a mental health counselor could advise reduced
access to a firearm for someone at high risk of commit-
ting suicide. A particular focus of our research is motor
vehicle crashes, a leading cause of death in rural agricul-
tural communities. Predictive models can aid prevention
efforts that target individual protective behaviors while
driving or operating agricultural equipment on road-
ways. Examples of protective behaviors include use of
seat belts, installation of rollover protective devices in
tractors and safe driving behaviors (following traffic
regulations). Notably, predictive models serve a different purpose
than causal (or associational) models and their resultant
measures like relative (ratio) measures of risks, rates,
odds or hazards. In multivariable modeling, causal or
associational measures (risk ratios, rate ratios, odds
ratios or hazard ratios) examine the partial relationship
(or association) between a single independent variable
with a health outcome (dependent variable) on a ‘popu-
lation level.’ Causal measures tell us for example the
effectiveness of population level interventions in impro-
ving public health or the population level impact of harm-
ful exposures and their resulting public health burden. Example data Predictive models were fit using multivariable logistic
regression for individual-level data. To accommodate
clustering at the crash level, generalized estimating
equations (GEEs) with an exchangeable working corre-
lation structure were employed to fit the models. Three
types of models were formulated. First, we added a set
of non-modifiable variables, which cannot be practically
intervened on, to determine changes in injury probabil-
ity given the non-modifiable factors (model 1). The
non-modifiable variables included state, season, weather,
time-of-crash, number of involved vehicles, equipment
type, and age and sex of the occupant. Next, we added
semi-modifiable factors that can be indirectly intervened
upon or are consequences of the modifiable factors
(model 2). These included ambient light, manner of
collision, vehicle action, number of occupants in the
vehicle, and the occupant type. Lastly, we added modi-
fiable factors, which may be directly intervened on, to
the model (model 3). These included driver contributing
circumstances and occupant protection. Non-modifiable,
semi-modifiable, and modifiable factors were classified
a-priori based on previous literature (Hughes and
Rodgman 2000; Peek-Asa et al. 2007; Pinzke and
Lundqvist 2004; Costello et al. 2003; Gerberich et al. 1996;
Jaarsma and De Vries 2014; Marlenga et al. 2006; Gkritza
et al. 2010; Maio et al. 1992; Stephan and Newstead 2014;
Russo et al. 2014), expert knowledge, and consensus
among the research team members. Transportation is the leading cause of agricultural-re-
lated death, and crashes with farm equipment on road-
ways present a burden for all roadway users (BLS 2013a,
b). Predictive
algorithms
in
transportation
research
could allow us to identify high crash risk scenarios and
provide opportunities to intervene. We used secondary
crash data from 2005 to 2010 to assess the risk of injury
or death in farm vehicle-related crashes (referred hereon
after as crash/ farm crash, unless otherwise mentioned),
given
crash-level,
vehicle-level,
and
individual-level
factors. The data were collected from the departments
of transportation (DOT) of Illinois (IL), Iowa (IA),
Kansas (KS), Minnesota (MN), Missouri (MO), Nebraska
(NE), North Dakota (ND), South Dakota (SD), and
Wisconsin (WI). The data include all police-reported
crashes, including death, injury, or property damage of
at least $500 to $1500, depending on the state. Methods
to identify and code farm vehicle crashes from these
data have been discussed previously (Harland et al. 2014;
Ranapurwala et al. 2016). Multilevel crash data include
the characteristics of crashes, involved vehicles, and their
occupants. Introduction Thus, causal models help in population-level decision
making, especially in formulating policy or even treatment
decisions that affect large populations (e.g., firearm safety
laws, laws mandating use of prescription monitoring pro-
grams or standardized treatment regimens for treating
cancers or HIV-AIDS). However, causal measures do not
quantify the risk of the adverse event for a particular indi-
vidual with a particular set of variable values. Predictive
models offer a complementary means to aid decision
making at the individual or situational level, which causal
(or associational) models are unable to address. For
example, while comparing a sample of individuals who
were exposed to a certain condition to another sample of
individuals who were not exposed to that condition, we We used farm vehicle equipment involved motor
vehicle crash (henceforth, farm crash) data from nine
Midwestern states in the United States from 2005 to
2010 to build models for forecasting the risk of injury or
death in a farm crash. For this case example, we employ
the logistic regression framework, as this regression
technique best addresses the nature of the data. How-
ever, the methods described herein can be easily repli-
cated in other datasets where outcome distributions
favor the use of alternative modeling frameworks. We demonstrate model building approaches for pre-
dictive models using prior knowledge of the substantive Ranapurwala et al. Injury Epidemiology (2019) 6:31 Ranapurwala et al. Injury Epidemiology (2019) 6:31 Page 3 of 11 Ranapurwala et al. Injury Epidemiology Page 3 of 11 (Hughes and Rodgman 2000; Peek-Asa et al. 2007; Pinzke
and Lundqvist 2004; Costello et al. 2003; Gerberich et al. 1996; Jaarsma and De Vries 2014; Marlenga et al. 2006;
Gkritza et al. 2010; Maio et al. 1992; Stephan and Newstead
2014; Russo et al. 2014). Vehicle type was classified as farm
vehicle or non-farm vehicle. Number of vehicle occupants
was categorized as single or multiple occupant. The
crash type was defined as either a single vehicle crash
(farm vehicle only), or a multiple vehicle crash (two
or more vehicles, one of them being a farm vehicle). area. We further demonstrate model fitting approaches,
model validation techniques using external datasets, and
the calculation and interpretation of predicted proba-
bilities. Additionally, we share two risk prediction tools
that can be used by lay and scientific audiences to
predict their own or hypothetical risks of being injured
should they be involved in a farm crash. Introduction Lastly, we dis-
cuss best practices and common limitations associated
with predictive models. Results From 2005 to 2010 there were 7094 farm crashes in the
nine states, of which 86% (n = 6119) were multiple
vehicle crashes and 14% (n = 975) were single vehicle
crashes. Of the 7094 crashes, 10 crashes had missing
injury or fatality information, and were subsequently
excluded from the analyses. A total of 12,936 vehicles
were involved in the 7084 crashes, of which 11,961 ve-
hicles were involved in multiple vehicle farm crashes. There were 14,834 occupants involved in the 7084
crashes, and of these, 2087 (14.1%) had been injured or
killed in the crash. The distribution of the crashes, injuries
and deaths by state and year are presented in Table 1. External validation We partitioned 2005–2010 data in three ways to obtain
a training dataset and a validation dataset: 1) 2005–2007
data were separated as training data and 2008–2010 as
validation data, 2) 2005–2008 data were separated as
training data and 2009–2010 as validation data, and 3)
2005–2009 data were separated as training data and
2010 as validation data. We fit models 1–3 using each of
the three training datasets and applied the estimated
model coefficients to obtain expected outcome counts in
the corresponding validation datasets. This allowed us to
compare the observed injury/death counts from the
validation dataset to the expected injury/death counts
from the same validation dataset using predictions based
on models 1–3 from the training datasets. Table 2 presents regression coefficients from three
fitted models: model 1 included non-modifiable factors,
model 2 added the semi-modifiable factors, and model 3
included two additional modifiable factors. Comparing
the QICs and c-statistics (estimated AUCs) from the
three nested models (models 1, 2 and 3), we see that
model 3 was the best fitting and was hence selected as
the final model. The comparisons of state-by-state expected injuries/
deaths to the observed injury/ deaths from also implies
that model 3 best characterized the observed data as
compared to models 1 and 2 (Table 3), thereby suggesting
that model 3 best estimated the overall number of injuries
and deaths. Example data The crash characteristic variables were year,
month, and date of the crash, day of the week, time of
the day, the state in which crash occurred, season, num-
ber of fatalities, number of injured occupants, number of
vehicles involved in the crash, manner of collision, ambient
lighting at the time of crash, and weather conditions. The
vehicle characteristics included vehicle type, vehicle action
at the time of the crash, number of occupants, and driver
contributing circumstances. The person-level characteris-
tics included age, sex, injury status (no injury or fatal and
non-fatal injury), occupant protection information, occu-
pant seating, and driver or passenger status. We did not include variables that were highly collinear
with other variables that better explained the outcomes. Seating was highly correlated with the driver status
(driver/ passenger), and month was highly correlated
with season; consequently, these two variables were
removed from the predictive model. Day of the week
was removed because it did not predict injury. Occupant
age in 10-year age categories provided better penalized
model fit than continuous or log-transformed age. Multiple imputation (five imputed datasets), using the
fully conditional specification methods for logistic re-
gression, was performed to impute missing values for
vehicular action (5.9%), driver circumstances (14.8%), and
occupant protection (21.4%) with no interactions. All Injury severity, coded by the reporting law enforcement
officer, was available at the individual-level, defined as no
injury, possible injury, non-incapacitating injury, incapaci-
tating injury, or fatality. We developed a new binary injury
variable for each individual, such that ‘any injury’ cor-
responds to non-incapacitating, incapacitating, or fatal
injury; and ‘no injury’ corresponds to no or possible injury. Discrete covariate categories were collapsed into broader
but meaningful categories based on a-priori knowledge Ranapurwala et al. Injury Epidemiology (2019) 6:31 Ranapurwala et al. Injury Epidemiology Page 4 of 11 Page 4 of 11 models were fit to each of the five imputed datasets. The
resulting regression coefficients from the imputed datasets
were then pooled to obtain the final coefficients that are
reported here. some crash scenarios as examples to demonstrate the
calculation and interpretation of predicted probability of
injury. In our application, the predicted probability (or
risk) of injury or death in a farm crash scenario was calcu-
lated for each occupant involved in the crash as: Internal validation P Injury or Death
ð
Þ ¼
Odds
1 þ Odds ¼
1
1 þ
exp −α þ β1C1 þ β2C2 þ ⋯þ βiCi
Model fit was assessed in three ways. First, by using the
quasi-likelihood information criterion (QIC), a variant of
AIC designed for models fit using GEEs (Pan 2001). Smaller values of QIC (or AIC or BIC) correspond to
models that provide better penalized fit (Akaike 1973;
Schwarz 1978). Second, by calculating the concordance
statistic (or c-statistic), which estimates the area under
the receiver operating characteristic curve (AUC). The
c-statistic ranges from 0.50 to 1, with higher values cor-
responding to models that provide better discrimination
between outcome occurrence and non-occurrence (Gail
et al. 2007; Quante et al. 2012). Lastly, model fit was
assessed by estimating an expected outcome count using
the model coefficients in the same dataset from which
the coefficients were obtained (Keys et al. 1972). A
chi-squared statistic was calculated to compare the
expected and observed outcome count to assess statis-
tical difference between the two. Note that all three of
these methods are of limited utility because they only
evaluate the fitted model based on the same dataset used
in the model construction (i.e., the “training data”), and
do not examine if the predictive models could accurately
predict outcomes in new data (i.e., in “validation data”). Hence validation of the predictive model in external data
is also needed. Here, P is the predicted probability or risk of injury or
death, C1, C2, … , Ci are the covariates in the logistic
regression model, and β1, β2, … , βi are the regression
coefficients for those covariates (Keys et al. 1972;
Kleinbaum et al. 1971). All analyses were conducted using SAS 9.4 (SAS
Institute, Cary, NC). Using the model coefficients, we
built two interactive risk prediction tools, one using
Microsoft Excel (Microsoft, Inc), and one using R-Shiny
(RStudio). The study was considered non-human subjects
by the Institutional Review Board at the University of Iowa
due to the de-identified nature of the secondary data. Predicted probabilities The observed and expected counts for the validation
data were most similar for models 2 and 3 (Table 4). This comparison revealed that our predictive models
could accurately predict the number injuries or deaths
in future farm crash data from the same nine US
states. Predicted probabilities for different regression models
can be readily obtained from standard statistical analyses
programs such as SAS, STATA, R, etc. For this example,
based on the estimated model coefficients from the final
fitted logistic regression model (model 3), we constructed Ranapurwala et al. Injury Epidemiology (2019) 6:31 Page 5 of 11 Table 1 Distribution of farm vehicle crashes and resulting
injuries and deaths by calendar year and state: 2005–2010
Farm vehicle
crashes
N (%)
Injuries and deaths
N (rate per 100
crashes)
Deaths
N (rate per 100
crashes)
Total
7084
2087 (29.5)
163 (2.3)
Calendar Year
2005
1166 (16.5)
338 (29.0)
21 (1.8)
2006
1114 (15.7)
318 (28.5)
29 (2.6)
2007
1198 (16.9)
336 (28.0)
26 (2.2)
2008
1160 (16.4)
316 (27.2)
22 (1.9)
2009
1196 (16.9)
407 (34.0)
37 (3.1)
2010
1250 (17.6)
372 (29.8)
28 (2.2)
State
Iowa
1178 (16.6)
421 (35.7)
35 (3.0)
Illinois
1214 (17.1)
421 (34.7)
27 (2.2)
Kansas
700 (9.9)
186 (26.6)
19 (2.7)
Minnesota
850 (12.0)
199 (23.4)
22 (2.6)
Missouri
1084 (15.3)
207 (19.1)
12 (1.1)
North Dakota
253 (3.6)
51 (20.2)
12 (4.7)
Nebraska
536 (7.6)
189 (35.3)
10 (1.9)
South Dakota
232 (3.3)
74 (31.9)
8 (3.4)
Wisconsin
1037 (14.6)
339 (32.7)
18 (1.7) Using predicted probabilities based on model 3, we con-
structed a hypothetical crash scenario to estimate the
probability of injury or death for the involved individuals
with specific demographic and driving characteristic
profiles. We consider the following non-modifiable
characteristics for the scenario: male driver of a farm
vehicle in Iowa, aged 25–34 years, was involved in a single
vehicle farm crash on a clear morning (6:00–11:59 am) dur-
ing the growing season (June–August). Semi-modifiable
characteristics: the manner of collision set to non-collision,
the vehicle action set to heading straight, and the presence
of a male passenger. Modifiable characteristics: Driver
contributing circumstances set to none and occupant
protection
set
to
all
occupants
wearing
seatbelts
(Table 5). In this scenario, the risk of injury or death
for the farm vehicle driver would be 16.7% and that for
a male passenger would be 17.8%. Predicted probabilities If, however, the
driver and passenger were not wearing seatbelts, and
the driver disregarded the traffic regulations, their
respective risks of injury or death would be 48.5% and
50.4% (Table 5). Suppose this scenario was altered to be
a multivehicle crash in which the farm vehicle (with the
same male driver and male passenger) was rear-ended
by a non-farm vehicle (with a male driver and a male
passenger). If all the individuals were wearing seat belts,
and both the drivers were following traffic regulations,
then the risk of injury or death in the crash would be
7.3% for the farm vehicle driver, 7.8% for the farm
vehicle passenger, 28.7% for the non-farm vehicle driver,
and 30.3% for the non-farm vehicle passenger. If these
individuals were not wearing seatbelts and the drivers did
not follow traffic regulations, then their respective risks
would be 26.9, 28.4, 65.3, and 67% (Table 5). Examining model fit as shown in Table 3, and validation
in an external dataset (external to the training dataset) as
shown in Table 4, are important pieces in addressing
potential underfitting or overfitting of the predictive
models. With an underfitted predictive model, within the
training data, the expected model outcome counts will
not accurately represent the observed counts due to bias. This may happen due to consideration of a limited num-
ber of predictors, or due to collapsing variables with mul-
tiple discrete categories into a few large categories that
may not represent the finer categories. An adequate model
fit will exhibit a close correspondence between the
expected and observed counts for the training data
(Table 3). On the other hand, one can include too
many predictors (and interactions of those predictors) to
achieve a nearly exact model fit, so that the expected
model outcome counts from the training data nearly
replicate the observed outcomes from the training data. When applied to external validation datasets, such overly
complex models may not be able to accurately estimate
the new observed outcome counts. These types of pre-
dictive models are referred to as overfitted or non-
generalizable. In this example, the models accurately
approximate the observed counts for both the training
dataset (Table 3) and the validation dataset (Table 4). The
propriety of the model fits is also represented by the AUCs. These risks can be easily estimated by deploying inter-
active tools that utilize model coefficients from the final
fitted model. Predicted probabilities We developed a Microsoft Excel-based tool
using the model 3 coefficients from 2005 to 2010 data that
can be downloaded at https://drive.google.com/drive/
folders/0B0B0TgPTSgJ8bjVZYjdGaF96UzQ. We also de-
veloped a similar internet-based tool using R-Shiny (Fig. 1);
the online tool can be found at https://gpcah-farmcrash-
predictive.shinyapps.io/predictiveapp/. These two tools are
freely available and can be used by anyone to estimate an
individual’s risk of injury in a farm crash. The risk
estimates in Table 5 were produced using these tools. Discussion In this manuscript, we demonstrate how the regression
coefficients obtained from predictive analyses can be
used to forecast the risk of a health outcome for indivi-
duals in specific farm crash scenarios. The predictive
models allow us to compare the risks in different sce-
narios, helping us to appreciate the change in risk with
varying circumstances as shown in Table 5. Specifically, Page 6 of 11 Ranapurwala et al. Injury Epidemiology (2019) 6:31 Ranapurwala et al. Injury Epidemiology Table 2 Injury / death status, and estimated regression coefficients for non-modifiable, non + semi-modifiable, and non + semi
+modifiable risk factors to predict the risk of injury or death in a farm crash: 2005–2010
Variables
Categories
Injured
or died
Model coefficients (std. Discussion Injury Epidemiology (2019) 6:31 Ranapurwala et al. Injury Epidemiology Table 2 Injury / death status, and estimated regression coefficients for non-modifiable, non + semi-modifiable, and non + semi
+modifiable risk factors to predict the risk of injury or death in a farm crash: 2005–2010 (Continued)
Variables
Categories
Injured
or died
Model coefficients (std. error)
Yes
No
Model 1a
Model 2b
Model 3c
Manner of collision
Non collision (referent)
294
1301
0
0
Head-on
127
415
0.40 (0.17)
0.40 (0.18)
Rear-end
716
2747
0.28 (0.13)
0.28 (0.13)
Angle, oncoming left turn
361
2343
0.04 (0.14)
0.10 (0.14)
Sideswipe, same direction
192
2698
−0.85 (0.15)
−0.73 (0.16)
Sideswipe, opposite direction
152
1462
−0.65 (0.16)
−0.55 (0.16)
Other
245
1781
−0.21 (0.14)
−0.21 (0.14)
Vehicle action
Heading straight (referent)
1510 6934
0
0
Turning
133
2489
−0.55 (0.11)
−0.62 (0.11)
Overtaking/ passing/ changing lanes
280
1750
−0.25 (0.09)
−0.34 (0.10)
Slowing/stopping
43
608
−0.89 (0.17)
−0.84 (0.18)
Other
121
966
−0.30 (0.11)
−0.35 (0.11)
Multiple passengers
No (referent)
1392 9908
0
0
Yes
695
2839
0.28 (0.08)
0.34 (0.08)
Driver
No (referent)
433
1710
0
0
Yes
1654 11,037
−0.16 (0.09)
−0.08 (0.09)
Modifiable factors
Driver contributing circumstance No contributing action (referent)
871
6508
0
Disregarded traffic regulation
302
1487
0.35 (0.09)
Reckless, careless, negligent, aggressive driving 407
2510
0.16 (0.08)
Inattentive/distracted driver
256
1093
0.25 (0.09)
Other contributing action
251
1149
0.25 (0.10)
Occupant Protection
None (referent)
870
4534
0
Seat belt
1052 7439
−1.19 (0.09)
Child safety restraint
59
384
−1.25 (0.20)
Other restraint/ protection
106
390
0.18 (0.16)
Quasi-likelihood Information Criterion (QIC)
10,844.4
10,077.4
9676.8
AUC (95% CI)
0.69 (0.68, 0.71) 0.75 (0.74, 0.76) 0.78 (0.76, 0.79)
Abbreviations: AUC Area under the receiver operating characteristic (ROC) curve
amodel 1 includes non modifiable factors Table 2 Injury / death status, and estimated regression coefficients for non-modifiable, non + semi-modifiable, and non + semi
+modifiable risk factors to predict the risk of injury or death in a farm crash: 2005–2010 (Continued) the change in modifiable and semi-modifiable factors may
allow us to understand the impact of intervening on these
factors on the individual’s risk of injury or death. outcome for every individual differently based on all
other individual or scenario-specific coefficients (Table 5). Discussion error)
Yes
No
Model 1a
Model 2b
Model 3c
Intercept
Intercept (constant)
2087 12,747 −1.75 (0.18)
−1.73 (0.24)
−0.97 (0.25)
Non-modifiable factors
State
Iowa
421
1858
0.39 (0.10)
0.46 (0.10)
0.42 (0.10)
Illinois
421
2230
0.19 (0.10)
0.20 (0.11)
0.33 (0.11)
Kansas
186
1291
−0.19 (0.12)
−0.21 (0.12)
−0.26 (0.13)
Minnesota
199
1688
−0.30 (0.11)
−0.24 (0.12)
−0.08 (0.12)
Missouri
207
1686
−0.09 (0.11)
−0.21 (0.12)
−0.34 (0.12)
North Dakota
51
431
−0.43 (0.21)
−0.50 (0.21)
−0.56 (0.21)
Nebraska
189
970
0.16 (0.12)
0.11 (0.12)
0.08 (0.12)
South Dakota
74
379
0.16 (0.17)
0.14 (0.18)
−0.06 (0.19)
Wisconsin (referent)
339
2214
0
0
0
Season
Winter (Jan-Mar) (referent)
192
1510
0
0
0
Planting (Apr-May)
341
2307
0.06 (0.12)
0.20 (0.12)
0.17 (0.12)
Growing (Jun-Aug)
635
3492
0.30 (0.11)
0.42 (0.11)
0.38 (0.11)
Harvesting (Sep-Dec)
919
5438
0.19 (0.10)
0.16 (0.10)
0.15 (0.10)
Weather at the time of crash
Clear (referent)
1618 9802
0
0
0
Cloudy
329
2081
−0.01 (0.08)
−0.09 (0.08)
−0.11 (0.08)
Rain
78
376
0.17 (0.15)
−0.03 (0.16)
0.03 (0.16)
Snow/sleet/hail/freezing rain/drizzle
22
289
−0.74 (0.28)
−0.89 (0.28)
−0.87 (0.28)
Fog/smog/smoke/other
40
199
0.15 (0.22)
0.01 (0.22)
−0.08 (0.23)
Time of crash
12:00–5:59 am (referent)
131
739
0
0
0
6:00–11:59 am
501
3478
−0.11 (0.13)
0.13 (0.14)
0.17 (0.14)
12:00–5:59 pm
885
6218
−0.14 (0.13)
0.08 (0.13)
0.08 (0.14)
6:00–11:59 pm
570
2312
0.41 (0.14)
0.12 (0.14)
0.11 (0.14)
Number of vehicles
Single vehicle
238
827
1.37 (0.10)
1.22 (0.13)
1.22 (0.14)
Two or more vehicles (referent)
1849 11,920 0
0
0
Equipment type
Farm vehicle/equipment
672
6875
−1.14 (0.06)
−1.02 (0.06)
−1.63 (0.08)
Non-farm vehicle (referent)
1415 5872
0
0
0
Age
< 16 years age
159
799
0.24 (0.11)
0.04 (0.13)
0.13 (0.14)
16–24 years age
388
2120
0.24 (0.09)
0.21 (0.09)
0.22 (0.10)
25–34 years age (referent)
234
1839
0
0
0
35–44 years age
288
1851
0.15 (0.10)
0.15 (0.10)
0.20 (0.10)
45–54 years age
325
2326
0.11 (0.09)
0.10 (0.10)
0.13 (0.10)
55–64 years age
278
1794
0.22 (0.10)
0.24 (0.10)
0.30 (0.10)
65+ years age
415
2018
0.49 (0.09)
0.51 (0.09)
0.59 (0.10)
Sex
Female (referent)
626
2663
0
0
0
Male
1461 10,084 −0.19 (0.06)
−0.14 (0.06)
−0.26 (0.06)
Semi-modifiable factors
h
l h ( f
) Table 2 Injury / death status, and estimated regression coefficients for non-modifiable, non + semi-modifiable, and non + semi
+modifiable risk factors to predict the risk of injury or death in a farm crash: 2005–2010 Table 2 Injury / death status, and estimated regression coefficients for non-modifiable, non + semi-modifiable, and non + semi
+modifiable risk factors to predict the risk of injury or death in a farm crash: 2005–2010 Page 7 of 11 Page 7 of 11 Ranapurwala et al. Discussion Average predicted probability, AUC Area under the receiver operating curve, QIC Quasi-likelihood information criteria this example, other characteristics were considered con-
stant for all the vehicle occupants, but they may be
changed easily (and will result in different risk esti-
mates) to determine risks for other individuals in
different scenarios. 2010; Maio et al. 1992; Stephan and Newstead 2014;
Russo et al. 2014). However, these studies focused on
reporting effect estimates (odds ratios or rate ratios) of
association between individual risk factors and injury or
death in a farm crash. Such models also assume that an
individual’s outcomes do not depend on another individ-
ual’s outcomes (no interference assumption, also known as
the stable unit treatment value assumption) (Schwartz et al. 2012). This is another differentiating feature, that while
causal (or associational) modeling assumes (rather requires)
no interference, predictive modeling embraces interference. The non-modifiable, semi-modifiable, and modifiable
factors used to predict the injury or death in our
example dataset have been previously identified as risk
factors for a farm crash (Ranapurwala et al. 2016;
Hughes and Rodgman 2000; Peek-Asa et al. 2007; Pinzke
and Lundqvist 2004; Costello et al. 2003; Gerberich et al. 1996; Jaarsma and De Vries 2014), and injury (Hughes
and Rodgman 2000; Marlenga et al. 2006; Gkritza et al. p
g
Using different combinations of predictors could allow
one to estimate the predictive capability (or sensitivity) Table 4 Validation of the predictive models
Validation
data years
(Training
data years)
Total
validation
data
occupants
(training
data
occupant)
Observed
injuries or
deaths in
validation
data (N)
Non-modifiable (Model 1)
Non + semi-modifiable
(Model 2)
Non + semi + modifiable
(Model 3)
Avg. pred. Prob. Expected injuries/
deaths (N)
Avg. pred. Prob. Expected injuries/
deaths (N)
Avg. pred. Prob. Expected injuries/
deaths (N)
2008-‘10
(‘05-‘07)
7624
(7210)
1095
0.1407b
1073
0.1445e
1102
0.1437h
1095
2009-‘10
(‘05-‘08)
5216
(9618)
779
0.1383c
721a
0.1405f
733
0.1397i
729
2010 (‘05-‘09)
2615
(12,219)
372
0.1424d
372
0.1410g
369
0.1401j
366
Abbreviations: Avg. pred. Prob. Average predicted probability
ap-value = 0.0327 (Chi Sq = 4.56, df = 1), suggesting that expected injuries and deaths (n = 721) were significantly different than the observed (n = 779). Discussion Predictive modeling is used to ultimately answer the
questions like “what is the probability (or risk) of an
injury for an individual given a crash and the combi-
nation of all circumstantial factors?” It is noteworthy
that the risks estimates presented in Table 5 do not
entirely depend on driver behavior. They also depend on
the total contribution of risk factors such as driver or
passenger (semi-modifiable factor), farm or non-farm
vehicle (non-modifiable factor), and single vehicle crash or
multivehicle crash (non-modifiable factor). In addition, in In predictive analyses, the estimated model coefficients
may be used to suggest that a certain variable is a strong
or weak predictor of the outcome; however, the coeffi-
cient estimate is not used to characterize the effect of a
variable on the outcome (which is the aim of causal
modeling). This fundamentally differentiates predictive
modeling from causal modeling, with the latter being
ubiquitous in the public health literature. In predictive
modeling, a coefficient affects the probability of the Ranapurwala et al. Injury Epidemiology (2019) 6:31 Page 8 of 11 Ranapurwala et al. Injury Epidemiology Table 3 Comparison of the expected (model-based) to observed number of injuries in the nine states from 2005 to 2010
State Total
occupants
(N)
Observed
injuries or
deaths (N)
Non-modifiable (Model 1)
QIC = 10,844.4
AUC = 0.69 (0.68, 0.71)
Non + semi-modifiable (Model 2)
QIC = 10,077.4
AUC = 0.75 (0.74, 0.76)
Non + semi + modifiable (Model 3)
QIC = 9676.8
AUC = 0.78 (0.76, 0.79)
Avg. pred. Prob. Expected injuries/
deaths (N)
Avg. pred. Prob. Expected injuries/
deaths (N)
Avg. pred. Prob. Expected injuries/
deaths (N)
IA
2279
421
0.1804
411
0.1820
415
0.1818
414
IL
2651
421
0.1600
424
0.1602
425
0.1594
422
KS
1477
186
0.1251
185
0.1246
184
0.1242
183
MN
1887
199
0.1008
190
0.1044
197
0.1017
192
MO
1893
207
0.1135
215
0.1169
221
0.1197
227
ND
482
51
0.1052
51
0.1066
51
0.1064
51
NE
1159
189
0.1634
189
0.1641
190
0.1619
188
SD
453
74
0.1549
70
0.1586
72
0.1592
72
WI
2553
339
0.1383
353
0.1367
349
0.1365
348
Total 14,834
2087
0.1408
2089
0.1419
2104
0.1415
2099
Abbreviations: Avg. pred. Prob. Discussion All other
expected to observed differences were non-significant
bAUC = 0.69 based on training data 2005–2007
cAUC = 0.70 based on training data 2005–2008
dAUC = 0.69 based on training data 2005–2009
eAUC = 0.75 based on training data 2005–2007
fAUC = 0.75 based on training data 2005–2008
gAUC = 0.74 based on training data 2005–2009
hAUC = 0.77 based on training data 2005–2007
iAUC = 0.77 based on training data 2005–2008
jAUC = 0.77 based on training data 2005–2009 Table 4 Validation of the predictive models Table 4 Validation of the predictive models Page 9 of 11 Page 9 of 11 Ranapurwala et al. Injury Epidemiology (2019) 6:31 Ranapurwala et al. Injury Epidemiology Table 5 Predicted probabilities of injury or death for drivers and
passengers in varying farm crash scenarios using the model 3
estimated coefficients from Table 1
The risk or injury or death in a farm crash in Iowa for a 25–34 year old,
male, in growing season, clear weather, between 6:00–11:59 am,
daylight, heading straight, passengers on board (for single vehicle crash,
manner of collision = non collision; for multiple vehicle crash, manner of
collision = rear end)
Vehicle/ occupant
type
Seat
belt
Driver
contributing
circumstances
Risk of injury or
death (%)
Single
vehicle
crash
Multiple
vehicle
crash
Farm vehicle
driver
Yes
None
16.7%
7.3%
Farm vehicle
passenger
17.8%
7.8%
Non-farm
vehicle driver
28.7%
Non-farm
vehicle
passenger
30.3%
Farm vehicle
driver
Yes
Disregarded traffic
regulations
22.2%
10.1%
Farm vehicle
passenger
23.6%
10.8%
Non-farm
vehicle driver
36.4%
Non-farm
vehicle passenger
38.2%
Farm vehicle
driver
No
None
39.8%
20.6%
Farm vehicle
passenger
41.6%
21.8%
Non-farm
vehicle driver
57.0%
Non-farm
vehicle
passenger
58.8%
Farm vehicle
driver
No
Disregarded traffic
regulations
48.5%
26.9%
Farm vehicle
passenger
50.4%
28.4%
Non-farm
vehicle driver
65.3%
Non-farm
vehicle
passenger
67.0% Table 5 Predicted probabilities of injury or death for drivers and
passengers in varying farm crash scenarios using the model 3
estimated coefficients from Table 1 simulate
potential
effects
of
evidence-based
inter-
ventions in well-defined populations and conduct cost-
benefit analysis for widespread policy initiatives. For
example,
if
an
intervention
is
known
to
improve
seat-belt compliance by 60% among a specific group of
low compliant drivers, it could be simulated using our
predictive model, which will allow us to estimate the
number of overall injuries and deaths prevented. Discussion We can
then estimate the cost of the prevented injuries and
deaths and compare that against the cost of implemen-
ting the intervention. Similar predictive models could be developed in other
research areas to help physicians make more informed
decisions about prescribing medications to their patients. For example, a risk prediction tool, like the one we devel-
oped, could inform physicians of their patient’s risk of
suffering from an opioid use disorder in the future due to
exposure to prescription opioid medication. Such in-
formation will help the physician to better titrate the
patient’s pain medications. Eventually, such tools may
not only aid physicians but also improve health out-
comes for patients. Now, with the advancement of
analytical approaches and superior computing power,
and their integration in medicine, such approaches
can be feasibly deployed and used. y
p
y
Some best practices to develop and improve predictive
models may involve the following, although this is not
an exhaustive list. First, the overall data sample should
be carefully selected so that it represents the relevant
source population. Second, the training dataset should
be large enough so as to observe all possible combi-
nations of predictors. Third, the validation dataset
should represent the same data generating mechanism
as the training dataset, which can be accomplished
through random selection of the observations for the
validation dataset from the overall sample. Fourth, for
quantitative variables, the most appropriate functional or
categorical representation must be utilized. For example,
age broken into 5-year categories may sometimes provide
better prediction than 10-year age categories or age
treated as a continuous variable. Fifth, when predictive
outcomes
exhibit
secular
time
trends,
appropriately
accounting for such trends in the modeling structure may
help improve the prediction. Sixth, when combining finer
categorical variables into larger categories, it is prudent to
combine finer categories that produce similar model
coefficients and avoid combining categories that vary
considerably in their model coefficients. The use of a
model selection criterion, such as QIC, AIC, or BIC, can
often facilitate the fourth, fifth and sixth objectives. Lastly,
for a predictive model to be utilized in practice, there
must be a temporal ordering between the predictors
and the outcome; specifically, all predictors must be
observed before the occurrence of the outcome. of the predictive models. Discussion For example, we identified real-
istic scenarios where the risk of injury given a farm crash
was as low as 3% for some occupants, and as high as
93% for others, a difference of up to 90% of injury and
death risk. This suggests good predictive capability of
the model, since the predictive model covers most of the
probability range between 0 and 1 (or 0 and 100%). Such
a sensitive tool may be useful for policy makers to Ranapurwala et al. Injury Epidemiology (2019) 6:31 Ranapurwala et al. Injury Epidemiology Page 10 of 11 (2019) 6:31 Fig. 1 Screenshot of the online tool to calculate risk of injury or death in a farm crash Fig. 1 Screenshot of the online tool to calculate risk of injury or death in a farm crash These individual-level predictive models are extended
further into more complex modeling frameworks like
agent-based models that predict systems level changes. Historical and existing data sources can be used to
develop predictive models, which can incorporate more
data over time to improve the model’s predictive capabi-
lities. Such iterative updating can be viewed as a type of
“machine learning” (Wicks et al. 2016). Advances in
computing have already taken predictive modeling to
the next stage, via machine learning and precision
medicine. Methods such as random forests, neural net-
works, and Q-learning utilize observational or experi-
mental data, superimpose predictive modeling theory,
and not only predict potential future outcomes, but also
allow us to make evidence-based decisions to optimize
the best possible outcomes in the future. However, some
machine learning methods have been characterized as
“black box” techniques that obfuscate rather than
illuminate the dynamics of the underlying phenomenon. A clear advantage of the predictive modeling approach
presented here is that it provides an interpretable and
transparent characterization of these dynamics (Keil and
Edwards 2018). US states. Second, the crashes included in these data are
those that were reported to the police. Some crashes may
never be reported, misreported as non-farm crashes, or be
excluded due to missing information. Similarly, mistakes
may materialize in the reporting of injury severity by the
responding police officers. Such exclusions and measure-
ment errors may lead to misclassification of the predicted
probabilities. Conclusion Predictive analysis offers elegant tools that may help us
understand the multidimensionality of the occurrence of
different health outcomes and allow individual-level risk
assessment. Algorithms and tools derived from such
analyses could support policymakers, physicians, and
environmental health practitioners in developing and
implementing tailored prevention strategies to improve
health outcomes for their patients or clients. Discussion Third, we developed the predictive models
using commonly available variables form the nine states;
however, there may be other unmeasured variables that
may predict the outcome and hence may have an impor-
tant role in determining the risk of injury for an indivi-
dual. Generalizability, misclassification, and unmeasured
predictors are three main limitations that may exist with
most predictive models. Limitations We encountered a number of limitations in our example
dataset that commonly arise in predictive modeling
regardless of the data source or substantive area of
research. First, these data were collected from nine US
states; hence, the results are not generalizable to other g
The authors thank the Great Plains Center for Agricultural Health and Dr.
Karisa Harland for their continued support and guidance in continuing this
work. We also thank Dr. Elizabeth T. Rogawski and Dr. Stephen W. Marshall
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North Carolina at Chapel Hill, 137 E Franklin St, Suite 500, CB# 7505, Chapel
Hill, NC 27599, USA. 2Department of Epidemiology, College of Public Health,
University of Iowa, Iowa City, IA, USA. 3Injury Prevention Research Center,
Department of Occupational and Environmental Health, College of Public
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published article in aggregate form (Tables 1 and 2) which can be easily
used to reproduce the study results. Line level data are available from
the Departments of Transportation of the nine states listed in this study,
but restrictions apply to the availability of these data, which were used
under license for the current study, and so are not publicly available. Data are however available from the authors upon reasonable request
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Intern Med. 2018; [Epub ahead of print]. Acknowledgements The authors thank the Great Plains Center for Agricultural Health and Dr. Karisa Harland for their continued support and guidance in continuing this
work. We also thank Dr. Elizabeth T. Rogawski and Dr. Stephen W. Marshall
for their review of earlier versions of this manuscript. Page 11 of 11 Page 11 of 11 Ranapurwala et al. Injury Epidemiology (2019) 6:31 Ranapurwala et al. Injury Epidemiology Ranapurwala et al. Injury Epidemiology (2019) 6:31 (2019) 6:31 Consent for publication The data was collected on human beings, however, it was collected as an
administrative data by law enforcement at the scene of a crash. The data
was pre-existing at the time of this research and the study team did not
receive and identifiable information on any individuals and there was no
direct contact with people whose data are included in the study. Hence, we
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youth tractor crashes on public roads. Inj Prev. 2006;12:46–51. Marlenga B, Doty BC, Berg RL, Linneman JG. Evaluation of a policy to reduce
youth tractor crashes on public roads. Inj Prev. 2006;12:46–51. Pan W. Akaike’s information criterion in generalized estimating equations. Biometrics. 2001;57(1):120–5. Peek-Asa C, Sprince NL, Whitten PS, et al. Characteristics of crashes with farm
equipment that increase the potential for injury. J Rural Health. 2007;23(4):339–47
Pinzke S, Lundqvist P. Slow-moving vehicles in Swedish traffic. J Agric Saf Health. 2004;10(2):121–6. Pan W. Akaike’s information criterion in generalized estimating equations. Biometrics. 2001;57(1):120–5. Peek-Asa C, Sprince NL, Whitten PS, et al. Characteristics of crashes with farm
equipment that increase the potential for injury. J Rural Health. 2007;23(4):339–47. References Akaike H. Information theory and an extension of the maximum likelihood principle. In: Proc. 2nd inter. symposium on information theory, 267–281, Budapest; 1973. American Heart Association (AHA). Atherosclerotic cardiovascular disease (ASCVD)
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Middle Jurassic black shales (Skrzypny Shale Formation) – palaeoenvironmental significance of one of the oldest deposits of the Pieniny Klippen Belt.
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Środkowojurajskie czarne łupki (formacja łupków ze Skrzypnego) – znaczenie
paleośrodowiskowe jednych z najstarszych osadów pienińskiego pasa skałkowego Katarzyna Górniak1, Krzysztof Bahranowski1, Adam Gaweł1,
Leszek Marynowski2 & Tadeusz Szydłak1
1 AGH University of Science and Technology, Department of Mineralogy, Petrography and Geochemistry,
30-059 Kraków, al. Mickiewicza 30, Poland;
e-mail: gorniak@agh.edu.pl
2 University of Silesia, Faculty of Earth Sciences, 41-200 Sosnowiec, ul. Będzińska 60, Poland
e-mail: marynows@wnoz.us.edu.pl Abstract: This paper deals with a fragment of geological history
of Middle Jurassic black spherosideritic shales outcropping in
upper part of the Homole Gorge (Skrzypny Shale Formation,
Niedzica succession). These important deposits belong to the
Flackenmergel facies, widespread in oceanic Tethyan deposits. This formation represents one of the oldest rocks of the Pieniny
Klippen Belt. Dark colour of these rocks (total organic carbon
content in them amounts to about 1 wt.%) and the occurrence of
siderite concretions indicate reducing conditions of their origin
but oxidizing environment of waters above them in sedimentary
basin. Oxic conditions of organic matter sedimentation are
confirmed by the elevated values of pristine to phytane ratio. Moreover the results based on n-alkanes indicate mixed
terrestrial/marine origin of organic matter with prevalence of
the last one These information are slightly obliterated because
of advanced thermal maturity of organic matter caused by post-
sedimentation processes. Microstructural features of deposits
of the Skrzypny Shale Formation and the composition of clay
minerals occurring in them indicate late diagenetic conditions. In SEM images we observe one-directional orientation of clay
minerals and rotation of mica flakes, often chloritized. Clay
minerals are represented by high-illitic illite/smectite, micas,
post-biotitic chlorite and subordinately kaolinite. As follows
from authors studies, these macroscopically nearly “mute” clay
rocks, little interesting in outcrops, particularly when they occur
in such attractive geological objects as the Homole Gorge, can
be the source of important information concerning the history of
sedimentation basins. Key words: Pieniny Klippen Belt, middle Jurassic, black shales,
Warszawa
Kraków
Warszawa
Kraków
Warszawa
Kraków
Warszawa
Kraków
Zakopane
Chabówka
Czarny Dunajec
Niedzica
Szczawnica
Nowy Targ Warszawa
Kraków
Warszawa
Kraków
Warszawa
Kraków
Warszawa
Kraków Zakopane
Chabówka
Czarny Dunajec
Niedzica
Szczawnica
Nowy Targ ukazujących się w niewielkim odsłonięciu w górnej części
wąwozu Homole (formacja łupków ze Skrzypnego, sukcesja
niedzicka). Te ważne utwory należą do facji Fleckenmergiel
szeroko rozpowszechnionej w oceanie Tetydy. Formacja
łupków ze Skrzypnego reprezentuje jedne z najstarszych skał
pienińskiego pasa skałkowego. Środkowojurajskie czarne łupki (formacja łupków ze Skrzypnego) – znaczenie
paleośrodowiskowe jednych z najstarszych osadów pienińskiego pasa skałkowego Ciemna barwa badanych skał
(zawartość całkowitego węgla organicznego około 1% wag.)
oraz występowanie w nich konkrecji syderytowych, wskazują na
warunki redukcyjne panujące w osadzie, natomiast na obecność
natlenionych wód nad osadem w środowisku ich sedymentacji. Sedymentację substancji organicznej w warunkach raczej
utleniających potwierdzają badania związków organicznych,
tj. wysokie wartości stosunku pristan/fitan. Wyniki tych badań
wskazują ponadto na mieszane, lądowo/morskie pochodzenie
substancji organicznej z przewagą ostatniej z wymienionych. Informacje te są nieco zatarte z powodu dużej dojrzałości
termicznej substancji organicznej spowodowanej procesami
postsedymentacyjnymi. Cechy mikrostrukturalne utworów
formacji łupków ze Skrzyponego oraz skład występujących w
nich minerałów ilastych wskazują na warunki odpowiadające
późnej diagenezie. W obrazach elektronomikroskopowych (SEM)
widoczna jest bowiem jednokierunkowa orientacja minerałów
ilastych i rotacja blaszek mik, często schlorytyzowanych. W składzie minerałów ilastych występuje zaś wysokoillitowy illit/
smektyt, miki, pobiotytowy chloryt i podrzędnie kaolinit. Z przeprowadzonych badań wynika, że makroskopowo niemal
„nieme” skały ilaste, niepozorne wyglądające w odsłonięciu,
szczególnie jeśli występują obok atrakcyjnych obiektów
geologicznych jak to ma miejsce w wąwozie Homole, są
źródłem ważnych informacji dotyczących historii basenów
sedymentacyjnych. Warto więc zwrócić na nie uwagę. Słowa kluczowe: Pieniński Pas Skałkowy jura środkowa czarne Warszawa
Warszawa
Warszawa
Warszawa Abstract: This paper deals with a fragment of geological history
of Middle Jurassic black spherosideritic shales outcropping in
upper part of the Homole Gorge (Skrzypny Shale Formation,
Niedzica succession). These important deposits belong to the
Flackenmergel facies, widespread in oceanic Tethyan deposits. This formation represents one of the oldest rocks of the Pieniny
Klippen Belt. Dark colour of these rocks (total organic carbon
content in them amounts to about 1 wt.%) and the occurrence of
siderite concretions indicate reducing conditions of their origin
but oxidizing environment of waters above them in sedimentary
basin. Oxic conditions of organic matter sedimentation are
confirmed by the elevated values of pristine to phytane ratio. Moreover the results based on n-alkanes indicate mixed
terrestrial/marine origin of organic matter with prevalence of
the last one These information are slightly obliterated because
of advanced thermal maturity of organic matter caused by post-
sedimentation processes. Microstructural features of deposits
of the Skrzypny Shale Formation and the composition of clay
minerals occurring in them indicate late diagenetic conditions. In SEM images we observe one-directional orientation of clay
minerals and rotation of mica flakes, often chloritized. Clay
minerals are represented by high-illitic illite/smectite, micas,
post-biotitic chlorite and subordinately kaolinite. Środkowojurajskie czarne łupki (formacja łupków ze Skrzypnego) – znaczenie
paleośrodowiskowe jednych z najstarszych osadów pienińskiego pasa skałkowego As follows
from authors studies, these macroscopically nearly “mute” clay
rocks, little interesting in outcrops, particularly when they occur
in such attractive geological objects as the Homole Gorge, can
be the source of important information concerning the history of
sedimentation basins. Abstract: This paper deals with a fragment of geological history
of Middle Jurassic black spherosideritic shales outcropping in
upper part of the Homole Gorge (Skrzypny Shale Formation,
Niedzica succession). These important deposits belong to the
Flackenmergel facies, widespread in oceanic Tethyan deposits. This formation represents one of the oldest rocks of the Pieniny
Klippen Belt. Dark colour of these rocks (total organic carbon
content in them amounts to about 1 wt.%) and the occurrence of
siderite concretions indicate reducing conditions of their origin
but oxidizing environment of waters above them in sedimentary
basin. Oxic conditions of organic matter sedimentation are
confirmed by the elevated values of pristine to phytane ratio. Moreover the results based on n-alkanes indicate mixed
terrestrial/marine origin of organic matter with prevalence of
the last one These information are slightly obliterated because
of advanced thermal maturity of organic matter caused by post-
sedimentation processes. Microstructural features of deposits
of the Skrzypny Shale Formation and the composition of clay
minerals occurring in them indicate late diagenetic conditions. In SEM images we observe one-directional orientation of clay
minerals and rotation of mica flakes, often chloritized. Clay
minerals are represented by high-illitic illite/smectite, micas,
post-biotitic chlorite and subordinately kaolinite. As follows
from authors studies, these macroscopically nearly “mute” clay
rocks, little interesting in outcrops, particularly when they occur
in such attractive geological objects as the Homole Gorge, can
be the source of important information concerning the history of
sedimentation basins. Słowa kluczowe: Pieniński Pas Skałkowy, jura środkowa, czarne
łupki, mikrostruktura, minerały ilaste Geoturystyka 2 (13) 2008: 19-24 Geoturystyka 2 (13) 2008: 19-24 Introduction Early Jurassic dark marly facies (of Fleckenmergel type)
are particularly interesting for researchers since in them there
are recorded the initial stages of evolution of the Pieniny basin
(Birkenmajer, 1986, Krobicki & Golonka 2008a and the refer-
ences in these papers). In Polish part of the Pieniny Klippen
Belt one of the oldest rocks belonging to this facies are dark
sphaerosideritic shale with Bositra bivalve (Skrzypny Shale
Formation) of Late Aalenian-Early Bajocian age (Krobicki Key words: Pieniny Klippen Belt, middle Jurassic, black shales,
mikrostructure, clay minerals Treść: Praca przedstawia fragment historii geologicznej
środkowojurajskich czarnych łupków sferosyderytowych 19 Middle Jurassic black shales (Skrzypny Shale Formation) Middle Jurassic black shales (Skrzypny Shale Formation) transporting oxygen-deficient waters are entering the conti-
nental slope. This model corresponds to the conditions
dominating in Middle Jurassic Pieniny basin. Dark marly
facies sediments of this age appear in rocks of the Niedzica
succession deposited on continental slope and in those of the
Czorsztyn succession accumulated on the slope of Czorsztyn
ridge. As already informed, the former are one of the oldest
deposits forming the fold of the Czajakowa Skała Klippe and
are observed in uppermost part of the Homole Gorge. The
dark shales of the Czorsztyn succession are exposed in the
Krupianka stream (Krobicki & Golonka, 2008a, Fig. 14A,
and references in this paper). Bathymetric conditions of for-
mation of sediments of the Fleckenmergel facies, estimated
by Tyszka (2001) on the ground of microfossil studies, cor-
respond to the slope zone. & Golonka, 2008a, Fig. 7). This formation is described as a
series of clay rocks of black and black-greenish colour, con-
taining sphaerosideritic concretions and sometimes ammo-
nites. The thickness of this formation amounts to approxi-
mately 5 m. These interesting deposits are outcropping in
upper part of the Homole Gorge, at the pass below Czajakowa
Skała Klippe (Krobicki & Golonka, 2008a, Fig. 12D). & Golonka, 2008a, Fig. 7). This formation is described as a
series of clay rocks of black and black-greenish colour, con-
taining sphaerosideritic concretions and sometimes ammo-
nites. The thickness of this formation amounts to approxi-
mately 5 m. These interesting deposits are outcropping in
upper part of the Homole Gorge, at the pass below Czajakowa
Skała Klippe (Krobicki & Golonka, 2008a, Fig. 12D). Outcrop at the pass below
Czajakowa Skała Klippe The Skrzypny Shale Formation outcropping here belongs
to the Niedzica succession. The deposits of this succession
occur in the form of a slice contacting tectonically with un-
derlying rocks. This slice represents an overturned fold,
strongly tectonically folded and forming the Czajakowa Skała
Klippe. The Skrzypny Shale Formation occurs in the bottom
of the Niedzica succession rock complex. A fragment of
Czajakowa Skała fold was displaced on a bed of these shales,
representing sliding plane, and actually they occur in a land-
slide in upper part of the Homole Gorge (Krobicki & Golon-
ka, 2008a, Fig. 12D). In this small outcrop the shales in
question represent fine slaty, dark gray-olive shales contain-
ing mica flakes up to 0.5 mm in size and brown sideritic
concretions. These shales are soft, poorly calcareous, contain-
ing no sand fraction. The size of sphaerosiderites does not
exceed a dozen cm. They are hard, containing fractures filled
with calcite. These carbonate concretions from other occur-
rence of these shales of the Niedzica succession in Kapuśnica
near Niedzica castle were examined in detail by W. Narębski
(Birkenmajer & Narębski, 1957). They were found to contain,
apart from sideroplesitic (Mg-sideritic) component, significant
admixture of calcite, indicating their deposition in rather
deep-water and more offshore marine environment than these
shales of the Czorsztyn and Branisko successions. The geo-
chemical character of concretions and shales containing them
clearly suggests their early diagenetic origin under conditions
of moderately reducing sideritic geochemical facies (Birken-
majer & Narębski, 1957). This conclusion is confirmed by
their loaf-shaped forms (Seilacher, 2001). The series of dark clayey rocks accumulating in oxygen-
deficient environments are usually but apparently monotonous. Complex sedimentologic, palaeontologic and geochemical
studies of the Lower Jurassic sediments in Yorkshire, United
Kingdom, allowed to distinguish three facies occurring in the
basins dominated by generally oxygen-deficient waters. They
have resulted in distinguishing normal, restricted and bitumi-
nous shales (Morris, 1979). This facial variability is caused
by cyclic change of position of the boundary between oxygen-
ated and oxygen-deficient zones in the water column above
the sediment. Lithological features of sediments of the
Skrzypny Shale Formation of the Niedzica succession outcrop-
ping at the pass below Czajakowa Skała Klippe represent the
normal shales facies. This conclusion results from the com-
parison of their properties with those of sediments reported
by Morris (1979) for individual facies distinguished. Outcrop at the pass below
Czajakowa Skała Klippe One-directional orientation of flakes of clay minerals bent on a
quartz grain (Q) due to compaction (SEM/EDX) • Jednokierunkowa
orientacja blaszek minerałów ilastych ugiętych na ziarnie kwarcu
wyniku kompakcji widoczna w badaniach elektronomikroskopo-
wych świeżego przełamu próbki. (SEM/EDX)
Q Fig. 4. One-directional orientation of flakes of clay minerals bent on a
quartz grain (Q) due to compaction (SEM/EDX) • Jednokierunkowa
orientacja blaszek minerałów ilastych ugiętych na ziarnie kwarcu
wyniku kompakcji widoczna w badaniach elektronomikroskopo-
wych świeżego przełamu próbki. (SEM/EDX) Fig. 4. One-directional orientation of flakes of clay minerals bent on a
quartz grain (Q) due to compaction (SEM/EDX) • Jednokierunkowa
orientacja blaszek minerałów ilastych ugiętych na ziarnie kwarcu
wyniku kompakcji widoczna w badaniach elektronomikroskopo-
wych świeżego przełamu próbki. (SEM/EDX) Because of complicated geological history of the Pieniny
Klippen Belt, not only organic matter but also primary
features of the Skrzypne Shale Formation could be signifi-
cantly changed by diagenetic processes and tectonic defor-
mations. The rocks of the Skrzypny Shale Formation of the
Niedzica succession, outcropping at the pass below the Cza-
jakowa Skała Klippe are composed mainly of clay minerals
and micas (about 77 wt.%). Clay minerals are represented by
high-illitic illite/smectite, illite, chlorite and kaolinite. Mo-
reover, these shales contain quartz (about 20 wt.%), trace
amounts of feldspars and calcite (about 3 wt.%) (Fig. 2). strongly chlorotized, mica flakes and kaolinite plates up to a
dozen or even several ten micrometers in size (Fig. 5),
exhibit rotation by 30 to 90º relative to one-directional ori-
entation of fine flakes of clay minerals (Fig. 6). According to
Weaver (1989) criteria, the above described microstructure,
clay mineral composition and the state of preservation of
organic matter indicate that the rocks studied were subjected
to processes characteristic of late stage of diagenesis. The
Skrzypny shales in this outcrop were till now not yet studied
in detail. Their geologic history can be reconstructed on the
ground of X-ray, geochemical and electron microscope stud-
ies using SEM/EDX technique. Compaction process resulted in one-directional orientation
of flakes of clay minerals (illite/smectite, illite) surrounding
fine quartz grains and rare feldspars and bioclasts (Fig. 3). Quartz and feldspar grains (Fig. 4) are very fine, up to a
dozen micrometers in size. Bioclasts are strongly destroyed,
showing numerous dissolution caverns (Fig. 3). Outcrop at the pass below
Czajakowa Skała Klippe Semi-quantitative mineral composition of rocks of the
Skrzypny Shale Formation from the outcrop at the pass below
Czajakowa Skała Klippe (Niedzica succession) based on standard
X-ray data • Półilościowy skład mineralny utworów formacji łupków
ze Skrzypnego w z odsłonięcia na przełęczy pod Czajakową Skałą
(sukcesja niedzicka) określony w oparciu o standardowe badania
rentgenogarficzne B
Fig. 3. Bioclasts (B) showing dissolution caverns surrounded by flakes
of clay minerals (SEM/EDX) • Bioklast z kawernami z rozpuszczania
otoczony przez blaszki minerałów ilastych. (SEM/EDX) Fig. 2. Semi-quantitative mineral composition of rocks of the
Skrzypny Shale Formation from the outcrop at the pass below
Czajakowa Skała Klippe (Niedzica succession) based on standard
X-ray data • Półilościowy skład mineralny utworów formacji łupków
ze Skrzypnego w z odsłonięcia na przełęczy pod Czajakową Skałą
(sukcesja niedzicka) określony w oparciu o standardowe badania
rentgenogarficzne Fig. 3. Bioclasts (B) showing dissolution caverns surrounded by flakes
of clay minerals (SEM/EDX) • Bioklast z kawernami z rozpuszczania
otoczony przez blaszki minerałów ilastych. (SEM/EDX) pH of pore waters in this sediments (Birkenmajer & Narębski
1957). Biomarkers are the indices of redox conditions in sedi-
mentation environment of rocks enriched in organic matter. They represent “molecular fossils” i.e. fragments of organic
matter which preserved the internal structure of initial biomol-
ecules. Two isoprenoids: pristine (Pr) and phytane (Ph) were
used to determination of water column conditions during
Skrzypny shales sedimentation. Elevated Pr/Ph ratio amounts
to 1.8 suggest rather oxic conditions, but this data should be
treated as preliminary. Taking into account relatively high TOC
content and rather oxic conditions of the organic matter sedi-
mentation, all this suggest that suboxic to anoxic conditions
took place during early stage of diagenesis (Fig. 1). Unfortu-
nately, due to relatively high thermal maturity of the samples
and their partial weathering such biomarkers as terpenoids and
steroids were not found. The prevalence of short-chain n-
alkenes, with maximum at n-C16 indicates rather marine
character of organic matter. However, in the range of long-chain
n-alkanes we observe slight prevalence of odd carbon chain
n-alkanes (n‑C27, n-C29, n-C31) what, in turn, indicates a ter-
restrial component of organic matter. Consequently, these data
indicate a mixed terrestrial/marine character of organic matter
with distinct prevalence of the marine one. Fig. 4. Outcrop at the pass below
Czajakowa Skała Klippe They
were formed under conditions when the boundary of oxidizing
and reducing conditions was situated within the sediment and
oxidized water above the latter. Such environment is favour
the development of epifauna and infauna and its accumulation
in the sediment (Fig.1). On the other side, in such geochemical
facies siderite-bearing concretions can be formed (Birkenma-
jer & Narębski, 1957; Potter et al., 2005). The sediments of the Skrzypny shale formation contain
about 1 wt.% of total organic carbon (TOC) and trace amounts
of sulphur. Taking into account weathering of the investigated
material, not altered TOC content seems to be higher, which
indicate oxygene depletion during deposition or early diagen-
esis. Increased content of calcite in them indicates rather high Dark colour of sediments initiating sedimentation in the
Pieniny basin suggests their deposition under oxygen-deficient
conditions enabling preservation of organic matter. Such
conditions are characteristic of a source rocks for oil and gas
deposits. In Demaison and Moore (1980) opinion, oxygen-
deficient environment is characterized by the content of less
than 0.5 ml oxygen in one litre of water. Such conditions are
formed when the request for oxygen in water is higher than
its supply. This request depends on the productivity of surface
waters and the supply of oxygen to sediments on the circula-
tion of currents. It is considered that oxygen-deficient condi-
tions were particularly common in Upper Jurassic and
Lower Cretaceous period because of global worming of cli-
mate and an increase of water level in the oceans. The source
rocks of the largest known oil and gas deposits are of this age. Moreover, anoxic conditions can be formed but locally. By
analogy to actual oceanic environment (continental slope zone
of the Indian Ocean) it is considered that oxygen-deficient
sediments are accumulating in the areas, where currents Fig. 1. Presentation of sedimentary conditions of deposits of
the Skrzypny Shale Formation based on lithologic and biomar-
ker data • Ilustracja warunków sedymentacji utworów formacji
łupków ze Skrzypnego w oparciu o badania cech litologicznych
i biomarkerów Fig. 1. Presentation of sedimentary conditions of deposits of
the Skrzypny Shale Formation based on lithologic and biomar-
ker data • Ilustracja warunków sedymentacji utworów formacji
łupków ze Skrzypnego w oparciu o badania cech litologicznych
i biomarkerów 20 Middle Jurassic black shales (Skrzypny Shale Formation) Fig. 2. Outcrop at the pass below
Czajakowa Skała Klippe Large, often As follows from the obtained results, the deposition of
sediments of the Skrzypny Shale Formation of the Niedzica
succession took place under conditions of oxidized waters 21 Middle Jurassic black shales (Skrzypny Shale Formation) Bt
Ko
Ch
Bt
e biotite (Bt) and chloritized biotite (Ch) flakes and
o) plates among fine flakes of high-illitic illite/smectite
EM/EDX) • Duże blaszki biotytu zubożonego w Fe,
wanego biotytu i płytki kaolinitu wśród drobnych bla-
oillitowego illitu/smektytu i illitu. (SEM/EDX)
Fig. 6. Rotation of large mica (M) and post-mica chlorite
plates by 30-90º relative to one-directional orienta-
tion of fine flakes of clay minerals (CM). F – feldspar
(SEM/EDX) • Rotacja dużych blaszek mik i chlorytu
pomikowego o kąt 30-90º w stosunku do jednokierun-
kowego ułożenia drobnych blaszek minerałów ilastych. (SEM/EDX)
F
CM
M
ediments but pore waters impoverished in oxygen. atter contained in them is of continental/marine
predominance of the latter. During late diagen-
mented by the composition of clay minerals and
tural features of the shales in question, organic
gained considerable thermal maturity. The occur-
igh-illitic illite/smectite, Fe-impoverished and
biotite and the microstructures, characterizing by
onal orientation of clay minerals and rotation of
itized mica flakes the result of post-sedimentary
under conditions of late diagenesis.
ledgements: This work was partially financial
by AGH University of Science and Technology
.140.158. Bt
Ko
Ch
Bt
d
e
e,
-
Fig. 6. Rotation of large mica (M) and post-mica chlorite
plates by 30-90º relative to one-directional orienta-
tion of fine flakes of clay minerals (CM). F – feldspar
(SEM/EDX) • Rotacja dużych blaszek mik i chlorytu
pomikowego o kąt 30-90º w stosunku do jednokierun-
kowego ułożenia drobnych blaszek minerałów ilastych. (SEM/EDX)
F
CM
M
n. e
-
d
c
-
d
y
f
y
l
y Fig. 5. Large biotite (Bt) and chloritized biotite (Ch) flakes and
kaolinite (Ko) plates among fine flakes of high-illitic illite/smectite
and illite (SEM/EDX) • Duże blaszki biotytu zubożonego w Fe,
schlorytyzowanego biotytu i płytki kaolinitu wśród drobnych bla-
szek wysokoillitowego illitu/smektytu i illitu. (SEM/EDX) Fig. 5. Large biotite (Bt) and chloritized biotite (Ch) flakes and
kaolinite (Ko) plates among fine flakes of high-illitic illite/smectite
and illite (SEM/EDX) • Duże blaszki biotytu zubożonego w Fe,
schlorytyzowanego biotytu i płytki kaolinitu wśród drobnych bla-
szek wysokoillitowego illitu/smektytu i illitu. (SEM/EDX) Bt
Ko
Ch
Bt above the sediments but pore waters impoverished in oxygen. Outcrop at the pass below
Czajakowa Skała Klippe Organic matter contained in them is of continental/marine
origin with predominance of the latter. During late diagen-
esis, documented by the composition of clay minerals and
microstructural features of the shales in question, organic
matter has gained considerable thermal maturity. The occur-
rence of high-illitic illite/smectite, Fe-impoverished and
chloritized biotite and the microstructures, characterizing by
one-directional orientation of clay minerals and rotation of
often chloritized mica flakes the result of post-sedimentary
processes under conditions of late diagenesis. Ko Bt Acknowledgements: This work was partially financial
supported by AGH University of Science and Technology
grant: 11.11.140.158. Fig. 6. Rotation of large mica (M) and post-mica chlorite
plates by 30-90º relative to one-directional orienta-
tion of fine flakes of clay minerals (CM). F – feldspar
(SEM/EDX) • Rotacja dużych blaszek mik i chlorytu
pomikowego o kąt 30-90º w stosunku do jednokierun-
kowego ułożenia drobnych blaszek minerałów ilastych. (SEM/EDX)
F
CM
M Fig. 6. Rotation of large mica (M) and post-mica chlorite
plates by 30-90º relative to one-directional orienta-
tion of fine flakes of clay minerals (CM). F – feldspar
(SEM/EDX) • Rotacja dużych blaszek mik i chlorytu
pomikowego o kąt 30-90º w stosunku do jednokierun-
kowego ułożenia drobnych blaszek minerałów ilastych
F
CM
M Fig. 6. Rotation of large mica (M) and post-mica chlorite
plates by 30-90º relative to one-directional orienta-
tion of fine flakes of clay minerals (CM). F – feldspar
(SEM/EDX) • Rotacja dużych blaszek mik i chlorytu
pomikowego o kąt 30-90º w stosunku do jednokierun-
kowego ułożenia drobnych blaszek minerałów ilastych. (SEM/EDX)
F
CM
M M Fig. 6. Rotation of large mica (M) and post-mica chlorite
plates by 30-90º relative to one-directional orienta-
tion of fine flakes of clay minerals (CM). F – feldspar
(SEM/EDX) • Rotacja dużych blaszek mik i chlorytu
pomikowego o kąt 30-90º w stosunku do jednokierun-
kowego ułożenia drobnych blaszek minerałów ilastych. (SEM/EDX) Streszczenie Środkowojurajskie czarne łupki
(formacja łupków ze Skrzypnego)
– znaczenie paleośrodowiskowe jednych
z najstarszych osadów pienińskiego
pasa skałkowego
Katarzyna Górniak, Krzysztof Bahranowski,
Adam Gaweł, Leszek Marynowski
& Tadeusz Szydłak w utworach tych zapisane są początkowe fazy rozwoju ba-
senu pienińskiego (Birkenmajer, 1986, Krobicki & Golonka,
2008 a i literatura tam cytowana). W polskiej części Pieniń-
skiego Pasa Skałkowego, jednymi z najstarszych skał nale-
żących do tych facji są czarne łupki sferosyderytowe z mał-
żami Bositra (formacja łupków ze Skrzypnego) wieku
późny aalen-wczesny bajos (Krobicki & Golonka, 2008a, Fig. 7). Formacja łupków ze Skrzypnego jest opisywana jako seria
skał ilastych o barwie czarnej i czarnozielonej z konkrecjami
syderytowymi w których spotyka się amonity. Miąższość
formacji określa się na około 5 m. Te interesujące utwory od-
słaniają się w górnej części wąwozu Homole, na przełęczy pod
Czajakową Skałą (Krobicki & Golonka, 2008a, Fig. 12D). Ciemne wczesnojurajskie facje margliste (typu Flecken-
mergiel) budzą szczególne zainteresowanie badaczy, ponieważ 22 Middle Jurassic black shales (Skrzypny Shale Formation) ka, 2008a, Fot. 14A i literatura tam cytowana). Warunki ba-
tymetryczne dla facji Fleckemergiel podane przez Tyszkę
(2001) w oparciu o badania mikroskamieniałości odpowia-
dają strefie zewnętrznego szelfu i skłonu. Przełęcz pod Czajakową Skałą – odsłonięcie utworów
formacji łupków ze Skrzypnego j
p
yp
g
Formacja łupków ze Skrzypnego odsłaniająca się na prze-
łęczy pod Czajakową Skałą należy do sukcesji niedzickiej. Utwory sukcesji niedzickiej w wąwozie Homole występują
w formie płatu kontaktującego z podłożem tektonicznie. Płat
ten tworzy fałd obalony, silnie wtórnie sfałdowany nazywa-
ny Czajakową Skałą. Formacja łupków ze Skrzypnego wy-
stępuje w spągu sukcesji niedzickiej. Fragment struktury
fałdu Czajakowej Skały został przemieszczony po warstwie
tych łupków stanowiących powierzchnię poślizgu i obecnie
widoczne są one w osuwisku znajdującym się w górnej czę-
ści wąwozu Homole (Krobicki & Golonka, 2008a, Fot. 12D). Utwory formacji łupków ze Skrzypnego w tym niewielkim
odsłonięciu wykształcone są jako drobnołupiące się, ciem-
noszarooliwkowe łupki z blaszkami miki o wielkości do
0.5 mm i z brązowymi konkrecjami syderytowymi. Łupki są
miękkie, słabo wapniste, bezpiaszczyste. Wielkość sferosy-
derytów nie przekracza kilkunastu centymetrów. Są one
twarde, pryskliwe, ze szczelinami zabliźnionymi kalcytem. Jak wynika ze szczegółowych badań W. Narębskiego (Bir-
kenmajer & Narębski, 1957) konkrecje te są zbudowane z
syderoplezytu (Mg-syderyt) ze znacząca domieszką kalcytu. Skład chemiczny konkrecji wskazuje, że powstały one w
czasie wczesnej diagenezy w słabo redukcyjnych warunkach
facji syderytowej (Birkenmajer & Narębski, 1957). Bochen-
kowaty kształt również wskazuje na ich wczesnodiagenetycz-
ną genezę (Seilacher, 2001). Streszczenie Serie ciemnych skał ilastych gromadzące się w środowi-
skach zubożonych w tlen zwykle pozornie są monotonne. Powiązanie badań sedymentologicznych, paleontologicznych
i geochemicznych przeprowadzonych dla utworów dolnoju-
rajskich (Yorkshire, Wielka Brytania) pozwoliło zdefiniować
trzy facje występujące w basenach zdominowanych przez
wody generalnie niedotlenione. Są to facje: łupków normal-
nych (normal shale), łupków słabo bitumicznych (restricted
shale) i łupków bitumicznych (bituminous shale) (Morris
1979). Przyczyną tego zróżnicowania facjalnego jest cyklicz-
nie zmieniające się położenia granicy między strefą natlenio-
ną i zubożoną w tlen w słupie wody na osadem. Cechy litologiczne utworów formacji łupków ze Skrzypne-
go sukcesji niedzickiej odsłaniające się na przełęczy pod
Czajakowa Skałą reprezentują fację łupków normalnych. Wy-
nika to z porównania ich cech z cechami osadów podanymi
przez Morrisa (1979) dla poszczególnych facji. Środowisko w
jakim powstaje facja łupków normalnych to: położenie grani-
cy między warunkami utleniającymi i redukcyjnymi w obrębie
osadu i natlenionych wód nad osadem. W takim środowisku
możliwy jest rozwój epifauny i infauny oraz nagromadzenie
jej w osadzie (Fig. 1). Jednocześnie są to warunki geochemicz-
ne w których mogą tworzyć się konkrecje syderytowe (Birken-
majer & Narębski, 1957; Potter et al., 2005). Badane utwory formacji łupków ze Skrzypnego zawiera-
ją około 1% całkowitego węgla organicznego (TOC) oraz
śladowe ilości siarki. Biorąc pod uwagę przypowierzchniowe
zwietrzenie badanego materiału, pierwotna ilość TOC była
zapewne wyższa, co wskazuje na sedymentację w warunkach
zubożonych w tlen. Wskaźnikiem warunków redoks panują-
cych w środowisku sedymentacji skał zasobnych w substan-
cje organiczną są biomarkery. Biomarkery pozwalają określić
również pochodzenie substancji organicznej nagromadzonej
w osadach. Biomarkery to „molekularne skamieniałości”
(molecular fossils) czyli fragmenty substancji organicznej,
które zachowują budowę wewnętrzna wyjściowych biomo-
lekuł. Stopień natlenienia kolumny wody podczas sedymen-
tacji łupków ze Skrzypnego określa stosunek pristanu do fi-
tanu czyli izoprenoidów obecnych w badanych próbkach. Wartość tego stosunku wynosi 1,8, co sugeruje raczej tleno-
we warunki sedymentacji substancji organicznej, przy czym
nie są to dane jednoznaczne i należy je traktować jako wyj-
ściowe do dalszych badań. Zatem, stosunkowo wysoka za-
wartość węgla organicznego w badanych skałach oraz raczej
tlenowe warunki sedymentacji, sugerują występowanie wa-
runków suboksycznych do anoksycznych już w osadzie na
wczesnym etapie diagenezy (Fig. 1). Z powodu stosunkowo
wysokiej dojrzałości termicznej materii organicznej oraz jej
częściowego utlenienia w próbkach nie stwierdzono terpeno-
idów i steroidów. Przewaga n-alkanów krótkołańcuchowych,
z maksimum przy n-C16 wskazuje na raczej morski charakter
materii organicznej. Streszczenie Jednak w zakresie n-alkanów długołań-
cuchowych uwidacznia się lekka przewaga n-alkanów nie-
parzysto węglowych (n-C27, n-C29, n-C31), co z kolei wskazu-
je na składnik lądowej materii organicznej. Z danych tych ą g
ę (
,
)
Ciemna barwa osadów rozpoczynających sedymentację
w basenie pienińskim sugeruje, że są to utwory deponowane
w warunkach niedoboru tlenu, umożliwiających zachowanie
w osadzie substancji organicznej. Z takimi niedotlenowymi
środowiskami wiąże się powstanie skał macierzystych dla
złóż ropy naftowej i gazu. Demaison i Moore (1980) uważa-
ją środowisko za niedotlenione jeśli w jednym litrze wody
rozpuszczone jest mniej niż 0.5 mililitra tlenu. Takie warun-
ki tworzą się wówczas kiedy zapotrzebowanie na tlen wodzie
jest większe niż jego dostawa. Zapotrzebowanie na tlen za-
leży od produktywności wód powierzchniowych, a dostawa
tlenu do osadu od cyrkulacji prądów. Uważa się, że warunki
zubożone w tlen szczególnie powszechnie panowały w póź-
nej jurze i wczesnej kredzie z powodu globalnego ocieplenia
klimatu i podniesienia poziomu wód w oceanach. Skały
macierzyste największych znanych złóż ropy i gazu są tego
wieku. Warunki anoksyczne mogą tworzyć się też lokalnie. Przez analogię do współczesnego środowiska oceanicznego
(strefa skłonu kontynentalnego Oceanu Indyjskiego) uważa
się, że osady niedotlenione gromadzą się w obszarach,
w których prądy niosące wody zubożone w tlen (nie więcej
niż 0.5 ml tlenu/litr wody) wkraczają na skłon kontynentalny. Model ten odpowiada warunkom panującym w basenie pie-
nińskim w środkowej jurze. Ciemne facje margliste tego
wieku pojawiają się wśród utworów sukcesji niedzickiej de-
ponowanej na skraju szelfu kontynentalnego i sukcesji czor-
sztyńskiej gromadzonej na skłonie grzbietu czorsztyńskiego. Pierwsze z wymienionych, jak wspomniano powyżej, są
jednymi z najstarszych utworów budujących fałd Czajakowej
Skały i widoczne są w najwyższej części wąwozu Homole,
drugie odsłaniają się w potoku Krupianka (Krobicki & Golon- 23 Middle Jurassic black shales (Skrzypny Shale Formation) wynika, że materia organiczna badanych skał jest mieszane-
go, lądowo/morskiego pochodzenia z wyraźną przewagą
morskiej substancji organicznej. wynika, że materia organiczna badanych skał jest mieszane-
go, lądowo/morskiego pochodzenia z wyraźną przewagą
morskiej substancji organicznej. Mikrostruktura, skład minerałów ilastych oraz stan za-
chowania substancji organicznej opisane powyżej wskazują,
zgodnie z kryteriami Weavera (1989), że badane utwory
formacji łupków ze Skrzypnego ulegały procesom charakte-
rystycznym dla późnego etapu diagenezy. Złożona historia geologiczna Pienińskiego Pasa Skałko-
wego powoduje, że nie tylko substancja organiczna ale
i pierwotne cechy utworów formacji łupków ze Skrzypnego
są w znacznym stopniu zmienione przez procesy diagenezy
i deformacje tektoniczne. Streszczenie Utwory formacji łupków ze
Skrzypnego sukcesji niedzickiej odsłaniające się na przełę-
czy pod Czajakową Skałą zbudowane są głównie z minerałów
ilastych i mik (około 77% wag.). Minerały ilaste są repre-
zentowane przez wysokoillitowy illit/smektyt, illit, chloryt
i kaolinit. Ponadto w badanych skałach występuje kwarc
(około 20% wag.), śladowo skalenie oraz kalcyt (około 3%
wag.) (Fig. 2). Utwory formacji łupków ze Skrzypnego z omawianego
odsłonięcia nie były jak dotąd przedmiotem tak szczegółowych
badań. Swoją historię geologiczną ujawniają po przeprowa-
dzeniu badań rentgenograficznych, geochemicznych i przy
użyciu mikroskopu elektronowego (SEM/EDX). Z badań tych
wynika, że sedymentacja utworów formacji łupków ze Skrzyp-
nego sukcesji niedzickiej przebiegała w warunkach natlenio-
nych wód nad osadem i zubożonych w tlen wód porowych. Substancja organiczna w nich zawarta jest pochodzenia lądo-
wo/morskiego z wyraźną przewagą ostatniej z wymienionych. W warunkach później diagenezy zamanifestowanej w składzie
minerałów ilastych i w cechach mikrostrukturalnych badanych
utworów substancja organiczna osiągnęła znaczną dojrzałość
termiczną. Występowanie wysokoillitowego illitu/smektytu,
zubożonego w Fe i schlorytyzowanego biotytu oraz mikro-
struktur charakteryzujących się jednokierunkowym ułożeniem
blaszek minerałów ilastych i rotacją blaszek mik, często schlo-
rytzowanych, jest skutkiem procesów posedymentacyjnych
odpowiadających warunkom późnej diagenezy. i Skutkiem procesu kompakcji jest jednokierunkowa orien-
tacja blaszek minerałów ilastych (illit/smektyt, illit) obleka-
jących drobne ziarna kwarcu oraz nieliczne skalenie i biokla-
sty (Fig. 3). Ziarna kwarcu (Fig. 4) i skaleni są bardzo drobne. Mają wielkość nie przekraczającą kilkunastu mikrometrów. Bioklasty są silnie zniszczone, z licznymi kawernami z roz-
puszczania (Fig. 3). Duże blaszki miki, często schlorytyzo-
wanej oraz płytki kaolinitu o wielkości osiągającej od kilku-
nastu do kilkudziesięciu mikrometrów (Fig. 5) wykazują
rotację o kąt od 30 do 90° w stosunku do jednokierunkowego
położenia drobnych blaszek minerałów ilastych (Fig. 6). Podziękowania: Praca była częściowo finansowana przez
Akademię Górniczo-Hutniczą, umowa nr 11.11.140.158. References (Literatura)
Birkenmajer, K., 1986. Stages of structural evolution of the Pieniny Klippen
Belt, Carpathians. Studia Geologica Polonica, 88: 7-32. Potter, P. E., Maynard, J.B. & Depetris, P.J., 2005. Mud and Mudstones. Springer 297pp. Moore, D.M. & Reynolds, R.C., Jr. 1997. X-ray diffraction and the identi-
fication and analysis of Clay Minerals.Oxford University Press. New
York, 378. l Birkenmajer, K. & Narębski, W., 1958. Konkrecje węglanowe łupków aale-
nu pienińskiego pasa skałkowego Polski. Rocznik Polskiego Towarzystwa
Geologicznego, XXVII, 51-74. Seilacher, A., 2001. Concretion morphologies reflecting diagenetic and
epigenetic pathways. Sedimentary Geology, 143, 41-57. Demaison, G. J. & Moore, G.T. 1980. Anoxic Environments and oil source
bed genesis. Streszczenie The American Association of Petroleum Geologists Bul-
letin, 64, 8: 1179-1209. Tyszka, J., 2001. Microfossil assambladges as bathymetic indicators of the
Toarcian/Aalenian “Fleckenmergiel” – facies in the Carpathian Pieniny
Klippen Belt. Geologica Carpathica, 52: 147-158. Krobicki, M. & Golonka, J., 2008. Geological history of the Pieniny Klip-
pen Belt and Middle Jurassic black shales as one of the oldest deposits
of this region – stratigraphical position and palaeoenvironmental sig-
nificance. Geoturystyka (ten tom). Weaver, C.E., 1989. Clays, muds and shales. Development in Sedimento-
logy 44. Elsevier, Amsterdam, 819pp. logy 44. Elsevier, Amsterdam, 819pp. Morris, K. A., 1979. A classifcation of Jurassic marine shale sequence: an
example from the Toarcin (Lower Jurassic) of Great Britain. Paleoge-
ography, Paleoclimatology, paleoecology, v.26:117-120.
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Traducción comentada de un acta notarial de celebración de matrimonio civil del español al árabe
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Anaquel de estudios árabes
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cc-by
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ARTÍCULOS
Anaquel de Estudios Árabes
ISSN: 1130-3964
https://dx.doi.org/10.5209/anqe.66701
Traducción comentada de un acta notarial de celebración de matrimonio
civil del español al árabe
Beatriz Soto Aranda1; Rabab Kabbour2
Recibido: 2 de diciembre de 2019 / Aceptado: 29 de febrero de 2020
Resumen. Las actas notariales españolas de celebración de matrimonio civil tienen su origen en la Ley
15/2015, de 2 de julio, de la Jurisdicción Voluntaria, que permite a los notarios, en tanto que juristas y
titulares de la fe pública, intervenir con plena efectividad y sin merma de garantías, en algunos de los
actos de jurisdicción voluntaria que hasta la fecha se encomendaban a los jueces, como es el matrimonio.
Esta circunstancia ha supuesto una diversificación del tipo de documentos que se traducen al árabe para
constatar el estado civil de un ciudadano residente en España, como son la certificación de matrimonio
expedida por el Registro Civil y el certificado de estar inscrito en el Registro de Uniones de Hecho,
cuya regulación es autonómica, no estatal.
El presente estudio presenta una propuesta de traducción comentada al árabe de esta tipología documental
de carácter jurídico-administrativo, con especial atención a los conocimientos lingüísticos, jurídicos y
culturales necesarios para abordar este tipo de traducción especializada, y las macroestructuras de los
textos original y meta. Por último, y en lo referente a la terminología y los aspectos formales exigidos
por la legislación del ordenamiento jurídico meta, habida cuenta las diferencias existentes entre los
ordenamientos jurídicos árabes, y al no tratarse de un estudio comparativo, se ha seleccionado Marruecos
como contexto de recepción de un hipotético encargo de traducción, por ser este el país árabe con mayor
número de inmigrantes en España.
Palabras clave: actas notariales, traducción jurídica, español, árabe.
[en] Annotated translation of a notarial act of civil marriage from Spanish into
Arabic
Abstract. The Spanish civil marriage notarial acts stem from Law 15/2015 of 2 July 2015 of the
Voluntary Jurisdiction. Up to then, voluntary jurisdiction acts could only be performed by judges,
however, since the entry into force of Law 15/2015, notary publics, in their capacity as jurists, can
intervene in some of these acts, such as a marriage act, with full effectiveness. This has led to a
diversification of the type of documents that are translated into Arabic to prove the marital status of a
citizen residing in Spain - documents such as marriage certificates, which are issued by the Civil
Registry, and de facto union certificates, which are a matter of Autonomous Communities competence,
and do not depend on state regulation.
This paper presents a commented translation into Arabic of this type of legal-administrative documents,
paying special attention to the linguistics, legal and cultural knowledge that this type of specialized
translation requires. And it focuses as well in the macro-structures of both the original and the target
texts. Finally, regarding the terminology and formal aspects stipulated by law in the target legal system,
1
2
Institución: Universidad Rey Juan Carlos
E-mail: beatriz.soto@urjc.es
Institución: Universidad Autónoma de Madrid
E-mail: kabbour.rabab@gmail.com
Anaquel estud. árabes 31 2020: 179-205
179
Soto Aranda, B.; Kabbour, R. Anaquel estud. árabes 31 2020: 179-205
180
given the differences between the Arab legal systems, and since this is not a comparative study, Morocco
has been selected as the reception context of a hypothetical translation order, as this is the Arab country
with the largest number of immigrants in Spain.
Keywords: notarial acts; legal translation; Spanish; Arabic.
Sumario: 1. Introducción. 2. Aproximaciones a la traducción jurídica árabe-español. 3. Conocimientos
para afrontar la traducción de un acta notarial de matrimonio del español al árabe. 4. Propuesta de
traducción comentada. 4.1. Menciones preliminares. 4.2. Comparecencia. 4.3. Exposición. 4.4.
Estipulaciones y otorgamiento. 4.5. Autorización. 5. Conclusiones.
Cómo citar: Soto Aranda, B., Kabbour, R. (2020): Traducción comentada de un acta notarial de
celebración de matrimonio civil del español al árabe, en Anaquel de Estudios Árabes 31, 179-205.
1. Introducción
Tanto el estudio de los textos relativos al derecho contemporáneo de los países árabes3 como aquellos dedicados al derecho comparado4 han tenido un importante desarrollo en las últimas décadas en España; ello a la vez que aumentaba el número de
investigaciones sobre la traducción jurídica del árabe al español, así como aquellos
con una orientación didáctica más específica5, junto con manuales de traducción jurídica y administrativa6 y diccionarios especializados7.
3
4
5
6
7
Pueden consultarse las obras que Caridad Ruíz-Almodóvar ha dedicado a diferentes aspectos del derecho de los
países árabes, en especial, RUÍZ ALMODÓVAR, Caridad, Códigos de estatuto personal: el derecho privado en
los países árabes, Granada, 2005. RUÍZ ALMODÓVAR, Caridad, “El testamento en los códigos de estatuto
personal de los países árabes”, en Anaquel de estudios árabes, 26, 2015, (DOI: https://doi.org/10.5209/rev_
ANQE.2015.v26.47696). Con anterioridad a la firma del acuerdo con las comunidades islámicas en materia de
matrimonio, ver FIERRO, Maribel (Coord.), Repertorio bibliográfico de derecho islámico, Murcia 1999.
Entre otros, puede consultarse BLÁZQUEZ RODRÍGUEZ, Irene, (2004) “Pluralidad de formas de celebración
y matrimonio musulmán. Una perspectiva desde el derecho internacional privado español”, en Boletín Mexicano de Derecho Comparado [En línea], 110, disponible en https://helvia.uco.es/xmlui/bitstream/handle/10396/2198/art1.pdf?sequence=1&isAllowed=y [Último acceso el 30 de octubre de 2019]; y ROCA, Encarna, ‘‘Las consecuencias del acuerdo con las comunidades islámicas en materia de matrimonio’’, en El islam
jurídico y Europa. A. Borrás y S. Meinissi (eds.) (1998) 245-464.
LAPIEDRA GUTIÉRREZ, Eva, “La traducción del derecho árabe: planteamientos y propuestas”, en Traducir
del árabe. M. de Epalza y Mª. J. Rubiera (coords.) (2004) 215-260. AGUESSIM EL GHAZOUANI, Abdellatif,
“La enseñanza de la traducción jurídica, económica y comercial en la combinación lingüística: árabe “c” /español”, en El Mundo profesional y las lenguas: enseñar según el MCER. M. Montoro (coord.) (2009) 149-166;
GOGAZEH, Ziyad, (2014) “La problemática de la traducción de la terminología jurídica en el libro Manual de
traducción jurídica árabe-español”, en Philologia Hispalensis 28/1-2, 2014 (Doi: http//dx.doi.org/10.12795.
PH.2014.v28.i01.02); ABDEL-AZIZ, Khaled, “Clasificación del texto jurídico árabe (desde el punto de vista de
la traducción jurídica)”, en Quaderns: Revista de traducción, 13 (2006) 169-179 y BOUTEFFAH AGHBALOU, Bouchra, La didáctica de las traducciones jurídica y jurada en la combinación lingüística árabe-español: propuesta de un manual didáctico. Málaga, 2017.
MOHAMED SAAD, Saad. Curso de traducción judicial, Vol. I, árabe/español, Madrid 2017. MOHAMED
SAAD, Saad. Curso práctico de traducción legal: Volumen I. Árabe/Español, Madrid 2015. MOHAMED
SAAD, Saad. Curso de traducción administrativa: Volumen I, Árabe-Español. Madrid 2010.
AGUESSIM EL GHAZOUANI, Abdellatif, Diccionario jurídico español/árabe. Granada 2006; FERIA GARCÍA, Manuel, Diccionario de términos jurídicos árabe-español. Barcelona 2006. KHAY, Sarah, Traducción
judicial en España y Marruecos. Propuesta de elaboración de un diccionario jurídico en materia penal árabefrancés-español, Alcalá 2016. EL KRIRH, Khatima. Sobre la traducción jurídica y judicial. Estudio de la variación aplicado a la terminología del Derecho penal y procesal penal en español, francés y árabe, Málaga
2016.
Soto Aranda, B.; Kabbour, R. Anaquel estud. árabes 31 2020: 179-205
181
Dentro de este marco general de tratamiento de la traducción jurídica en la dirección árabe-español, destacan los estudios dedicados al derecho de familia y a los
estatutos árabes de código personal8 y, en particular, aquellos centrados en la traducción de documentación relativa a la filiación expedida por los registros civiles9, así
como las actas de matrimonio10, y, más recientemente, las sentencias judiciales de
divorcio11. Ello se debe a que es en este ámbito del derecho donde parece generarse
mayor número de anisomorfismos culturales y asimetrías terminológicas entre el
ordenamiento jurídico español y los de los países árabes12. Ello supone un reto para
el traductor que debe procurarse amplios conocimientos de las culturas y ordena8
9
10
11
12
Véase, entre otros, EL-MADKOURI MAATAOUI MAATAOUI MAATAOUI, Mohamed, “Los aspectos lingüísticos y culturales de la traducción jurídica árabe (El caso del estatuto personal marroquí)”, en Revista del
Instituto Egipcio de Estudios Islámicos, 39 (2011) 75-96. ALLOUCHI, Outman, “Nociones de derecho marroquí de familia imprescindibles para abordar el estudio de la traducción de sentencias judiciales marroquíes en
materia de disolución del matrimonio”, en Tonos digital: Revista de estudios filológicos [En línea] Nª 30, disponible en http://hdl.handle.net/10201/47882 [Último acceso el 30 de octubre de 2019]. BENYAICH, Sokaina
(2014), Estudio terminológico de la Mudawana (Código de Familia Marroquí). Máster Universitario en Comunicación Intercultural, Traducción e Interpretación en los Servicios Públicos, Universidad de Alcalá. FERIA
GARCÍA, Manuel, “Reflexiones en torno a las últimas reformas del derecho de familia en Marruecos”, en
Hesperia culturas del Mediterráneo 2 (2005) 21-36.
AGUESSIM EL GHAZOUANI, (2013) Abdellatif, “Aproximación a la traducción jurídico-administrativa de
documentos del registro civil marroquí: certificaciones de nacimiento” en Tonos digital: Revista de estudios filológicos [En línea] Nª 25, disponible en https://digitum.um.es/digitum/handle/10201/38087 [Último acceso el
30 de octubre de 2019]
Sobre la traducción de actas de matrimonio egipcias, puede consultarse ABDEL-AZIZ, Khaled, (2005) “El
lenguaje jurídico del derecho egipcio de familia (un enfoque descriptivo)” en Sendebar: Revista de la Facultad
de Traducción e Interpretación [En línea]. Nº 16, disponible en https://revistaseug.ugr.es/index.php/sendebar/
article/view/1049/1247 [Último acceso el 30 de octubre de 2019] y ABDEL-AZIZ, Khaled, Un estudio descriptivo de la Ley egipcia de Estatuto Personal número 100 de 1985: un enfoque traductológico. Barcelona 2003.
Sobre la traducción jurada de actas de matrimonio marroquíes, véase FERIA GARCÍA, M.C. “La traducción
jurada de actas matrimoniales marroquíes”, en Traducir para la justicia. M. Feria García (ed.) (1999) 221-258;
FERIA GARCÍA, Manuel, La traducción fehaciente del árabe: fundamentos históricos, jurídicos y metodológicos. Málaga 2002; y FERIA GARCÍA, Manuel, “Convivencia e interacción de tradiciones jurídicas en Marruecos: la fe pública: sofrim, notarios latinos y adules”, en Awraq: Estudios sobre el mundo árabe e islámico
contemporáneo, 24 (2007), 241-286. Con relación a la traducción de actas de matrimonio tunecinas, véase BEN
OTHMAN, Fathen, “Análisis lingüístico-pragmático y cultural de problemas de traducción en un documento
jurídico (árabe-español). Acta de matrimonio”. Actas del XXIV Congreso Internacional de AESLA [Recurso
electrónico]: aprendizaje de lenguas, uso del lenguaje y modelación cognitiva : perspectivas aplicadas entre
disciplinas, 2007.
ALLOUCHI, Outman, La sentencia de divorcio (árabe/español): Estudio jurídico y textual preliminar. Granada
2010. ALLOUCHI, Outman, (2016) “Nociones de derecho marroquí de familia imprescindibles para abordar el
estudio de la traducción de sentencias judiciales marroquíes en materia de disolución del matrimonio” en Tonos
digital: Revista de estudios filológicos [En línea], Nº 30, disponible en en http://www.tonosdigital.es/ojs/index.
php/tonos/article/view/1413 [Último acceso el 30 de octubre de 2019]. EL-MADKOURI MAATAOUI, Mohamed, “La traducción del árabe al español y el exequátur en materia de divorcio”, Anaquel De Estudios Árabes, 30, 2019 (Doi: https://doi.org/10.5209/ANQE.62685).
A cerca de los parámetros culturales que condicionan los documentos jurídicos y administrativos poscoloniales,
expedidos por países con población musulmana, véase MAYORAL, Roberto, Translating Official Documents
(Translation practicies explained), Londres 2003. Con relación a la hibridación terminológica de los documentos africanos, véase El-MADKOURI MAATAOUI, Mohamed y SOTO ARANDA, Beatriz (2012) “Aspectos
lingüísticos y extralingüísticos de la traducción jurídico-administrativa de documentos africanos al francés”, en
Synergies Espagne [en línea], Nº 5, 111-128, disponible en http://gerflint.fr/Base/Espagne5/el_madkouri.pdf
[Último acceso el 30 de octubre de 2019]. KARIM, Hasna, La traducción de los anisomorfismos culturales en
el ámbito jurídico: aplicaciones al derecho marroquí en comparación con el derecho francés y español. Málaga
2010. EL JIRARI, Abderrahim, “La influencia de las asimetrías institucionales en la traducción jurídica y administrativa: estudio de caso: Marruecos y España”, en FITISPos international journal: public service interpreting
and translation, 5 (2018) 159-169.
182
Soto Aranda, B.; Kabbour, R. Anaquel estud. árabes 31 2020: 179-205
mientos jurídico-administrativos asociados a las lenguas que el proceso traductor
pone en contacto, además de desarrollar una capacidad de documentación adecuada.
Como señala El-Madkouri Maataoui, incluso la competencia de un traductor cuya L1
es el árabe:
depende de si traduce documentos de países cuya legislación, instituciones y terminología domina y que suelen coincidir con sus países de origen, tránsito o estudios. […] Las dificultades se dan en este caso no por desconocimiento de la lengua
árabe, sino por no poder aprehender el mundo conceptual y referencial de las palabras13.
En este sentido, cabe recordar que los ordenamientos jurídicos son propios de
cada país, y distintos del resto de ordenamientos de otros países, con independencia
de que la lengua vehicular y sus bases sean comunes.
Junto a ello, los procesos migratorios, en general, y la residencia y el arraigo de
la población migrante en los países de acogida, en particular, llevan consigo necesariamente la interacción entre las administraciones del país de origen y de acogida,
que resultarían imposibles sin la mediación de la traducción14. Así, la labor traductora constituye una herramienta imprescindible en trámites judiciales, administrativos
y notariales para los inmigrantes15. Lapiedra señala sobre esta cuestión que:
Hay que tener también en cuenta que el mayor contacto entre el derecho de los
países árabes y el derecho tanto español como europeo en general se da justamente en el ámbito del Derecho de Familia. […] Y es lógico que así sea, ya que el
derecho tiene un componente territorial sobradamente conocido, al que escapa sin
embargo el Estatuto Personal. Así lo dispone el art. 9 del Código civil español: “la
ley personal correspondiente a las personas físicas es la determinada por su nacionalidad. Dicha ley regirá la capacidad y el estado civil, los derechos y deberás de
la familia y la sucesión por causa de muerte…”16.
En este contexto social, y lejos de visiones románticas de la traducción que cuestionan la traducción inversa realizada por no nativos17, el dominio de la traducción
13
14
15
16
17
EL-MADKOURI MAATAOUI MAATAOUI, Mohamed, “Aspectos lingüísticos y extralingüísticos de la traducción jurídico-administrativa del árabe al español”, en Estudios de lingüística y traductología árabe. S. M.
Saad (coord.) (2010), p. 236.
Véase p. e. el convenio hispano-marroquí de cooperación judicial en materia civil, mercantil y judicial firmado
el 30 de mayo de 1997 (BOE 151, de 25 de junio de 1997), disponible en https://www.boe.es/boe/dias/1997/06/25/
pdfs/A19583-19587.pdf, [Último acceso el 30 de octubre de 2019], que incluye un artículo sobre la traducción
de documentación judicial. EL-MADKOURI MAATAOUI MAATAOUI, Mohamed, “El discurso del lenguaje
jurídico-administrativo español: análisis y perspectivas.”, en Estudio multilingüe e implicaciones culturales. Mª
M. Errutia (coord.) (2016)127-164.
NEBOT, ROSER, Nicolás. “El lenguaje jurídico en español y en árabe. Una misma situación con enfoques divergentes. Una misma necesidad de traducción con soluciones diferentes”, en Recursos documentales y tecnológicos para la traducción del texto jurídico. G. Corpas Pastor (ed.) (2003) 297-309.
LAPIEDRA GUTIÉRREZ, Eva, op. cit. p. 217.
KOCIJANČIČ POKORN, Nike, Challenging the Traditional Axioms: Translation into a Non-Mother Tongue.
Amsterdam 2005. Kelly, Dorothy, “The Wrong Way Round? Consideraciones sobre la cuestión de la direccionalidad en la traducción profesional y la formación de traductores”, en Experiencias de traducción. Reflexiones
desde la práctica traductora. C. García de Toro e I. García Izquierdo (eds.) (2005), 129-146. CAMPBELL,
Stewart: Translation into the Second Language, Londres 1998.
Soto Aranda, B.; Kabbour, R. Anaquel estud. árabes 31 2020: 179-205
183
hacia el árabe es una necesidad formativa para los estudiantes de traducción en un
mercado laboral cada vez más especializado, y en constante cambio a nivel social,
económico y político18.
2. Aproximaciones a la traducción jurídica español-árabe
La traducción inversa en la combinación lingüística español-árabe ha recibido cierta
atención académica en España, con especial referencia a la conocida como Ley de
Extranjería19 y a un tipo de acta notarial de dominio para reanudación del tracto sucesivo, esto es, un requisito para la transmisión de derechos o bienes20. En ambos casos,
se trata de documentación con un importante calado social; la traducción de una ley
es relevante porque todo ciudadano con independencia de su estatus debe conocer sus
derechos y obligaciones, y ese es un paso clave en el proceso de integración social; la
traducción de un acta notarial lo es porque los instrumentos notariales relacionados
con compra-venta de inmuebles y herencias da cuenta de los procesos de integración
social de la población inmigrada21, sin olvidar que la asistencia de los traductores-intérpretes en las notarías es obligatoria por ley cuando el cliente no tiene la competencia lingüística suficiente en la lengua vehicular. En los dos estudios, queda patente que
la traducción es un acto de comunicación intercultural22, y que la labor del traductor
debe regirse por la deontología profesional, máxime cuando la agencia de este tipo de
traducciones corresponde principalmente a la figura del traductor jurado.
Las investigaciones realizadas sobre la calidad tanto de la traducción jurídica
como de la traducción inversa coinciden en señalar que el éxito de una traducción no
depende tanto de la competencia lingüística en ambas lenguas, que se le presupone a
todo estudiante que se enfrenta a una traducción especializada, como del desarrollo
de las competencias extralingüística, de transferencia y documental. Siguiendo la
taxonomía propuesta por Borja23, la competencia extralingüística incluye conocimientos enciclopédicos del mundo, conocimientos jurídicos y del lenguaje jurídico,
del derecho de las lenguas A y B, de derecho comparado y de derecho internacional.
Esta descripción es más amplia que la definición propuesta por el Grupo PACTE,
esto es, “conocimientos acerca de la organización del mundo en general y de ámbitos
particulares”24, y está más en consonancia con el concepto de competencia intercul18
19
20
21
22
23
24
HUSNI, Ronak y Newman, Daniel, Arabic-English-Arabic Translation: Issues and Strategies, Londres, 2015, XIV.
AGUESSIM EL GHAZOUANI, Abdellatif, Traducción jurídico-administrativa e inmigración. Propuesta de
traducción al árabe de la ley de extranjería en vigor. Granada, 2004. https://hera.ugr.es/tesisugr/1540576x.pdf
[Último acceso el 30 de octubre de 2019]; y AGUESSIM EL GHAZOUANI, Abdellatif, “Traducción jurídicoadministrativa e inmigración: propuesta de traducción al árabe de la Ley de extranjería en vigor”, Traducción
como mediación entre lenguas y culturas = Translation as mediation or how to bridge linguistic and cultural
gaps [Recurso electrónico] / Carmen Valero Garcés (ed. lit.) (2005), 272-279.
NEBOT, ROSER, Nicolás. op. cit.
DEL POZO TRIVIÑO, Maribel (2008) “La interpretación en las notarías españolas”, en Actas del III Congreso
Internacional de la Asociación Ibérica de Estudios de Traducción e Interpretación. La traducción del futuro:
mediación lingüística y cultural en el siglo XXI. L. Pegenaute, (ed.) (2008) vol. 2, 491-504. disponible en: http://
www.aieti.eu/wp-content/uploads/AIETI_3_MPT_Interpretacion.pdf [Último acceso el 30 de octubre de 2019].
HURTADO ALBIR, A. Traducción y traductología. Madrid 2001, p. 607.
BORJA ALBI, Anabel, “Organización del conocimiento para la traducción jurídica a través de sistemas expertos basados en el concepto de género textual”, en El género textual y la traducción. Reflexiones teóricas y
aplicaciones pedagógicas. I. García Izquierdo (ed.) (2005), 37-67.
Grupo PACTE, “La competencia traductora y su adquisición” en Quaderns. Revista de traducció 6, (2001), 41.
184
Soto Aranda, B.; Kabbour, R. Anaquel estud. árabes 31 2020: 179-205
tural, en el sentido de que ha de incluir conocimientos de ambas culturas, además de
los enciclopédicos y los temáticos, puesto que no se trasladan palabras, sino discursos, que cumplen una función concreta en el contexto sociocultural de partida, al
contexto de llegada con una función equivalente25. Sin olvidar que la traducción jurídica es un caso particular de traducción de textos especializados, pues como señala
El-Farahty, el vocabulario legal es específico de cada cultura y está vinculado al
propio sistema cultural. Así, “el trabajo del traductor jurídico no consiste solo en
transcodificar el significado legal, sino en transferir también su efecto legal”26.
A su vez, la competencia de transferencia puede definirse como la capacidad de
comprensión y reexpresión de los textos, teniendo en cuenta la finalidad de la traducción y el destinatario de esta. Esta incluye la competencia textual formada por el
conocimiento de las taxonomías textuales de partida y de llegada, los aspectos formales exigidos por la legislación en cada ordenamiento jurídico, función y eficacia
jurídica de los géneros en cada ordenamiento jurídico, así como la fraseología y
terminología propias de cada texto27. En cuanto a la competencia documental, Sales
la define como la capacidad de búsqueda, recuperación y tratamiento de información
necesaria para llevar a cabo una traducción28. Resulta llamativo que los estudios para
la combinación español-árabe antes citados señalen como retos para la traducción
jurídica, cuestiones ligadas directamente con estas tres competencias:
1. Ausencia de diccionarios especializados.
2. Falta de unificación de términos jurídicos en los países árabes.
3. Importancia de la relación forma-contenido en el lenguaje jurídico árabe.
4. Conocimiento de las diferencias entre los ordenamientos jurídicos árabes y la
terminología empleada en cada uno de ellos.
5. Conocimiento tanto de las bases religiosas del derecho de los países árabes
como de los ordenamientos occidentales que han influido en ellos.
6. Dificultad del propio discurso jurídico en cualquier idioma pues se trata de un
discurso culturalmente marcado.
7. Estrategias para la traducción de los anisomorfismos.
8. Conocimiento de textos de partida y de llegada, Aspectos formales exigidos
por la legislación en cada ordenamiento jurídico.
Así, el árabe es lengua oficial de veintidós países con bagajes históricos y culturales muy diversos, a los que cabe sumar algunos países como Chad que tienen el
árabe como lengua cooficial29. En muchos casos, se trata de ordenamientos jurídicos
híbridos30, característicos de sociedades poscoloniales que han heredado el derecho
positivo de las ex metrópolis o de países con un destacado peso histórico, como es el
25
26
27
28
29
30
El-MADKOURI MAATAOUI, Mohamed y SOTO ARANDA, Beatriz, op. cit., p. 127.
EL-FARAHATY, Hanem. International Journal of the Semiotics of Law 29 (2016), https://doi.org/10.1007/
s11196-016-9460-2, p. 475.
Aunque existen otras definiciones, la propuesta por Anabel Borja Albí (2005), en su artículo “Organización del
conocimiento para la traducción jurídica a través de sistemas expertos basados en el concepto de género textual”, ya citado, es muy útil y completa desde el punto de vista de la didáctica de la traducción jurídica.
SALES SALVADOR, Dora, Documentación aplicada a la traducción: presente y futuro de una disciplina, Gijón 2006.
SHIYAB, Said “The dilema of legal terminology in the Arab World”. Handbook of Terminology. Vol. 2 Terminology in the Arab World. Abied Alsulaiman y Ahmed Allaithty (eds), (2019), p. 140.
EL-MADKOURI MAATAOUI, Mohamed y SOTO ARANDA, Beatriz, op. cit. p. 121.
Soto Aranda, B.; Kabbour, R. Anaquel estud. árabes 31 2020: 179-205
185
caso de Francia en Egipto; derecho, de influencia anglosajona o continental, que
conjugan con algunos principios islámicos. En palabras de Nebot, “los ordenamientos legales vigentes en estos países son transpolaciones, a veces simples traducciones, de los códigos occidentales, adaptadas a las condiciones dispuestas por algunos
de los principios islámicos, pero no todos”31. Abdel-Aziz coincide con esta afirmación cuando señala que, en el caso egipcio,
Actualmente, las leyes del derecho islámico tienen poca influencia en el sistema
jurídico egipcio (el derecho egipcio actual ha conservado algunos aspectos de alšarî‘a al-islamiyya, que se manifiestan básicamente en la aplicación del derecho de
familia (Código de Estatuto Personal) y lo que llamamos el espíritu de las leyes
islámicas, a través de islamizar algunas de las leyes civiles, no en su contenido
sino en su forma externa; por ejemplo, al- šarî‘a al-islamiyya prohíbe la usura; de
hecho, las leyes civiles evitaron mencionar el término usura a la hora de egipcializar el derecho francés32.
En este sentido, se puede afirmar que el derecho francés ha influido notoriamente
en los ordenamientos jurídicos árabes por dos vías: directamente, como es el caso de
la Ley libanesa de 9 de marzo de 1932 relativa al Código de obligaciones y contratos33, aún en vigor, que no solo se basa en el código civil francés sino también en la
doctrina y jurisprudencia francesas, siendo que el texto original se redactó en francés
y el texto en árabe fue una traducción posterior34. Otra, indirecta, merced al hecho de
que el código civil egipcio de 1948 elaborado por al-Sunhûrî, de inspiración francesa, sirvió de modelo para el código sirio (1949), el de Iraq (1953), el de Libia (1954),
el de Argelia (1975) y el de Jordania (1977). Asimismo, las disposiciones jurídicas
que regulan las obligaciones en el código civil egipcio fueron incorporadas al Código de Comercio de Kuwait en 194835.
Otros autores como Fakhri36 van más allá, y afirman que el lenguaje jurídico marroquí ha sufrido un cambio sustancial por influencia del francés, pues mientras los textos
jurídicos tradicionales seguían una estructura narrativa, la organización actual es cronológica, con oraciones y cláusulas que dan cuenta de una secuencia de hechos y circunstancias ordenados temporalmente, junto con personajes y sus funciones. En un
trabajo posterior, Fakhri sostiene que las sentencias del Tribunal de Casación marroquí
reflejan prácticas retóricas híbridas, que han tomado prestado modelos occidentales37.
Así, ... من قانون المسطرة المدنية543 ( بناء على أن الفصلVisto el artículo 345 del Ley de Enjui31
32
33
34
35
36
37
NEBOT ROSER, Nicolás. op. cit., p. 300
ABDEL-AZIZ, Khaled, op. cit, (2003), p. 176.
https://www.ilo.org/dyn/natlex/docs/ELECTRONIC/57070/134590/F-755173354/LBN57070%20ARA_FR.pdf
[Último acceso el 21 de noviembre de 2011].
EL CHAZLI, Karim. “Le role des langues dans la connassaince et le devéloppement des droits des pays árabes”,
en General Reports of the XIXth Congress of the International Academy of Comparative Law. Martin Schaouer
y Bea Verschraegen (eds.) Dordrecht (2017), p. 598.
ALUFFI, Roberta, “Unity and varieties of Arabic as a legal language. Practices of interpretation and translation”, en Handbook of communication in the Legal sphere, Jacqueline Visconti (ed.), Der Gruyter La Haya
(2018) p. 431.
FAKHRI, Ahmed, “Borrowing Discourse patterns: French rhetoric in Arabic legal texts”. Dilworth B. Parkinson, Elabbas Benmamoun (eds.) (2002), p. 161.
FAKHRI, Ahmed, “Citations in Arabic Legal Opinion. `Iftaa´versus qadaa´”. Perspectives on Arabic Linguistics Dilworth. B. Parkinson (ed.) (2008), p. 126
Soto Aranda, B.; Kabbour, R. Anaquel estud. árabes 31 2020: 179-205
186
ciamiento Civil …) corresponde a expresiones del francés jurídico como Vu l´article
1382 du code civil, mientras que من قانون االلتزامات والعقود929 ( عمالً بمقتضيات الفصلEn
aplicación de lo estipulado en el artículo 929 del Código de Obligaciones y Contratos)
corresponde a expresiones como En application des articles R. 142-1 et R. 142-18 du
code de la securité.
Desde una perspectiva complementaria, Lapiedra subraya el hecho de que la
šarî‘a es un conjunto de normas más propiamente religiosas o morales, y que, debido
a su carácter no jurídico, las normas de contenido jurídico que se fundamentan en
elementos de la šarî‘a, tendrán en cada país la fuerza que su ordenamiento jurídico
haya establecido: desde una remisión directa, a la plasmación gradual en leyes, decretos, etc.38. Estas diferencias se observan, por ejemplo, en las constituciones saudí
y marroquí. En la primera, aprobada en 1992 y modificada en 2005, denominada
النظام األساسي للحكم بالمملكة السعودية, sus artículos 1 y 8 dicen:
ودستورها كتاب هللا، دينها اإلسالم، ذات سيادة تامة، المملكة العربية السعودية دولة إسالمية.المادة األولى
)...( .تعالى وسنة الرسول صلى هللا عليه وسلم
Art. 1. El Reino de Arabia Saudí es un Estado islámico, totalmente soberano, su
religión es el islam, y su constitución es el Libro de Allah, Todopoderoso, y la Sunnah del Profeta, la paz sea con él. (...)
يقوم الحكم في المملكة العربية السعودية على أساس العدل والشورى والمساواة وفق الشـريعة.٨ المادة
.اإلسالمية
Art. 8 – El Gobierno del Reino de Arabia Saudí se basa en la justicia, la consulta
y la igualdad de acuerdo con la šarî‘a islámica.
Por su parte, en la constitución marroquí de 2011, denominada دستور المملكة المغربية,
los artículos 3, 6 y 16 fueron redactados de la siguiente forma:
. والدولة تضمن لكل واحد حرية ممارسة شؤونه الدينية، اإلسالم دين الدولة.3 الفصل
Art. 3. El islam es la religión del Estado y es el Estado el que garantiza a cada uno
la libertad para practicar sus cultos religiosos.
بما فيهم، أشخاصا ذاتيين أو اعتباريين، والجميع. القانون هو أسمى تعبير عن إرادة األمة.6 الفصل
. وملزمون باالمتثال له، متساوون أمامه،السلطات العمومية
Art. 6. La Ley es la expresión suprema de la voluntad de la nación. Toda persona,
física o jurídica, incluidas las autoridades públicas [los poderes públicos], son iguales ante ella y están obligados a cumplirla.
تعمل المملكة المغربية على حماية الحقوق والمصالح المشروعة للمواطنات والمواطنين.61 الفصل
في إطار احترام القانون الدولي والقوانين الجاري بها العمل في بلدان،المغاربة المقيمين في الخارج
.)...( االستقبال
Art. 16. El Reino de Marruecos protegerá los derechos e intereses legítimos de los
ciudadanos marroquíes que residen en el extranjero, en el marco del respeto al derecho internacional y las leyes vigentes en los países de acogida (…).
38
LAPIEDRA, Eva, op. cit. p. 216.
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187
Así, mientras Arabia Saudí fundamenta sus leyes sobre una determinada lectura
del Corán y de la tradición profética (Sunnah)39, otros países como Marruecos, aun
reconociendo el islam como religión del Estado, supeditan sus normas al derecho
internacional y al de los países donde viven ciudadanos marroquíes.
El lenguaje jurídico árabe se caracteriza pues por la pluralidad de fuentes del
derecho (continental, Common Law, islámico, consuetudinario), la diversidad de sistemas de terminología legal, todo ello acentuado por las influencias externas en
cuanto a préstamos, y el hecho de que otros organismos internacionales como la
ONU y la OTAN también producen su propia terminología en árabe, basada en criterios locales y la preferencia de los traductores40. Por ejemplo, el término `artículo´
se traducirá por مادةsi la traducción ha de surtir efecto en Jordania, Siria, Irak, Egipto y países del Gofo, mientras que también se puede utilizar el término فصلpara
Marruecos, como se puede apreciar en los ejemplos extraídos de las constituciones.
En el caso de ´garante´, es كفيلen Jordania, Siria o Irak, y ضا ِمنen Egipto y el Magreb41.
No obstante, sí hay un acuerdo en que el lenguaje jurídico en lengua árabe presenta características comunes como la existencia de términos técnicos como قضية
(causa), ( دعوىdemanda) y ( محكمةtribunal), semitécnicos como ( تعويضcompensación) y ( دليلprueba), junto con palabras comunes como ( طرفparte), ( ملفexpediente) y ( تقريرinforme), que adoptan una acepción especializada en este tipo de textos.
Asimismo, son frecuentes las metáforas y otros recursos estilísticos. En cuanto a
arcaísmos, El-Farahaty señala términos como ( الحالة المعتبرة شرعاcumpliendo todos
los requisitos legales) o ( المذكور أعالهarriba mencionado), aunque otros han caído en
desuso como ( المزبورel arriba mencionado) y ( المسفورdeclarado).
Igualmente se caracteriza por el uso de la nominalización y de dobletes. Según
Fakhri, la nominalización cumple dos funciones en las sentencias judiciales: una
defensa sólida y una referencia anafórica concisa, como se observa en la siguiente
expresión formularia característica de los fallos del Tribunal Supremo marroquí,
para indicar que considera nulo y sin efecto el fallo de un Tribunal inferior. يعيب الطالب
. سوء ونقصان التعليل الموازي النعدامه... ( على القرار المطعون فيهSe rechaza la solicitud impugnada… por error de interpretación y falta de fundamento equivalente a su ausencia)42.
En cuanto a los culturemas, tenemos términos como ( عدةperíodo de espera antes de
contraer nuevas nupcias) o ( خلعdivorcio a petición de la esposa).
De lo dicho cabe extraer tres principios importantes para la traducción al árabe.
Por un lado, a pesar de la variabilidad, existen un conjunto de rasgos comunes que
caracterizan los textos legales y que deben tenerse en cuenta, en la medida de lo
posible. Por otro, a lo largo de la Historia, el lenguaje jurídico ha consolidado la
aplicación de una serie de procedimientos léxicos para dar cuenta de los cambios
producidos en las sociedades árabes; y por ende, la incorporación de términos extranjeros al lenguaje jurídico árabe tiene lugar preferentemente dentro de la tradición
gramatical de la lengua árabe43:
39
40
41
42
43
EL-FARAHATY, Hanem. op. cit. (2016), 473
DARWISH, Ali Terminology and Translation. A Phonological-Semantic Approach to Arabic Terminology, Melbourne 2009, p. 24.
SHIYAB, Said, op. cit. p. 142.
FAKHRI, Ahmed, “Nominalization in Arabic Discourse: A Genre Analysis perspective”. Arabic Language and
Linguistics. R. Bassiouney y E. G. Katz (eds.) (2012), 145-157
Procedimientos de acuñación e incorporación de terminología jurídica al árabe según Roser NEBOT, op. cit.
188
Soto Aranda, B.; Kabbour, R. Anaquel estud. árabes 31 2020: 179-205
– Derivación léxica:
a. Recuperación de términos jurídicos en desuso, e investirlos de una nueva
acepción.
b. Enriquecimiento polisémico de un vocablo jurídico homologado con un
nuevo significado según el contexto.
c. Creación de vocablos derivados de términos que comparten un campo semántico.
– Traducción explicativa:
a. Doblete de nombre y adjetivo, recoge el significado de la expresión original y no sus componentes léxicos inmediatos.
Ejemplos de incorporación de términos mediante alguna de estas técnicas son las
voces ( دستورconstitución), ( جنسيةnacionalidad) y ( مواطنةciudadanía). El primer término fue adoptado por primera vez en Túnez (1861), y se tomó prestado del persa,
referido a persona que ejerce la autoridad, y a una regla o regulación que conlleva
una pena44. El segundo término se tomó prestado del griego yenos, en la que indicaba
`raza´. El tercer término procede de ( مواطنciudadano), derivado de وطن, que en origen significaba `lugar de nacimiento o residencia´.
Por otro, la preferencia por estos procedimientos, frente a la paráfrasis o a la mera
transcripción, cuando ello es posible, es correlativa a la importancia que se le otorga
a la corrección formal de los enunciados jurídicos45, si bien en el caso de culturemas,
se tiende a recurrir a estas dos técnicas, además de la nota a pie de página. En este
sentido, el traductor debe prestar atención no solo a la correcta transmisión del contenido y a la terminología empleada en la traducción, sino también la redacción y
adecuación estilística del texto meta, al objeto de que la administración destinataria
la acepte. Así, el objetivo de una traducción especializada como esta es la de producir un texto meta que desempeñe la misma función legal y produzca el mismo efecto
legal46, por lo que además de informar, tienen una función performativa47.
En lo concerniente a textos relacionados con los códigos de estatuto personal, y
si bien es cierto que son los que mayor número de anisomorfismos presentan debido
a la influencia que los principios de la šarî‘a ejercen sobre ellos, hay que observar
esta cuestión con cierta precaución. Sin ir más lejos, Abdel-Aziz apunta que el propio término ‘estatuto personal’ “no es un término legal propio del derecho islámico
ni de la literatura religiosa árabe-islámica tradicional, sino un término jurídico traducido literalmente del inglés Personal Status y del francés Statut Personnel, según
han demostrado ‘Abd-Allah y Qamhawl (1986)”48. Esta traducción fue acuñada por
Muhammad Qadrî Bâšâ en 1875 y asumida por el resto de los países árabes49.
En el marco de un discurso culturalmente marcado por su inspiración religiosa,
los términos se adaptan, se reinterpretan o se crean, en función de los intereses y
objetivos del legislador. Para ello se recurre al iytihad, la potestad interpretativa que
tienen los especialistas en el fiqh, la jurisprudencia islámica. Ello ocurre, bien por
44
45
46
47
48
49
ALUFFI, Roberta, op. cit. p. 426
NEBOT ROSER, Nicolás, op. cit. p. 301
EL-FARAHATY, Hanem. op. cit. p. 473.
MEDIOUNI, Mohamed, “Towards a functional approach to Arabic-English legal translation: The role of comparable /parallel texts”. New insights into Arabic translation and interpreting. M. Taibi (ed,) (2016), 115-160.
ABDEL-AZIZ, Khaled, (2003) op. cit., p. 176.
ALUFFI, Roberta, op. cit. p. 426
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189
impulsos sociales internos -tal es el caso de los movimientos feministas-, bien por
cuestiones derivadas de la aplicación del derecho internacional; aunque también
existe otro factor a tener en cuenta, como es el peso y repercusiones de la emigración
a países europeos50. Un claro ejemplo de ello es la redacción del Código de Familia
marroquí (en adelante, CF) aprobado en 200451.
En lo concerniente a las cuestiones de fondo, el término ( وليtutor matrimonial),
uno de los requisitos para la validez del matrimonio islámico, deja de ser una figura
obligatoria. Así, el nuevo CF ha hecho opcional una condición hasta ahora obligatoria para la validez del matrimonio islámico según la antigua Mudawwana, como es
la tutela matrimonial para la mujer ejercida por el wali (tutor), y le ha dado a la mujer
mayor de edad la capacidad total de decidir casarse. Ella conserva la posibilidad de
ser asistida por un tutor, pero ahora es libre de usar esta facultad o no. Esto ha supuesto que la referencia al wali desaparezca de la mayoría de las actas matrimoniales
que se expiden en los últimos años.
En lo concerniente a las cuestiones de forma, según el nuevo texto, el matrimonio
de los ciudadanos marroquíes en el extranjero puede llevarse a cabo siguiendo las
formas del derecho local. En este caso, el CF no requiere que se cumplan las condiciones de validez de un matrimonio dentro de Marruecos, salvo la presencia de dos
testigos (en las actas marroquíes esa función es desempeñada por los dos notarios
que deben levantar acta). Las actas españolas también incluyen testigos, pero, en el
caso de que no sean varones musulmanes, para que sean aceptadas por el consulado
correspondiente, será requerida un acta complementaria de testificación que se levanta en el propio consulado.
3. Conocimientos previos para afrontar la traducción de un acta notarial de
matrimonio civil al árabe
Para traducir un acta notarial de matrimonio, debe tenerse en cuenta la tipología
textual, las características de las actas notariales en el contexto meta y el lenguaje
que las caracterizan.
Los instrumentos notariales se estudian dentro de los lenguajes de especialidad,
en concreto, en el marco del lenguaje jurídico. En cuanto al género, se trata de documentos públicos, esto es, autorizados, expedidos o intervenidos por un funcionario
público competente, y entran dentro de la categoría de textos de aplicación del Derecho, junto con contratos, testamentos, poderes de representación, etc.52. Borja incluye dentro de los textos jurídicos tanto aquellos que se utilizan en las relaciones entre
el poder público y los ciudadanos como aquellos textos codificados, empleados para
regular las relaciones entre particulares con transcendencia jurídica (que dan lugar a
50
51
52
La realidad de la inmigración también ha influido en otros cambios legislativos como el art. 65, cap. II de la Ley
de nacionalidad consolidada a 26.11.2011., por el que la nacionalidad se obtiene por vía de padre y madre, indistintamente. Antes de la reforma, la nacionalidad por vía materna para los hijos de ciudadanos residentes en
el extranjero solo se obtenía en casos de padre desconocido o apátrida.
Siguiendo la terminología empleada por Estaban de la Rosa et al. en su traducción comentada del nuevo Código
de Familia marroquí, y teniendo en cuenta los sustanciales cambios de fondo que éste presenta respecto del
Código del Estatuto Personal y de las Sucesiones, más conocido como antigua Mudawwana (1957-58), optamos
por referirnos al primero por sus siglas CF. Véase JUNTA DE ANDALUCÍA. La nueva Mudawwana marroquí:
entre tradición y modernidad (Traducción comentada del Código de Familia de 2004), Sevilla 2009.
BORJA, Anabel, op.cit, 2005.
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190
contratos, testamentos o poderes)53. El documento propuesto pertenece al segundo
grupo.
Desde el punto de vista árabe54, el acta objeto de este estudio se puede clasificar
del siguiente modo:
Acta notarial de celebración de matrimonio civil
Género
Documento notarial
Fuente emisora
Notario
Receptor
Administración
Función
Instructiva y expositiva, con la finalidad de establecer y legalizar las relaciones
particulares según lo establecido y guardar los derechos a largo plazo.
Modalidad
Escrito para ser leído en silencio
Tenor
Estándar fosilizado
Elaborado a partir de la propuesta de Abdel-Aziz sobre categorización de tipologías textuales jurídicas egipcias
(2006)
En lo referente a la función de la traducción, hay que tener presente que este tipo
de textos han de surtir efecto en una comunidad lingüística y cultural y un ordenamiento jurídico propio, distintos de aquella en la que se originaron, y funcionar de
facto como un original55, demostrando el vínculo matrimonial entre dos personas.
Como se observará más adelante, para que esto ocurra y el destinatario -en este caso
la administración marroquí-, el traductor deberá prestar especial atención al lenguaje
empleado y la corrección terminológica.
Frente a otros países árabes donde los documentos matrimoniales suelen ser certificados expedidos por un juez o un funcionario autorizado, en el caso de Marruecos
se emiten en forma de actas notariales. La razón de esta singularidad puede deberse a
que la institución que se encarga de la fe pública en Marruecos, el notariado, destaca
por un desarrollo histórico propio, diferenciado del de los países europeos, y de otros
países árabes. Marruecos no formó parte del Imperio otomano, mientras que el resto
de los países árabes conjugaron las estructuras administrativas heredadas de la época
colonial con el sistema jurídico otomano, cuya codificación del derecho de familia
comienza con la Ley Otomana de Derecho de Familia promulgada en 191756, que
estableció la posibilidad de que las cuestiones relacionadas con la familia se desarrollaran bajo la jurisdicción de tribunales de derecho civil o de derecho islámico.
Marruecos siguió un sistema notarial propio influido por el desarrollo que este
había tenido en al-Ándalus, y por la jurisprudencia desarrollada por la escuela malikí. Desde un punto de vista histórico, el notariado adular tiene sus raíces en la re53
54
55
56
BORJA, Anabel, El texto jurídico inglés y su traducción al español. Barcelona, Ariel, 2000
ABDEL-AZIZ, Khaled, “Clasificación del texto jurídico árabe (desde el punto de vista de la traducción jurídica”, Quaderns: Revista de traducción, (2006), vol. 13, 169-179.
MAYORAL ASENSIO, Alberto, “Las fidelidades del traductor jurado: una batalla indecisa”, en Traducir para
la justicia. M.C. Feria García (ed.) (1999). Disponible en: http://www.ugr.es/~greti/puentes/puentes2/07-articulo.pdf [Último acceso el 21 de noviembre de 2019].
TORRES GARCÍA, Ana. op. cit. p. 380.
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191
cepción de las primeras revelaciones del Corán, por las que ordena a los musulmanes
formalizar sus actos y negocios jurídicos en dos azoras (2: 282 y 24: 33) 57. Así, su
creación tuvo por objeto proteger y defender los derechos y bienes de los individuos,
regular y otorgar seguridad jurídica a las relaciones familiares mediante instrumentos públicos y dejar constancia de ellas de forma escrita. Para Nebot58, el origen coránico del lenguaje de especialidad de la jurisprudencia en árabe determina que los
campos semánticos, así como los términos jurídicos especializados, tengan que preservar una relación íntima e indestructible de significado con el lenguaje coránico,
así como sus aplicaciones y derivaciones prácticas.
Si los instrumentos notariales hasta el siglo X se caracterizaron por ser cortos,
concisos y resumidos, a partir de esa fecha, empiezan a presentar grandes cambios
en al-Ándalus y Marruecos, siendo la característica más importante de los instrumentos de aquella época las anotaciones marginales y las explicaciones jurisprudenciales, aunque estas desaparecen en el siglo XIII d. C., apareciendo publicaciones
que describen el objeto de cada instrumento.
Los Protectorados francés y español59 reconfiguraron el sistema judicial vigente
en Marruecos hasta principios del siglo XX con el fin de afianzar su control sobre el
territorio, hecho que también afectó al sistema notarial, al añadir a los notariados
islámico y rabínico ya existentes, el notariado latino, con el objeto de cubrir las necesidades de los colonos. Los gobiernos coloniales justificaron su creación alegando
la necesidad de modernizar la administración indígena, lo que, en la práctica, favoreció a los súbditos europeos que vivían en Marruecos y se sometían a la competencia de los tribunales franceses, españoles y mixtos —en el caso de Tánger que era
una zona internacional—. La creación de esta institución paralela a la del notariado
tradicional islámico cabe inscribirse dentro de la política colonial de sustitución y
suplantación de las lenguas vernáculas, particularmente por lo que hace referencia a
los países del Magreb, que en el momento de su colonización ya disponían de una
lengua de uso jurídico y administrativo, el árabe.
En el caso francés, este carácter intervencionista alcanzaría su máxima expresión
con la firma el 16 de marzo de 1930 del denominado dahir bereber por el que se
pretendía dividir el territorio marroquí bajo el mando de Francia en dos zonas jurídicamente diferenciadas independientes: la parte urbana y arabizada se regiría por la
šarî‘a —la ley islámica—, estaría bajo el control del sultán, y los crímenes serían
juzgados por el Tribunal Cheránico, mientras que, en las zonas rurales, de mayoría
bereber, pasaría a aplicarse el derecho consuetudinario, con el objetivo de ir introduciendo las leyes francesas. Tras las protestas, en 1936 se firma un dahir derogando el
artículo 6 del primero, que instauraba el sistema legal francés en las zonas bereberes.
Con el dahir de 7 julio de 1914, a la vez que se regulaba la profesión del notariado
adular o islámico, se crea de facto el notariado latino para tratar los asuntos de los colonos, y de este modo y al igual que ocurrió en otras colonias, los asuntos relacionados
con la familia, la herencia, etc., quedaron relegados a los tribunales de jurisdicción
religiosa. Durante la época colonial, la organización del notariado adular se fue regulando a través de diversos dahires o decretos reales (1938, 1943) hasta el dahir de 7 de
57
58
59
.2014 ) (مجلة إلكترونية، المسطرة المدنية- القانون المدني، عدنان،المرابط
ROSER NEBOT, Nicolás . op.cit.
Para un análisis del Protectorado español y de los traductores, véase, entre otros, FERIA, Manuel, “Conflictos
de legitimidad jurídica en Marruecos. El impacto del Protectorado”, El protectorado español en Marruecos:
gestión colonial e identidades, F. Rodríguez y H. de Felipe (eds.) (2002).
192
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febrero de 1944, por el que se hizo obligatoria la incorporación de una nota marginal
con las referencias necesarias (tomo, folio, asiento y fecha) al instrumento original.
La normativa colonial no se modificó hasta 1982, veinte años después de haber
alcanzado la independencia, cuando se incluyó como novedades la forma de otorgar
y redactar los testimonios, mientras que el dahir de 1983 regulaba el nombramiento
de los adules, la supervisión de su trabajo, la custodia y redacción de los instrumentos, así como los honorarios de los adules, entre otras. El Dahir 16.03, en vigor desde
el día 03 de marzo de 2006, es la que regula el acceso y el ejercicio de la profesión,
la competencia territorial de los adules, sus responsabilidades y la creación de un
colegio nacional de adules y colegios regionales. Por último, desde enero de 2018,
las mujeres ya pueden ejercer como notarias en el marco del notariado islámico.
El actual notariado marroquí es por lo tanto heredero del sistema colonial y está
formado por una estructura bipartita: el notariado confesional, que se encarga de
asuntos relativos al estatuto personal y sucesorio, y el notariado de derecho civil,
encargado de contratos relativos al derecho común. Dentro del notariado confesional
se distinguen a su vez tres categorías60:
– El notariado adular, competente para los asuntos religiosos de la población
musulmana. Para tener validez, las actas adulares necesitan de la firma de dos
notarios y la homologación de un juez.
– El notariado que se encarga de la fe pública rabínica, que sigue las disposiciones de la del Talmud y el código Caro61. Los instrumentos notariales deben
estar firmados por dos soffrims y homologados por el juez rabínico62.
– El notariado latino, que es competente para los actos de personas que no pertenecen a los grupos anteriores, también denominado notariado europeo. Se
encargan de la redacción de contratos notariales y no necesitan de homologación alguna.
En el proceso de construcción nacional, y en aras de la unión nacional, las cuestiones relativas a la familia y a la religión fueron asumidas como parte de las bases de la
reafirmación identitaria y un factor legitimador del Estado, por lo que se consideraron
prioritarias y trataron de blindarse institucionalmente tanto por parte de la élite nacionalista del partido Istiqlal como por la élite religiosa63; de hecho, el Código de Estatuto Personal y de Sucesiones (1957-1958), la antigua Mudawwana, fue aprobado con
anterioridad a la constitución marroquí, que data de 196264, y su contenido y orientación fue el resultado de las deliberaciones de un comité de diez expertos entre los que
figuraban entre autoridades religiosas y funcionarios del ministerio de Justicia65.
60
61
62
63
64
65
Para un análisis exhaustivo del Notariado marroquí, véase FERIA GARCÍA, Manuel, “Convivencia e interacción de tradiciones jurídicas en Marruecos: la fe pública: sofrims, notarios latinos y adules”, en Awraq: Estudios
sobre el mundo árabe e islámico contemporáneo, 24 (2007) 241-286.
Para un estudio más detallado del notariado rabínico, ZAFRANI, Haïm, Deux mille ans de vie juive au Maroc:
histoire et culture, religion et magie, Casablanca 2000.
En el caso de Casablanca, es la cámara hebrea del tribunal de primera instancia la competente para su homologación.
TORRES GARCÍA, Ana, op. cit. p. 380.
EL HAMOUD, Omar, “La constitución marroquí desde el punto de vista de los jóvenes”, en LEX SOCIAL-Revista de los Derechos Sociales 1 (2011) 228.
MERNISSI, Fatima, Beyond the Veil. Male-Female Dynamics in Modern Muslim Society. Revised edition,
Bloomington 1987.
Soto Aranda, B.; Kabbour, R. Anaquel estud. árabes 31 2020: 179-205
193
En él se impuso una visión conservadora de la sociedad y una lectura restrictiva
de la jurisprudencia malikí respecto de los principios que sostienen la familia y el
papel de la mujer, que era diametralmente opuesto al papel que habían desempeñado
las mujeres en la lucha por la independencia, organizadas en torno a la sección femenina del partido Istiqlal y de la asociación Akhawat Saffa.
Las actas matrimoniales se regulan por este código, por lo que las modificaciones
sufridas por la Mudawwana en 1993 y la nueva redacción del CF de 2004 han afectado en distinto grado a las actas notariales de matrimonio y a su expedición. Así, la
reforma de 1993 introdujo la obligatoriedad de registrar el matrimonio (Art. 43) para
su reconocimiento oficial y reguló el procedimiento administrativo a seguir. Asimismo, introdujo la obligatoriedad de registrar el divorcio (Arts. 60, 80 y 81) para su
reconocimiento oficial66. Ya la Mudawwana de 1958, en su Art. 81 decía:
1º. El acta de repudio debe mencionar, para cada uno de los esposos, su nombre,
filiación, domicilió y su identificación mediante el documento individual o un certificado administrativo de identidad. 2º Debe hacer referencia al acta de matrimonio, con indicación del número, folio y fecha, precisando que esta acta se encuentra debajo o detrás del acta de repudio.
Esto supuso de facto que muchos matrimonios anteriores a la promulgación de la
Mudawwana tuvieran que reconocerse mediante actas alternativas, tales como el
acta testifical de continuidad en matrimonio.
En 1958, la antigua Mudawwana estableció que las actas notariales de matrimonio debían incluir la siguiente información:
1. Los nombres, apellidos, filiación, domicilio e identidad completa de los esposos, con mención que están en posesión de sus facultades, así como el nombre
del Wali.
2. La fecha del acta de matrimonio, el lugar de realización, con indicación de que
los esposos y del tutor actúan con conocimiento de causa.
3. Todas las referencias útiles relativas a la edad de la esposa, si es virgen o no,
si tiene o no padre, provista o no de un tutor testamentario, si es divorciada o
viuda, y si ha observado la idda.
4. La mención del certificado administrativo con su número de orden.
5. La cantidad de la dote67, precisando lo que debe entregarse, si se entrega delante de los adules o si solo hay reconocimiento ante estos de una entrega anterior.
6. La firma de los adules y la homologación del juez con su sello.
El Art. 43, por su parte, señalaba que el acta de matrimonio se consignaría en el
registro que existe para tal efecto en el tribunal. Un ejemplar de esta acta debía enviarse al Registro Civil: el original del acta se debía enviar a la esposa o a su repre66
67
TORRES CALZADA, Katjia, “Poder y religión en los 50 años del Reino de Marruecos”, 50 años del Reino de
Marruecos: análisis sobre el Marruecos Actual. K. Torres Calzada (ed.), (2006), p. 22.
La dote es uno de los requisitos para que el matrimonio islámico sea válido. Se trata de un regalo con el que debe
obsequiar el esposo a la esposa para formalizar la unión matrimonial. Puede aplazarse la entrega de parte ; este
hecho deberá mencionarse expresamente en el acta de matrimonio. El término صداقse traduce por dote en español, aunque en puridad se trata de un anisomorfismo, pues en la cultura popular española, la dote corresponde
al ajuar que lleva la esposa al matrimonio).
194
Soto Aranda, B.; Kabbour, R. Anaquel estud. árabes 31 2020: 179-205
sentante en un período máximo de 15 días a contar desde su fecha. El esposo tenía
derecho a tener una copia del acta.
En el CF de 2004, en el capítulo denominado Título VI. De los procedimientos
administrativos y formalidades para la conclusión (establecimiento) del acta matrimonial, indica que el expediente para el matrimonio debe ser autorizado por el juez
de familia correspondiente, quien autorizará a los notarios del partido judicial en el
que se va a registrar el acta, a levantarla. En ella, los notarios deberán consignar68:
1. La referencia a la autorización del juez, su número y fecha, así como el número de orden del expediente que contiene los documentos proporcionados
para el matrimonio y el Tribunal en el que se encuentran en depósito.
2. El apellido y el nombre de los dos esposos, el domicilio o el lugar de residencia de cada uno ellos, el lugar y la fecha de nacimiento, los números de su
carné de identidad nacional o lo que proceda y su nacionalidad.
3. El nombre del tutor cuando proceda.
4. El consentimiento mutuo de los dos contrayentes que gozan de capacidad, de
discernimiento y la libertad de elegir.
5. En caso de poder otorgado para concluir un matrimonio, el nombre del mandatario, el número de su carné de identidad nacional y la fecha y el lugar en
el que se otorgó el poder.
6. La referencia a la situación jurídica del que o de la que ya haya contraído un
matrimonio.
7. El importe de la dote cuando se fija, precisando la parte pagada de antemano
y aquella al vencimiento de un plazo, y si su percepción tuvo lugar ante los
adules o por reconocimiento. Las cláusulas estipuladas por las dos partes.
8. Las firmas de los esposos y del tutor, cuando proceda.
9. El apellido y nombre de los adules y la firma de cada uno ellos y la fecha a la
en la cual concluyeron el acta.
10. La homologación por el juez, con su sello, del acta de matrimonio. La lista
de los documentos constitutivos del expediente del acta de matrimonio, así
como su contenido, pueden ser modificados y completados por decreto del
Ministro de Justicia.
A ello hay que añadir referencias que suelen aparecer en las actas notariales a los
certificados de estado civil y médico que han de presentarse para completar el expediente judicial. Por último, el Art. 68 señala que el contenido del acta de matrimonio
ha de transcribirse en el registro existente a tal efecto, en la Sección de Familia. Un
testimonio de este se remitirá al Encargado del Registro Civil del lugar de nacimiento de los esposos, acompañado de un acuse de recibo, en el plazo de quince días a
partir de la fecha de homologación del matrimonio por el juez. No obstante, si uno
de los dos esposos o los dos no hubieran nacido en Marruecos, dicho testimonio se
remitirá al Procurador del Rey del Tribunal de Primera Instancia de Rabat. El Encargado del Registro Civil debe incluir todas las menciones del testimonio como anotaciones marginales de la inscripción de nacimiento de cada uno de los cónyuges. La
forma, el contenido del registro previsto en el primer párrafo (arriba), así como las
menciones antes citadas, son fijados por resolución del Ministro de Justicia.
68
Se ha utilizado la traducción de la Junta de Andalucía (2009).
Soto Aranda, B.; Kabbour, R. Anaquel estud. árabes 31 2020: 179-205
195
El lenguaje notarial árabe marroquí comparte con el lenguaje notarial español69
una serie de rasgos, entre los que cabe mencionar los siguientes:
Uso construcciones nominales del tipo ( المنتصبين لإلشهادasignados para [recibir]
las declaraciones), ( الحامل للبطاقة الوطنيةtitular del documento nacional de identidad),
( تامة العقلcon sus facultades mentales) y locuciones prepositivas, como )من والده(ها
(hijo/a de), ( على صداق مباركcon una dote…).
En lo referente al léxico, destaca el uso de tecnicismos como نسخة رسم ثابت مستقل
(copia de una certificación auténtica e independiente), ( صداقdote) y ( وليtutor),
como de palabras de uso común que en este tipo de documentos adquieren un significado especializado, tal es el caso de ( قبل البناءantes de la consumación del matrimonio) y ة/( الساكنdomiciliado/a). El término الكتابse refiere al Corán.
Es habitual el recurso a la sinonimia (total o parcial), es el caso de expresiones
como ( الحل للنكاحapta para el matrimonio), así como al uso de dobletes y tripletes para
reforzar el significado de una determinada expresión. Es el caso de بإذنها ورضاها
( وتوكيلهاcon la autorización de la esposa), ( بعد باإليجاب والقبولacuerdo y aprobación).
Fórmulas de respeto. Es el caso de ( سيدseñor/D.) para el esposo, ( آنسةseñorita) y
( سيدةseñora) para la esposa; expresiones formularias del tipo ( عبد ربه تعالىlit. siervo
de su Señor, Todopoderoso), para referirse a los notarios con respeto.
Por el contrario, en los instrumentos notariales se usa el tiempo perfectivo, que
generalmente tiene valor de pasado (ej.: حضر،)تلقى, si bien suelen traducirse en función de los requerimientos de la lengua meta. También es habitual que las actas no
hagan uso de signos de puntuación como la coma, el punto y coma y el punto, como
puede observarse en el siguiente ejemplo extraído de un acta fechada en 2010:
الحمد هلل وحده بناء على اإلذن بتوثيق عقد الزواج من قاضي األسرة المكلف بالزواج رقم *** بتاريخ
ملف بمستندات الزواج رقم *** المودع بقسم قضاء األسرة بالمحكمة االبتدائية بالحسيمة27/01/2010
نحن *** و *** العدالن المنتصبان لإلشهاد بدائرة المحكمة المذكورة تلقينا بمذكرة الحفظ ألول شهيديه رقم
على الساعة الرابعة والنصف عشيا من يوم األربعاء حادي عشر صفر152 صفحة167 تحت عدد32
)...( الخير
aunque las actas expedidas por el Consulado de Marruecos sí hacen uso de ello,
como puede observarse en el siguiente ejemplo extraído de un acta de 2001.
) نهار يوم الجمعة رابع محرم عام اثنين و عشرين و أربعمائة وألف10( ففي الساعة العاشرة،الحمد هلل وحده
تزوج على بركة هللا تعالى السيد *** المولود بابني،للهجرة الموافق لثالثين مارس سنة ألفين وواحد للميالد
حسب بطاقة شخصية للحالة المدنية لموجز عقد،*** والدته ميلودة،1975 أكتوبر7 حديفة الحسيمة بتاريخ
)...( ،.*** المؤرخة في،*** بطاقة الوطنية رقم، جنسيته مغربي،الوالدة رقم ***؛ مهنته أجير باسبانيا
En ocasiones, los notarios de tipo tradicional o adular —ya que trabajan en parejas— recurren a descripciones físicas de los contrayentes de tono arcaizante como
las siguientes: ( وصفها مائلة للبياض أسيلة متوسطة االمتالء والطول محجوجبةsu descripción:
blanca, de nariz erguida, de talla mediana y con cejas pronunciadas).
Por los motivos antes expuestos, era habitual que el tenor de las actas notariales
soliera tener numerosas referencias religiosas, como citas del Corán, aunque dichos
elementos intertextuales varían, no tanto en función del año de emisión del acta,
69
ETXEBARRIA AROSTEGUI, Maitena (1997), “El lenguaje jurídico y administrativo. Propuestas para su modernización y normalización”, en Revista Española de Lingüística [En línea]. Nº 27, 2, disponible en: http://
www.sel.edu.es/pdf/jul-dic-97/27-2-Etxebarria.pdf [Último acceso el 21 de noviembre de 2019].
196
Soto Aranda, B.; Kabbour, R. Anaquel estud. árabes 31 2020: 179-205
como del estilo particular adoptado por los notarios en cada caso. Así, hay actas recientes que sí las incluyen y otras que no.
Las actas notariales de matrimonio venían caracterizándose por la inclusión de la
basmala70 (en el nombre de Dios, el Clemente, el Misericordioso), como elemento de
apertura, rasgo que las diferencia de las sentencias judiciales, cuyo encabezamiento
es ( باسم جاللة الملك وطبقا للقانونen nombre su majestad el Rey y de conformidad con la
Ley). No obstante, este elemento singularizador no aparece de forma sistemática en
todas ellas, siendo que, en algunos casos como las actas expedidas por el Consulado
de Marruecos en Madrid, aparecen en forma de logo debajo del escudo del país.
Del mismo modo, se recurre a fraseologismos de marcado carácter religioso, entre otros, وهللا سبحانه يؤلف بينهما, (que Dios Todopoderoso les congenie), en referencia a
los cónyuges, y por el uso de expresiones formularias del tipo ( الحمد هلل وحدهloor a
Dios Único). En el caso de esta última, por ejemplo, Mayoral se refiere a ella como
una fórmula de salutación, una referencia intertextual significativa en un texto árabe,
pero cuya referencia se pierde al traducirla en un contexto cultural no islámico, por
lo que muchos traductores omiten su traducción71.
Este tipo de referencias religiosas en forma de basmala, citas del Corán, etc., caracterizadoras de las actas marroquíes, no aparecen en las certificaciones de matrimonio de otros países como es el caso de Egipto, aunque el Código de Estatuto
Personal egipcio también tenga una fundamentación religiosa, luego se trata de un
elemento discursivo, meramente intertextual en este tipo de documentos.
4. Propuesta de traducción comentada
Las escrituras públicas, según el artículo 144-2 del Reglamento Notarial, tienen
como contenido las declaraciones de voluntad negociables de los interesados, los
actos jurídicos que impliquen prestación de consentimiento, los contratos y negocios
jurídicos de todas las clases. Su finalidad principal es la dación de consentimiento.
En el caso propuesto, los notarios tienen potestad para casar y divorciar en virtud de
la Ley 15/2005, de 12 de julio, de la Jurisdicción Voluntaria que:
opta por atribuir el conocimiento de un número significativo de asuntos que tradicionalmente se incluían bajo la rúbrica de la jurisdicción voluntaria a operadores
jurídicos no investidos de potestad jurisdiccional, tales como los secretarios judiciales, Notarios y registradores de la propiedad y Mercantiles, compartiendo con
carácter general la competencia para su conocimiento. Estos profesionales que
aúnan la condición de juristas y de titulares de la fe pública reúnen sobrada capacidad para actuar, con plena efectividad y sin merma de garantías, en alguno de los
actos de Jurisdicción Voluntaria que hasta ahora encomendaban a los Jueces […]
El acta de matrimonio civil tiene una estructura fija formada por los siguientes
apartados:
70
71
Se trata de una fórmula de invocación que constituye el primer versículo de todas las azoras del Corán, excepto
en el caso de la núm. 9.
MAYORAL ALONSO, Roberto, Translating Official Documents (Translation Practices Explained), Londres,
2003.
Soto Aranda, B.; Kabbour, R. Anaquel estud. árabes 31 2020: 179-205
197
– Menciones preliminares. En este apartado se hace constar el tipo de instrumento público, el número de protocolo, el lugar y la fecha de su autorización
y la identificación del notario que otorga la fe pública.
– Comparecencia. En él, se hacen constar los datos personales de los otorgantes
y, en su caso, de los demás intervinientes (testigos, abogados, etc.). Incluye
también la identificación de éstos por los medios establecidos en la legislación
notarial, así como la reseña y juicio de suficiencia de la representación alegada
y el juicio de capacidad notarial para el acto documentado.
– Exposición. Este apartado hace referencia a los antecedentes necesarios para
la documentación del acto. Por ejemplo, en caso de que contenga bienes inmuebles, estos deben ser descritos conforme lo dispuesto en la legislación
notarial, hipotecaria y especial.
– Estipulaciones y otorgamiento. Es el apartado más importante de las escrituras, ya que contiene las declaraciones de voluntad, así como los pactos, cláusulas y condiciones que las partes desean elevar a público.
– Autorización. Contiene la referencia a las advertencias y reservas legales y
fiscales, la lectura del documento (por el notario o por la parte otorgante), el
consentimiento de los otorgantes y la cláusula de autorización notarial. Al final, se transcriben la firma de los otorgantes y la firma, signo, rúbrica y sello
del notario que da fe de la escritura pública.
A continuación, presentamos una propuesta de traducción comentada, vertebrada
en torno al desarrollo de: a) la competencia extralingüística (con especial atención a
los conocimientos jurídicos y culturales) y b) la competencia de transferencia (macroestructura del TO y del TM; terminología y aspectos formales exigidos por la legislación del ordenamiento jurídico meta), a partir de la propuesta de Borja (2005)
sobre competencias para el ejercicio la traducción jurídica.
4.1. Menciones preliminares
Texto original:
ESCRITURA DE CELEBRACIÓN DE MATRIMONIO CIVIL -------------------NÚMERO (Número de Protocolo)72 -----------------------------------------------------En Madrid, a (Fecha de autorización). ---------------------------------------------------Ante mí, IÑIGO CASLA URIARTE, Notario de Madrid y de su Ilustre Colegio
con residencia en esta Capital, --------------------------
Propuesta de traducción:
---------------------------------------------------------------------------- عقد زواج مدني رسمي
-------------------------------------------------- . من سجل الحفظ00000000 ض ِّمن تحت عدد
ُ ------------------------------------------------------------------------ .) في (تاريخ تلقي العقد،مدريد
----- ، والمقيم بنفس هذه المدينة، الموثق بهيئة الموثقين في مدريد، نحن األستاذ إينييغو كاسال أوريارتي،أمامنا
72
Los elementos marcados en cursiva en el texto original tienen carácter orientativo para el notario o su(s)
ayudante(s) a la hora de elaborar un instrumento. En la versión árabe, estos elementos irán de igual modo en
cursiva, pero en algunos casos, irán entre paréntesis para evitar confusiones.
Soto Aranda, B.; Kabbour, R. Anaquel estud. árabes 31 2020: 179-205
198
Se ha optado por una traducción funcional del título, en la que se refleja en árabe
que se trata de un “matrimonio civil” ( )زواج مدنيcelebrado de forma oficial mediante
“escritura pública” ()عقد رسمي. Como ya se ha indicado, En Marruecos, de las certificaciones de matrimonio se encargan los notarios, también denominados adules,
requiriendo la autorización previa y la homologación posterior de un juez, luego se
consideran de por sí escrituras notariales; a diferencia del resto de países árabes, que
han optado por modelos semejantes a la certificación de matrimonio expedida por el
Registro Civil español. Como ya se ha mencionado, el CF marroquí (2004)73, permite a los ciudadanos marroquíes residentes en el extranjero contraer matrimonio según la forma prevista por las leyes del país de residencia, incluyendo el matrimonio
civil74.
4.2. Comparecencia:
Texto original:
------------------------------------- COMPARECEN ------------------------------------Los contrayentes: ---------------------------------------------------------------------------*, mayor de edad, nacido en *, el día *, hijo de don * y doña *, inscrito en el Registro Civil Central *, al tomo *, página *, de nacionalidad *, estado civil *, vecino
de *, con domicilio en calle *, número *. -----------------------------------------------Me exhibe NIF/CIF número *. -----------------------------------------------------------*, mayor de edad, nacido en *, el día *, hijo de don * y doña *, inscrito en el Registro Civil Central *, al tomo *, página *, de nacionalidad *, estado civil *, vecino
de *, con domicilio en calle *, número *. -----------------------------------------------Me exhibe NIF/CIF número *. ------------------------------------------------------------
Propuesta de traducción:
---------------------------------------------- حضر------------------------------------------------------------------------------------------------------------------------------------ :الزوجان
،) من والده (اسم األب،)الوالدة/ بتاريخ (تاريخ االزدياد،) المولود في (المدينة/ المزداد، الراشد،* السيد
،* الحامل للجنسية،* صفحة عدد،* كناش عدد،* المقيّد في السجل المدني المركزي،)ووالدته (اسم األم
--------------------- .* رقم،* سكناه في شارع،* المقيم في،) أرمل/ مطلق/ حالته العائلية * (عازب
-------------------------------------- .* رمز التعريف الضريبي رقم/ وأدلى برقم التعريف الضريبي
من والدها (اسم،)الوالدة/ بتاريخ (تاريخ االزدياد،) المولودة في (المدينة/ المزدادة، الراشدة،* والسيدة
الحاملة،* صفحة عدد،* كناش عدد،* المقيّدة في السجل المدني المركزي،) ووالدتها (اسم األم،)األب
------- ، * رقم،* سكناها في شارع،* المقيمة في،) أرملة/ مطلقة/ حالتها العائلية * (عازبة،*للجنسية
-------------------------------------- .* رمز التعريف الضريبي رقم/ وأدلت برقم التعريف الضريبي
Para proceder a la traducción de este apartado hay que tener en cuenta, en primer lugar, los valores asimétricos de los tiempos verbales en español y en árabe.
Las actas notariales en español recurren al presente simple para indicar que, en el
73
74
Puede consultarse en Junta de Andalucía: La nueva Mudawwana marroquí: entre tradición y modernidad. Traducción comentada del Código de Familia marroquí). https://www.juntadeandalucia.es/export/drupaljda/1_2030_
nueva_mudawwana_marroqui.pdf
FOBLETS, Mª Claire y LOUKILI, Mohamed, “Mariage et divorce dans le nouveau Code marocain de la famille: quelles implications pour les marocains en Europe?”, en Revue Critique de droit international privé 3
(2006) p. 530.
Soto Aranda, B.; Kabbour, R. Anaquel estud. árabes 31 2020: 179-205
199
momento de levantar el acta, las personas concernidas o sus representantes legales
se encontraban presentes físicamente ante el notario. Sin embargo, la norma de
redacción de las actas marroquíes es el uso del pasado الماضيcon un valor perfectivo, que indica que las personas concernidas han hecho acto de presencia ante el
notario.
En segundo lugar, hay que tener en cuenta las peculiaridades de la terminología
marroquí, sobre todo en cuanto a términos como كناشy المزدادcuyo uso es más habitual que دفترy المولود, respectivamente. También se opta por la expresión )من والده(ها
para traducir `hijo/a de´, por ser de uso habitual en Marruecos. En cuanto a abreviaturas como “NIF” y “CIF”, se ha optado por una traducción explícita para diferenciar
entre Número y Código, aunque en España desaparece el denominado “Código de
Identificación Fiscal”, mediante la Orden EHA/451/2008, de 20 de febrero, por la
que se regula la composición del número de identificación fiscal de las personas jurídicas y entidades sin personalidad jurídica.
Texto original:
Y como testigos instrumentales idóneos, designados por los contrayentes: --------------------------------------------------------------------------------------------------------** ---------------------------------------------------------------------------------------------** ---------------------------------------------------------------------------------------------Identifico a los comparecientes por el medio supletorio del apartado c) del artículo
23 de la Ley Orgánica del Notariado, resultando sus circunstancias personales de
sus propias manifestaciones.
Propuesta de traducción:
----------------------------------------------------- : اللذان عينهما الزوجان،وشاهدا العقد المناسبان
---------------------------------------------------------------------------------------------- **
---------------------------------------------------------------------------------------------- **
على التعريف باألطراف الحاضرة أمامنا بالطريقة االستثنائية المنصوص عليها في، نحن الموثق،ونشهد
وأن ظروفهم الشخصية هي المستمدة من، من القانون التنظيمي المتعلق بالتوثيق32 النقطة (ج) من المادة
.تصريحاتهم
Los términos jurídicos más destacados en este apartado son testigos instrumentales
y medio supletorio. Respecto del primer término, hay que tener en cuenta que toda
reglamentación que organiza una sociedad o las relaciones entre sus miembros —el
derecho privado— dimana del marco cultural que le sirve de base, que en el caso del
texto meta es el islam. Así, a pesar de tener una equivalencia funcional en árabe, por la
que optamos en la traducción, hay que saber que el término testigo difiere conceptualmente en el derecho marroquí (inspirados en el Derecho islámico), pues los testigos
instrumentales ( )شهود العقدhan de ser dos hombres o un hombre y dos mujeres, mientras
que en el Derecho español no se establece esta distinción por razón de sexo. Por ello,
en el momento de presentar el acta ante la administración marroquí, será requerida la
presencia y declaración de dos testigos varones, que se adjuntará al expediente administrativo correspondiente, pues su presencia “es indispensable ya que se trata de una
exigencia ad solemnitatem y no de una simple cuestión formal”75 para que el acto
75
BLÁZQUEZ RODRÍGUEZ, Irene, op. cit. p. 171.
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tenga validez jurídica . En el segundo caso, se ha optado por una traducción funcional,
“”بالطريقة االستثنائية المنصوص عليها, para desambiguar el significado.
Texto original:
INTERVIENEN: En su propio nombre e interés. --------------------------------------JUICIO DE CAPACIDAD: A los efectos prevenidos en el artículo 98.1 de la Ley
24/2001 de 27 de diciembre de Medidas Fiscales, Administrativas y de Orden
Social, yo, el Notario, hago constar, manifestando ambos contrayentes que ninguno de ellos está afectado por anomalías o deficiencias psíquicas o sensoriales que
afectan a sus capacidades cognitivas, volitivas o sensoriales necesarias para prestar el consentimiento matrimonial, que tienen, a mi juicio, bajo mi fe y responsabilidad, en el concepto en que intervienen, capacidad legal, legitimación y facultades suficientes para otorgar la presente ESCRITURA DE CELEBRACIÓN -----------------------------------------
Propuesta de traducción:
-------------------------------------------------------- . باسمهم ولحسابهم الخاص:وحيث يتصرفون
--- ديسمبر27 الصادر بتاريخ،24/2001 من القانون رقم98.1 طبقا ً ألحكام المادة:تقييم األهلية القانونية
تحت مسؤوليتنا وبنا ًء على تصريح، نحن الموثق، نشهد، واإلدارية واالجتماعية،بشأن التدابير الضريبية
، على أنهما ال يعانيان من أي تشوهات أو عاهات نفسية أو حسية قد تؤثر على قدراتهما اإلدراكية،الزوجين
يتمتعان باألهلية، بصفتهما المذكورة،واإلرادية والحسية الضرورية إلصدار اإليجاب والقبول وأنهما
--------------------------- ، ولهذا الغرض، والمشروعية والصالحيات الكافية إلبرام هذا العقد،القانونية
El término `intervención´ refiere a la calidad en la que intervienen las partes en el
otorgamiento de la escritura; se traduce por أهلية التصرفy no debe confundirse con
( أهلية األداءcapacidad de ejercicio) ni con ( أهلية الوجوبcapacidad de goce). Se trata de
conceptos jurídicos distintos, contemplados en los códigos civiles de España y Marruecos. En cuanto al término jurídico `juicio de capacidad´, se ha traducido por una
perífrasis, al no existir un término equivalente funcionalmente en la cultura meta.
Todo notario —sea de Marruecos o cualquier otro país— está obligado a asegurarse
de la capacidad de actuación, أهلية التصرف, de las personas que comparecen ante ellos,
aunque en Marruecos no se incluya en este tipo de documentos un título explícito
bajo el que el notario tiene que asegurar que según su criterio los intervinientes tienen esta capacidad. En cuanto al último término jurídico, consentimiento matrimonial, se ha traducido por un fraseologismo característico del lenguaje notarial marroquí, صدور اإليجاب والقبول.
4.3. Exposición
Texto original:
---------------------------------------- EXPONEN: ---------------------------------------I.- Que DON * y DOÑA *, han tramitado expediente matrimonial previa acreditación de la concurrencia de los requisitos legales necesarios para contraer matrimonio, en el Registro Civil Único de Madrid, y autorizado por Auto*, que es firme, del
Magistrado Encargado del citado Registro Civil Único de Madrid, el día *, donde
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resultó acreditada la concurrencia de los requisitos legales de capacidad para contraer matrimonio, así como la ausencia de impedimentos, su dispensa en su caso, o
cualquier otro obstáculo para la celebración del matrimonio proyectado.Me exhiben testimonio del auto que autoriza el matrimonio, que incorporo a la
presente matriz. -----------------------------------------------------------------------------II.- Que el expediente matrimonial se encuentra vigente a la fecha del otorgamiento de la presente escritura. -----------------------------------------------------------------III.- Que DON * y DOÑA * se ratifican en todo lo manifestado y acreditado en
dicho expediente, Y MANIFIESTAN que, después de concluido el mismo, no ha
sobrevenido causa ni circunstancia alguna modificativa de todo lo declarado. ----IV.- Los testigos instrumentales MANIFIESTAN, bajo su responsabilidad, que
conocen a los contrayentes, que les consta su libertad de estado y la aptitud legal
de los mismos para celebrar el matrimonio proyectado. ------------------------------V.- Que DON * y DOÑA *, MANIFIESTAN expresa y libremente ante los testigos instrumentales, su VOLUNTAD de celebrar el matrimonio proyectado, con
arreglo a lo dispuesto en la ley española. ---------------------------------------VI.- Y expuesto cuanto antecede, de conformidad con lo dispuesto en el artículo
58 del Código Civil y 52 de la ley del Notarial. ----------------------------------------
Propuesta de traducción:
-------------------------------------------- يصرحان------------------------------------------- أنجزا ملف مستندات الزواج بعد اإلدالء بما يثبت استيفاءهما للشروط القانونية،* أن السيد * والسيدة-.1
الذي أصبح،* المأذون به بموجب القرار، في السجل المدني الوحيد في مدريد،الضرورية إلبرام الزواج
والمثبت الستيفاء،* بتاريخ، الصادر عن القاضي المكلف بالسجل المدني الوحيد في مدريد،غير قابل للطعن
أو االستثناء من أحد تلك الموانع إن، وكذا انتفاء موانعه،الشروط القانونية المتعلقة باألهلية إلبرام الزواج
---------------------------------- . وعدم وجود أي عائق آخر إلبرام الزواج موضوع هذا العقد،وجدت
------------------ . الذي نرفقه بأصل العقد،وأدلى الزوجان بنسخة مصادق عليها من قرار اإلذن بالزواج
------------------------- . أن ملف مستندات الزواج ما زال ساري المفعول عند تاريخ تلقي هذا العقد-.2
بعد االنتهاء من، ويصرحان أنه، أن السيد * والسيدة * أكدا على تصريحاتهما الثابتة في الملف المذكور-.3
------------------------------------ . لم يقع أي سبب أو ظرف يؤدي إلى تعديل ما صرحا به،إجراءاته
وأنهما على علم بعدم ارتباطهما، أنهما يعرفان الزوجين، تحت مسؤوليتهما، أن شاهدي العقد يصرحان-.4
------------- .بآصرة زواج أخرى في الوقت الحالي وبأهليتهما القانونية إلبرام الزواج موضوع هذا العقد
على، عن رغبتهما في الزواج، صراحة وبكل حرية أمام شاهدي العقد، أن السيد * والسيدة * يعبران-.5
---------------------------------------------------------------------- .ضوء أحكام القانون اإلسباني
-------- . من قانون التوثيق25 من القانون المدني والمادة85 وطبقا ً ألحكام المادة، وبنا ًء على ما سبق-.6
En este apartado se observan una serie de términos jurídicos como `expediente
matrimonial´, `Registro Civil Único´, `Auto´, `Magistrado Encargado del Registro
Civil´, `capacidad para contraer matrimonio´, `impedimentos´, `dispensa´, `Exhibición´, `testimonio de auto´, `ratificación´, `libertad de estado´ y `aptitud legal´, que
varían en su clasificación entre administrativos y jurídicos, y en el grado de equivalencia, total o parcial, en la lengua meta. Asimismo, aparecen algunos términos ambiguos, como es el caso de testimonio de auto y libertad de estado, que se refieren a “una
copia de la sentencia que ha sido sellada y autenticada por el Letrado de la Administración de Justicia” y a “si el/la contrayente es soltero/a, divorciado/a o viudo/a”,
respectivamente. Como excepción, hemos optado por traducir literalmente el término
“Registro Civil Único” literalmente por السجل المدني الوحيد, en vez de utilizar el término
usado en Marruecos, مكتب الحالة المدنية, para evitar una posible confusión.
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Otro término que puede plantear dudas es el de dispensa. Según el DRAE, se
entiende como “excepción graciosa de lo ordenado por las leyes generales”. El artículo 48 del Código Civil español establece que:
el Juez podrá dispensar, con justa causa y a instancia de parte, mediante resolución
previa dictada en expediente de jurisdicción voluntaria, los impedimentos de muerte
dolosa del cónyuge o persona con la que hubiera estado unida por análoga relación
de afectividad a la conyugal y de parentesco de grado tercero entre colaterales. […].
Al entender que la dispensa es una situación excepcional en la que se autoriza
judicialmente la celebración de matrimonio, a pesar de la existencia de un impedimento para ello, se ha optado por traducirlo mediante la paráfrasis االستثناء من أحد تلك
الموانع إن وجدتque da cuenta del sentido del término. Por su parte, el término `Auto´
puede plantear un problema de traducción, porque a pesar de ser un término jurídico,
aquí no tiene aquí el significado de قرارo حكم, sino más bien el sentido de إذن, que es
el término utilizado en las actas adulares marroquíes.
4.4. Estipulaciones y otorgamiento
Texto original:
---------------------------------------- OTORGAN ---------------------------------------PRIMERO.- Yo, Notario procedo en alta voz a la lectura de los artículos 66, 67 y
68 del Código Civil que dicen lo siguiente: ---------------------------------------------“Artículo 66: --------------------------------------------------------------------------------Los cónyuges son iguales en derechos y deberes. -------------------------------------Artículo 67: ---------------------------------------------------------------------------------Los cónyuges deben respetarse y ayudarse MUTUAMENTE y actuar en interés de
la familia. ------------------------------------------------------------------------------------Artículo 68: ---------------------------------------------------------------------------------Los cónyuges están obligados a vivir juntos, guardarse fidelidad y socorrerse mutuamente. Deberán, además, compartir las responsabilidades domésticas y el cuidado y atención de ascendientes y descendientes y otras personas dependientes a
su cargo.” ------------------------------------------------------------------------------------SEGUNDO.- Preguntados por mí, el Notario, a cada uno de los contrayentes si
consiente en contraer matrimonio con el otro y si efectivamente lo contraen en este
acto, contestan ambos AFIRMATIVAMENTE. ----------------------------------------Así lo dicen y otorgan los comparecientes, habiendo realizado yo, Notario, las
reservas y advertencias legales. -----------------------------------------------------------
Propuesta de traducción:
----------------------------------------- : يتفقان على ما يلي------------------------------------------ : ونصها، من القانون المدني بصوت مرتفع86 و76 ،66 في تالوة المواد، نحن الموثق، بدأنا-.ًأوال
----------------------------------------------------------------------------------------- :66 «المادة
----------------------------------------------------------- .يتساوى الزوجان في الحقوق والواجبات
--------------------------------------------------------------------------------------- :76 المادة
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----------------- .يجب على الزوجين احترام ومساعدة بعضهما البعض والعمل من أجل مصلحة األسرة
--------------------------------------------------------------------------------------- :68 المادة
ويجب عليهما أيضا ً تقاسم. ويخلصا ويقدما المساعدة لبعضهما البعض،ًيتعين على الزوجين أن يعيشا معا
المسؤوليات المنزلية والرعاية واالهتمام باألصول والفروع وغيرهم من األشخاص الذين تقع مسؤولية
---------------------------------------------------------------------------- .»إعالتهم على عاتقهما
، كل واحد من الزوجين إذا كان موافقا ً على إبرام هذا الزواج وفي مجلس هذا العقد، نحن الموثق، سألنا-.ًثانيا
--------------------------------------------------------------------- .فصدر منهما اإليجاب والقبول
. بعد إشعارهما بالتحفظات والتحذيرات القانونية ذات الصلة، نحن الموثق،وتلك إرادة الحاضرين أمامنا
Según el DRAE, en su tercera acepción, “otorgar” significa “Disponer, establecer, ofrecer, estipular o prometer algo […] por lo común cuando interviene solemnemente la fe notarial”. Al no encontrar una equivalencia funcional en la lengua meta,
se ha optado por traducirlo teniendo en cuenta el significado de “acordar y estipular”,
un concepto usado en contratos árabes, اتفقا على ما يلي. A nivel fraseológico, se ha optado por una traducción funcional para las expresiones “así lo dicen y otorgan” y
“reservas y advertencias legales”, esto es, وتلك إرادة الحاضرين أمامناyالتحفظات والتحذيرات
القانونية, respectivamente.
4.5. Autorización
Texto original:
------------------------------------ AUTORIZACIÓN -----------------------------------Después de haber escuchado claramente a ambos contrayentes su expresa y libre
voluntad, y su determinación para contraer matrimonio ante mí, Notario, y en
presencia de los testigos, manifestando que consienten y efectivamente contraen
matrimonio con el otro en este acto, yo, Notario, de acuerdo con las potestades que
me han sido conferidas por el Ordenamiento Jurídico DECLARO UNIDOS EN
MATRIMONIO a DON * y DOÑA *. --------------------------------------------------SOLICITAN AMBOS COMPARECIENTES del Señor Encargado del Registro
Civil la práctica de la inscripción correspondiente, autorizando la presentación de
la presente escritura en el Registro Civil por ellos mismos o por un tercero. -------------------------------------Yo, el notario, remitiré una vez firmada esta escritura copia autorizada al Registro
Civil Único d Madrid. -----Y yo, Notario, DOY FE de la identidad de los otorgantes, de que, tienen a mi juicio, en el concepto en que intervienen, capacidad legal y legitimidad necesaria
para otorgar esta escritura, y de que el presente otorgamiento se adecua a la legalidad y a la voluntad debidamente informada de los otorgantes e intervinientes,
prestando libremente su consentimiento. ------------------------------------------------Advierto a los señores comparecientes de su derecho a leer este instrumento por sí,
y no lo usan; les realizo su íntegra lectura en alta voz; se ratifican en su total contenido; y firman conmigo. ------------------------------------------------------------------
Propuesta de traducción:
-------------------------------------------- موافقة--------------------------------------------- في هذا المجلس وبحضور، نحن الموثق،وبعد تلقي اإليجاب والقبول بشكل صريح من الزوجين أمامنا
، وبموجب الصالحيات التي يخولها لنا القانون، وتعبير كل منهما عن رغبته في الزواج من اآلخر،الشاهدين
-------------------------------------------------- .نعلن أن السيد * والسيدة * مرتبطان برباط الزواج
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ويوافقان،ويلتمس الطرفان الحاضران أمامنا من المكلف بالسجل المدني إدخال القيد المناسب في السجل
--------------------------------على تقديم هذا العقد إلى السجل المدني شخصيا ً أو عن طريق طرف ثا
----- . نسخة مطابقة لألصل من هذا العقد إلى السجل المدني الوحيد في مدريد، نحن الموثق،وسوف نحيل
، بصفتهما المذكورة أعاله، وأنهما يتمتعان، على تحديد هوية طرفي العقد والتحقق منها، نحن الموثق،ونشهد
باألهلية القانونية والمشروعية إلبرام هذا العقد وأن تلقي هذا العقد متوافق مع أحكام القانون وإرادة األطراف
----------------------------------------------- . المعبر عنها صراحة،المبرمة للعقد والمتدخلة في ذلك
فتنازال عن ممارسة هذا الحق؛،ً الحاضرين أمامنا بحقهما في تالوة هذا العقد شخصيا، نحن الموثق،وأشعرنا
---------------------- .وتلونا عليهما مضمون هذا العقد بصوت مرتفع؛ فوافقا على مضمونه ووقعاه معنا
En este apartado, cabe mencionar la traducción de términos como `expresa y libre
voluntad y determinación para contraer matrimonio´, traducidos por تلقي اإليجاب والقبول
بشكل صريح من الزوجينy تعبير كل منهما على رغبته في الزواج من اآلخر, respectivamente. Aquí
resulta necesaria una paráfrasis para hacer el mensaje más legible en la lengua meta,
respetando también el estilo utilizado en las actas adulares marroquíes. En el caso
del término ordenamiento jurídico (se trata del conjunto de leyes que tiene un país),
se entiende que el notario se refiere con ello a “la Ley”, por lo que traducido por ]...[
التي يخولها القانون. En el caso de las expresiones DECLARO UNIDOS EN MATRIMONIO, práctica de la inscripción y copia autorizada, se han traducido por equivalencias funcionales que hacen el texto más legible en la lengua meta.
La segunda parte de este apartado refiere a la Ley de Protección de Datos, y el
tenor del original es el siguiente:
Texto original:
PROTECCIÓN DE DATOS: -----------------------------------------------------------De acuerdo con lo establecido en la Ley Orgánica 15/1999, de 13 de Diciembre,
de protección de datos de carácter personal, y en el Real Decreto 1720/2007, que
aprueba su Reglamento de desarrollo, los intervinientes aceptan la incorporación
de sus datos y la copia del documento de identidad a los ficheros de la Notaría con
la finalidad de realizar las funciones propias de la actividad notarial y efectuar las
comunicaciones de datos previstas en la Ley a las Administraciones Públicas y, en
su caso, al Notario que suceda al actual al actual en la plaza. Puede ejercer sus
derechos de acceso, rectificación, cancelación y oposición en la notaría. En caso
de que se incluyan datos de personas distintas a los intervinientes, estos deberán
haberles informado, con carácter previo, del contenido de este párrafo. -----------Los comparecientes se dan por satisfactoriamente atendidos e informados por mí,
Notario, sobre el contenido, efectos y consecuencias del presente instrumento. ------De todo lo contenido en este instrumento público, extendido sobre COMPOSICIÓN PAPEL TIMBRADO, DOY FE. -----
Propuesta de traducción:
----------------------------------------------------------------------------------- :حماية البيانات
والمرسوم الملكي، المتعلق بحماية البيانات الشخصية،15/1999 طبقا ً ألحكام القانون التنظيمي
يوافق الطرفان الموقعان على هذا العقد على إدراج، الذي يصادق على نظامه التنفيذي،1720/2007
بياناتهم الشخصية ونسخة من بطاقة تعريفهم في الملفات التي يمسكها هذا الديوان ألغراض القيام بالمهام
للموثق الذي، وعند االقتضاء،الخاصة بنشاط الموثقين وتبليغ البيانات بمقتضى القانون لإلدارات العامة
وإلغائها، والموافقة عليها، ويحق لهم ممارسة حقوقهم في الولوج إلى بياناتهم.سيحل محل هذا الموثق
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وعند إدراج بيانات تخص أشخاص غير األشخاص المتدخلين في هذا.واالعتراض عليها أمام هذا الديوان
. ينبغي اطالعهم مسبقا ً بمحتوى هذه الفقرة،العقد
واآلثار المترتبة، أنهم تلقوا المعلومات الكافية حول مضمون هذا العقد، نحن الموثق،وأكد الحاضرون أمامنا
.عنه
---------------------------- . وبه اإلشهاد.وحرر هذا العقد الرسمي في * صفحات من الورق المدموغ
El acta también incluye una mención a la protección de datos de carácter personal. Para la traducción de términos relacionados con la Ley Orgánica 15/1999, de
Protección de Datos de Carácter Personal, se ha optado por llevar a cabo una labor
de documentación en las leyes marroquíes, y precisamente la denominada القانون رقم
المتعلق بحماية األشخاص الذاتيين تجاه معالجة المعطيات ذات الطابع الشخصي80.90, puede servir de
referencia para realizar una traducción acorde con la terminología utilizada en la
lengua meta.
4. Conclusiones
La documentación relacionada con el derecho privado como los documentos concernientes al matrimonio, al divorcio, o la herencia, están notoriamente marcados por el
contexto cultural que los produce, y su traducción es puente entre dos ordenamientos
jurídicos que responden a diferentes concepciones del mundo y de las relaciones
sociales76.
En el caso de la traducción en la combinación lingüística árabe-español-árabe, el
futuro traductor ha de tener unos conocimientos básicos de los respectivos marcos
legales, prestando especial atención a las variedades diatópicas que singularizan los
documentos árabes según su procedencia geográfica.
Junto a ello, el traductor debe tener presente que la traducción de documentos
jurídicos está destinada a surtir los mismos efectos legales que los originales, de ahí
que deba cuidarse la correspondencia formal y fehaciente de los mismos. Para llevar
a cabo satisfactoriamente esta tarea, requiere de una competencia intercultural, en
tanto en cuanto debe trasladar el discurso a un contexto meta con una función equivalente. Desde el punto de vista didáctico, la enseñanza de la traducción de este tipo
de textos debe estructurarse a partir del sistema referencial de dichos textos, los aspectos culturales, junto con sus anisomorfismos.
76
GUTIÉRREZ ARCONES, David (2015) “Estudio sobre el texto jurídico y su traducción: características de la
traducción jurídica, jurada y judicial” en Miscelánea Comillas [en línea]. Nº 73, disponible en https://revistas.
upcomillas.es/index.php/miscelaneacomillas/article/viewFile/5493/5303 [Último acceso el 21 de noviembre de
2019], p. 142.
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Role of Nitrate Reductase in NO Production in Photosynthetic Eukaryotes
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Received: 16 January 2019; Accepted: 8 February 2019; Published: 6 March 2019 Abstract: Nitric oxide is a gaseous secondary messenger that is critical for proper cell signaling
and plant survival when exposed to stress. Nitric oxide (NO) synthesis in plants, under standard
phototrophic oxygenic conditions, has long been a very controversial issue. A few algal strains contain
NO synthase (NOS), which appears to be absent in all other algae and land plants. The experimental
data have led to the hypothesis that molybdoenzyme nitrate reductase (NR) is the main enzyme
responsible for NO production in most plants. Recently, NR was found to be a necessary partner in a
dual system that also includes another molybdoenzyme, which was renamed NO-forming nitrite
reductase (NOFNiR). This enzyme produces NO independently of the molybdenum center of NR
and depends on the NR electron transport chain from NAD(P)H to heme. Under the circumstances
in which NR is not present or active, the existence of another NO-forming system that is similar to
the NOS system would account for NO production and NO effects. PII protein, which senses and
integrates the signals of the C–N balance in the cell, likely has an important role in organizing cell
responses. Here, we critically analyze these topics. Keywords: nitric oxide; nitrate reductase; NOFNiR; nitrogen metabolism Keywords: nitric oxide; nitrate reductase; NOFNiR; nitrogen metabolism Role of Nitrate Reductase in NO Production in
Photosynthetic Eukaryotes Manuel Tejada-Jimenez
, Angel Llamas
, Aurora Galván
and Emilio Fernández *
Departamento de Bioquímica y Biología Molecular, Campus de Rabanales y Campus Internacional de
Excelencia Agroalimentario (CeiA3), Edif. Severo Ochoa, Universidad de Córdoba, 14071 Córdoba, Spain;
manuel.tejada@uco.es (M.T.-J.); bb2llaza@uco.es (A.L.); bb1gacea@uco.es (A.G.)
* Correspondence: bb1feree@uco.es; Tel.: +34-957-218-591 Manuel Tejada-Jimenez
, Angel Llamas
, Aurora Galván
and Emilio Fernández *
Departamento de Bioquímica y Biología Molecular, Campus de Rabanales y Campus Internacional de
Excelencia Agroalimentario (CeiA3), Edif. Severo Ochoa, Universidad de Córdoba, 14071 Córdoba, Spain;
manuel.tejada@uco.es (M.T.-J.); bb2llaza@uco.es (A.L.); bb1gacea@uco.es (A.G.)
* Correspondence: bb1feree@uco.es; Tel.: +34-957-218-591 Manuel Tejada-Jimenez
, Angel Llamas
, Aurora Galván
and Emilio Fernández *
Departamento de Bioquímica y Biología Molecular, Campus de Rabanales y Campus Internacional de
Excelencia Agroalimentario (CeiA3), Edif. Severo Ochoa, Universidad de Córdoba, 14071 Córdoba, Spain;
manuel.tejada@uco.es (M.T.-J.); bb2llaza@uco.es (A.L.); bb1gacea@uco.es (A.G.)
* Correspondence: bb1feree@uco.es; Tel.: +34-957-218-591 Plants 2019, 8, 56; doi:10.3390/plants8030056 plants plants www.mdpi.com/journal/plants 1. Introduction Nitric oxide (NO) is a gaseous secondary messenger in humans, animals, plants, fungi, and
bacteria. In plants, NO is involved in important physiological processes, such as growth, development,
metabolism, leaf senescence, biotic and abiotic stress, defense processes, and plant–pathogen
interactions, which have been extensively reviewed [1–5]. In particular, in algae, such as the green alga
Chlamydomonas reinhardtii, NO also participates in fundamental cell functions, such as the regulation
of N-metabolism, N- and S-starvation stress, chloroplast biogenesis, programmed cell death, and
responses to darkness, hypoxia, or salt stress [6–11]. In the last two decades, it has been clarified that NO is a signaling molecule in plant defense during
plant–pathogen interactions [12,13]. Since then, different strategies have been used to understand NO
biosynthesis in plant cells, and this subject has not been short of controversies [5,14], with some aspects
yet to be understood. Two main pathways, reductive and oxidative, appear to explain NO synthesis
in plants. One is based on the reduction of nitrite, and the other involves the oxidation of aminated
molecules, such as the amino acid arginine [15]. In spite of the seminal work of Foresi and collaborators, who identified the first NO synthase
(NOS) from the plant kingdom in the green alga Ostreococus taurii [16], the existence of a plant NOS
that has the characteristics of the animal NOS has been puzzling [14,17] since no plant genome contains
such a conserved gene. In fact, Jeandrof and collaborators analyzed over 1000 species of land plants
and algae and found no typical NOS sequences in the 1087 sequenced transcriptomes of land plants, www.mdpi.com/journal/plants 2 of 13 Plants 2019, 8, 56 but they did find said sequences in 15 of the 265 algal species. Thus, it was concluded that land plants
had evolved a mechanism to synthesize NO in a manner that is different from that used in animals [18]. In this review, the different biosynthetic processes of nitric oxide formation are critically analyzed,
together with their physiological relevance. 2. Nitrite: The Substrate for Reductive NO Production Nitrite is a product of nitrate reductase (NR)-catalyzed nitrate reduction within the nitrate
assimilation pathway. Nitrogen acquisition is a fundamental process for living beings, including plants
in crops, in which N is usually a limiting factor that determines crop productivity [19]. Nitrate used to
be the preferred form of inorganic N that was available in soils and, thus, was used in fertilizers [20]. The incorporation of nitrogen from nitrate first requires its acquisition from the medium
by specific transporters, which are responsible for the sensing, uptake, storage, and distribution
of nitrate among plant tissues. Plant nitrate transporters belong to several families: the nitrate
transporter 1/peptide transporter/nitrate peptide transporter family (NRT1/PTR/NPF), NRT2/nitrate
nitrite porter (NRT2/NNP), chloride channels (CLC), slow anion channel-associated 1 homolog 3
(SLAC1/SLAH), and aluminum-activated malate transporters (ALMT). These have all been reviewed
in detail [21–25]. In the Chlamydomonas alga, this complexity is less but still significant, reflecting
the importance of this step. Here, we highlight the three families of transporter proteins found in
Chlamydomonas: NRT1/NPF, NRT2, and NAR1 (Figure 1). NRT1 has been described in Arabidopsis
as a dual-affinity nitrate/nitrite transporter, and NRT2 (with the accessory protein NAR2) mediates
the high-affinity transport (HAT) of nitrate and nitrite. Plants 2019, 8, x FOR PEER REVIEW
3 of 13 Figure 1. The schematic model for the coordinated regulation of Nitric Oxide (NO) synthesis and N
metabolism. Blue arrows indicate activation and red lines indicate inhibition by trans-nitrosylation. Dashed lines represent hypothetical steps. The NOS-like component represents the L-Arg-dependent
NOS activity reported in different plant species. Figure 1. The schematic model for the coordinated regulation of Nitric Oxide (NO) synthesis and N
metabolism. Blue arrows indicate activation and red lines indicate inhibition by trans-nitrosylation. Dashed lines represent hypothetical steps. The NOS-like component represents the L-Arg-dependent
NOS activity reported in different plant species. Figure 1. The schematic model for the coordinated regulation of Nitric Oxide (NO) synthesis and N
metabolism. Blue arrows indicate activation and red lines indicate inhibition by trans-nitrosylation. Dashed lines represent hypothetical steps. The NOS-like component represents the L-Arg-dependent
NOS activity reported in different plant species. Figure 1. The schematic model for the coordinated regulation of Nitric Oxide (NO) synthesis and N
metabolism. Blue arrows indicate activation and red lines indicate inhibition by trans-nitrosylation. Dashed lines represent hypothetical steps. The NOS-like component represents the L-Arg-dependent
NOS activity reported in different plant species. Figure 1. 2. Nitrite: The Substrate for Reductive NO Production The schematic model for the coordinated regulation of Nitric Oxide (NO) synthesis and N
metabolism. Blue arrows indicate activation and red lines indicate inhibition by trans-nitrosylation. Dashed lines represent hypothetical steps. The NOS-like component represents the L-Arg-dependent
NOS activity reported in different plant species. Figure 1. The schematic model for the coordinated regulation of Nitric Oxide (NO) synthesis and N
metabolism. Blue arrows indicate activation and red lines indicate inhibition by trans-nitrosylation. Dashed lines represent hypothetical steps. The NOS-like component represents the L-Arg-dependent
NOS activity reported in different plant species. 3. Nitrate Reductase is a Multidomain Protein
NR reduces nitrate to nitrite using electrons from NAD(P)H. The plant enzyme is about 200
KDa and contains two subunits, each bearing three prosthetic groups: FAD, heme b557, and
molybdenum. In an NR subunit, molybdenum is bound to a tricyclic pyranopterin and chelated by a
dithiolene, which is named the molybdenum cofactor (Moco). These domains are joined by two
Nitrite in the cytosol, either produced from nitrate or absorbed from the medium, has to be
transported to the chloroplast by a HAT system. In Chlamydomonas, HAT of nitrite is facilitated
by NAR1, which belongs to the FNT family and is absent in land plants. In higher plants, nitrite
transport to chloroplasts is typically mediated by members of the CLC family [26], and it is
exported from the chloroplast by a transporter from the NRT1 family, as is found in cucumber, 3 of 13 Plants 2019, 8, 56 Cucumis sativus [27,28]. Regardless, nitrite concentrations in the cytosol are maintained at very low
levels (micromolar range) [29] to prevent nitrite toxicity in the cell [30]. Once in the chloroplast,
nitrite is reduced to ammonium in a reaction catalyzed by nitrite reductase (NiR). All of these
genes (NRT2/NAR2/NR/NAR1/NiR) in Chlamydomonas are controlled by the master regulatory
gene for nitrate assimilation:
NIT2 [31]. Orthologous regulatory genes in land plants—NLP
genes—show a similar structural organization and signaling for nitrate [32–34]. Finally, ammonium
is incorporated into C-skeletons in the form of glutamate by the glutamine synthetase/glutamate
synthase (GS/GOGAT) cycle [35] (Figure 1). It is important to point out here that in contrast to the low cytosolic concentrations of nitrite,
those of nitrate are high (1–6 mM). Cytosolic nitrate levels are also more stably maintained than
vacuolar concentrations (5–75 mM), considering that external nitrate concentrations may change by
about 10,000-fold [36–38]. 3. Nitrate Reductase Is a Multidomain Protein NR reduces nitrate to nitrite using electrons from NAD(P)H. The plant enzyme is about 200 KDa
and contains two subunits, each bearing three prosthetic groups: FAD, heme b557, and molybdenum. In an NR subunit, molybdenum is bound to a tricyclic pyranopterin and chelated by a dithiolene,
which is named the molybdenum cofactor (Moco). These domains are joined by two protease-sensitive
hinge regions. The domains are redox centers, and electrons flow from NAD(P)H→FAD→heme
→Moco, which is within the active site for nitrate reduction [43,44]. Interestingly, the enzyme has
two partial activities, which can be assayed in vitro: diaphorase, which catalyzes the reduction of
artificial acceptors (ferricyanide or cytochrome c with NAD(P)H), and terminal-NR, which catalyzes
nitrate reduction using electrons supplied by FAD, FMN, viologens, or bromophenol blue, chemically
reduced by dithionite [43,44]. The crystal structure of the dimerized form of NR and Moco domains
was solved [45]. NR reduces nitrate to nitrite using electrons from NAD(P)H. The plant enzyme is about 200 KDa
and contains two subunits, each bearing three prosthetic groups: FAD, heme b557, and molybdenum. In an NR subunit, molybdenum is bound to a tricyclic pyranopterin and chelated by a dithiolene,
which is named the molybdenum cofactor (Moco). These domains are joined by two protease-sensitive
hinge regions. The domains are redox centers, and electrons flow from NAD(P)H→FAD→heme →Moco, which is within the active site for nitrate reduction [43,44]. Interestingly, the enzyme has
two partial activities, which can be assayed in vitro: diaphorase, which catalyzes the reduction of
artificial acceptors (ferricyanide or cytochrome c with NAD(P)H), and terminal-NR, which catalyzes
nitrate reduction using electrons supplied by FAD, FMN, viologens, or bromophenol blue, chemically
reduced by dithionite [43,44]. The crystal structure of the dimerized form of NR and Moco domains
was solved [45]. →Moco, which is within the active site for nitrate reduction [43,44]. Interestingly, the enzyme has
two partial activities, which can be assayed in vitro: diaphorase, which catalyzes the reduction of
artificial acceptors (ferricyanide or cytochrome c with NAD(P)H), and terminal-NR, which catalyzes
nitrate reduction using electrons supplied by FAD, FMN, viologens, or bromophenol blue, chemically
reduced by dithionite [43,44]. The crystal structure of the dimerized form of NR and Moco domains
was solved [45]. 2. Nitrite: The Substrate for Reductive NO Production This is important for ensuring efficient nitrate assimilation, together with
proper nitrate signaling in the tissues [38,39]. Nitrate homeostasis is the result of the membrane
transporter-mediated supply of nitrate from vacuoles and the outer medium, as well as by nitrate
efflux transporters such as NAXT1 [40]. NAXT1 belongs to the NRT1 family. In addition, the
NR-catalyzed reaction has an effect, facilitating the conversion of nitrate into nitrite [37,38,41]. In the
yeast Hansenula polymorpha, the sulfite transporter SSU2 and the nitrite transporter NAR1 have been
characterized as essential components of the nitrate/nitrite efflux system [42]. 4. Does NR Catalyze Nitrite Reduction to NO? In agreement with this, Moco-deficient mutants are deficient in molybdoenzymes, as described in
barley with a thermo-sensitive, wilty phenotype [53]. The most used Arabidopsis NR mutant to
support the involvement of NR in NO production is the double mutant deficient in NIA1 and NIA2
genes. This nia1/nia2 double mutant has only 0.5% of the activity of wild-type NR and grows very
poorly on medium with nitrate as the only nitrogen source [54]. As described below, NR is in fact
involved in NO production but not through its Moco-dependent activity. All five molybdoenzymes in plants (nitrate reductase, xanthine oxidase reductase (XOR), aldehyde
oxidase (AO), sulfite oxidase (SO), and mARC) are able to catalyze the one-electron reduction of nitrite
to NO. The molybdoenzymes are classified depending on how Moco binds to the enzyme’s active site:
either covalently through an enzyme cysteine thiol group (NR, SO, and mARC) or with inorganic sulfur
(XOR and AO) [55,56]. All of these enzymes show nitrite reductase activity to produce NO in vitro
and in anaerobic conditions [44]. The four known human molybdenum-containing enzymes are the
same as those in plants, except for NR, and they can also function as nitrite reductases under hypoxic
conditions [56]. In mammals, two known pathways for NO formation are known: arginine oxidation
under normoxic or aerobic conditions and nitrite reduction during hypoxia or anaerobiosis [56]. Plant SO seems to have a less potent nitrite reductase activity than human XOR and AO [57]. Whereas
plant AO participates in the synthesis of phytohormones and contributes to reactive oxygen species
(ROS) production, there is no information about its in vivo NO-producing activity [5]. Other processes that might produce NO from nitrite are associated with the plasma membrane-bound
NR, which appears to be related to the mycorrhizal colonization of tobacco roots [58] and the mitochondrial
electron transport chain (mETC), as demonstrated in several plants [59–62]. The mitochondrial complex
III and IV are primarily implicated in the nitrite reaction (Km of 175 µM), which requires anaerobic
conditions since oxygen is a strong inhibitor. Therefore, this reaction can occur in plant tissues
exposed to hypoxia, such as roots, and its occurrence might be important to the plant by protecting
the respiratory chain and mitochondrial metabolism when oxygen is lacking [63]. Recently, a role for
Alternative Oxidase (AOX) in the production of large amounts of NO, observed under hypoxia, has
been shown. 4. Does NR Catalyze Nitrite Reduction to NO? The experimental data have led to the proposal that the molybdoenzyme NR is the main enzyme
responsible for NO production in most plants. This proposal was based on the experimental findings
described below. The first evidence linking NR and NO production was their co-elution by NADH from Blue
Sepharose columns loaded with Soybean extracts with both NR and NO(X) evolution activities. In their main conclusion, the authors inferred their linkage from the fact that inhibiting the partial
activities of NR and NO(X) evolution activities led to the same pattern. However, they went further
by indicating that the terminal molybdenum-containing portion of NR is involved in the reduction
of nitrite to NO(X) [46]. Other studies showed that several plant species emitted in vivo NO when
there was nitrate in the soil, and the function was abolished in all plants in the study when they were
grown on ammonium-containing soil, indicating a role for NR [47]. In addition, isotopically labeled
15N-nitrate resulted in the emission of 15NO [48]. Moreover, using NR from corn led to the production
of significant amounts of NO from both nitrite and nitrate [49]. Those findings reinforced the idea that
NR reduces nitrate to nitrite and further converts de novo-generated nitrite into NO. The proposal
would be feasible if it could account for several facts. First, the nitrite-reducing activity of NR is very
low (only 1% of the nitrate-reducing activity), and the Km of nitrite is about 10 times higher than that of
nitrate, in agreement with the competitive character of nitrite (Ki = 50 µM) in nitrate reduction [50,51]. Plants 2019, 8, 56 4 of 13 Taking into consideration the intracellular concentrations of nitrite (in the micromolar range) and
nitrate (in the millimolar range), this nitrite-reducing activity would be irrelevant. Another piece of evidence indicating the involvement of NR in NO production originated from
the use of tungstate—an NR inhibitor—and NR-deficient mutants. In addition to its uncontrolled
effects, such as metal toxicity, tungstate is a very unspecific inhibitor of Mo-enzymes. When exchanging
Moco for the inactive tungsten cofactor (Wco) [52], all molybdoenzymes are inactivated, including
mitochondrial amidoxime-reducing component (mARC), which is presented below as the most
important enzyme in NO production under phototrophic conditions. Thus, tungstate is a clear
inhibitor of NO production because of the resulting mARC inhibition and not because of NR inhibition. 4. Does NR Catalyze Nitrite Reduction to NO? In this pathway of NO production, AOX has a role in scavenging the NO and ROS linked
to the hemoglobin–NO cycle, thus increasing energy efficiency without contributing to the formation
of toxic peroxynitrite [64]. The implication of mitochondria in NO production from nitrite has also
been shown in Chlamydomonas in the presence of high concentrations of nitrite [65]. In light of the biological importance of NO production, one would expect this process to be
efficiently and finely regulated. Some of the Moco-dependent enzymes described above can mediate
NO production under certain conditions, though it is difficult to currently envisage how to control this
diversity of processes; even mitochondrial NO production requires anaerobiosis, suggesting a burst of
NO synthesis from nitrite when this condition appears. 5. NR Does Not Catalyze In Vivo Nitrite Reduction to NO but Provides the Needed Electrons Recently, NR was shown to be a necessary partner for NO production in a dual system,
which, besides NR, includes another molybdoenzyme, mARC, since renamed NO-forming nitrite
reductase (NOFNiR). 5 of 13 Plants 2019, 8, 56 Plants 2019, 8, 56 mARC has been extensively characterized in prokaryotic and eukaryotic organisms [66–69]. mARC proteins are about 35 KDa and require two electron transport proteins—NADH-cytochrome b5
reductase and cytochrome b5—to be functional. mARC is a very efficient reductase [70] for a number of
N-oxygenated compounds, some of which are toxic or mutagenic [71–73]. This is why mARC has been
related to cell detoxification processes. Both human mARC isoforms are associated with mitochondria,
but mARC could also be located in peroxisomes because its two partners, NADH-cytochrome b5
reductase and cytochrome b5, were found in this organelle using a proteomic approach [74]. mARC
was also connected to NO metabolism because of its activity on Nω-hydroxy-Nδ-methyl-L-arginine, an
intermediate in the L-arginine-dependent biosynthesis of NO using NADH-cytochrome b5 reductase
and cytochrome b5 [75]. In addition, human mARCs have nitric oxide synthase activity from nitrite
with NADH and its two above-indicated partners [76]. In the eukaryotic alga Chlamydomonas, NO synthesis is carried out by a dual system comprising
NR and NOFNiR (mARC). These two components are closely connected at both the transcriptional
and activity levels, so mutants lacking one of them overexpress the other [77]. NR supplies NADH
electrons to NOFNiR for the reduction of nitrite to NO much more efficiently than NADH-cytochrome
b5 reductase and cytochrome b5. Both NR and NOFNiR are located in the cytosol [77]. NR mutants were found to be unable to provide electrons both in vitro and in vivo for NO
synthesis. Some exceptions are NR mutants such as Chlamydomonas strain 301, whose NR lacks
nitrate-reducing activity since its Moco domain is affected; however, it has functional diaphorase
activity with intact FAD and heme domains [77]. So, the dual complex NR:NOFNiR produces NO
independently of the molybdenum center of NR and depends on the NR electron transport chain from
NAD(P)H to heme. NR:NOFNiR has been proposed to be the main system producing NO during standard
phototrophic, oxygenic plant growth, which corresponds to most of its aerial part [77]. In tissues
exposed to hypoxia, such as roots, other molybdoenzymes or mETCs could be involved in the synthesis
of NO, as discussed above. 6. NO Levels in the Cells Are Regulated The first line of control of NO levels is the regulation of its synthesis and degradation within the
NO cycle (Figure 1). Members of the hemoglobin (HB) superfamily can oxygenate NO to nitrate, as
was shown for Chlamydomonas THB1 [78], a class 3 truncated hemoglobin (THB) [79]. In different
plant species, nitrate, nitrite, and NO upregulate HB expression [80,81]. In maize roots, the coordinated
expression of both NR and HB also occurs [82]. Similarly, in Chlamydomonas, the expression of
two truncated HBs, THB1 and THB2, respond selectively to N signals (nitrate, nitrite, and NO) and,
interestingly, also to NIT2, the major regulatory gene of the nitrate assimilation pathway [78,83]. This regulatory gene is also essential for NR upregulation by nitrate [19]. The activity of THB1 requires
electrons to be supplied by the NADH-diaphorase of NR, and the electron flow is likely from NADH
to FAD [78]. Similarly, the activity of NOFNiR also requires the NADH-diaphorase of NR, but the
electron flow is now from NADH to heme b [77]. Thus, nitrate through NIT2 would stimulate NO
production because of NR’s increased expression (NOFNiR is not under NIT2 control), and in turn,
NO degradation would occur as a result of stimulating both THB1 and NR. The homeostasis of NO is
controlled by the activities of NR, NOFNiR, THB1, and THB2, which, in turn, depend on the relative
concentrations of nitrate, nitrite, and NO, as well as NIT2. ,
,
,
So, just-synthesized NO, which is highly reactive, can react with different targets. Glutathione
(γ-glutamylcysteinylglycine, GSH) is an essential metabolite in plants that participates in important
functions, such as primary metabolism, redox signaling, and defense and detoxification processes [84]. GSH can react with NO to produce S-nitrosylated glutathione (GSNO). As a result, the half-life of
NO in tissues available as a free radical gas changes from seconds to a few minutes. Thus, there are very
sensitive mechanisms for regulating cellular processes. GSNO, which is considered the main reservoir
for NO, provides NO signals to proteins. Protein S-nitrosation is considered the most important mode So, just-synthesized NO, which is highly reactive, can react with different targets. Glutathione
(γ-glutamylcysteinylglycine, GSH) is an essential metabolite in plants that participates in important
functions, such as primary metabolism, redox signaling, and defense and detoxification processes [84]. GSH can react with NO to produce S-nitrosylated glutathione (GSNO). 6. NO Levels in the Cells Are Regulated As a result, the half-life of
NO in tissues available as a free radical gas changes from seconds to a few minutes. Thus, there are very So, just-synthesized NO, which is highly reactive, can react with different targets. Glutathione
(γ-glutamylcysteinylglycine, GSH) is an essential metabolite in plants that participates in important
functions, such as primary metabolism, redox signaling, and defense and detoxification processes [84]. GSH can react with NO to produce S-nitrosylated glutathione (GSNO). As a result, the half-life of
NO in tissues available as a free radical gas changes from seconds to a few minutes. Thus, there are very
sensitive mechanisms for regulating cellular processes. GSNO, which is considered the main reservoir
for NO, provides NO signals to proteins. Protein S-nitrosation is considered the most important mode 6 of 13 Plants 2019, 8, 56 of action of NO. This is the covalent binding of NO to the thiol group of protein cysteine residues, and
the creation of these bonds modifies the protein and can alter gene expression and/or lead to metabolic
changes, all of which ultimately translate into physiological responses. Thousands of nitrosation
sites have been identified in proteins [85]. GSNO is metabolized with GSNO reductase (GSNOR1) to
convert it to glutathione disulfide (GSSG) and ammonia (NH3) [86,87]. GSNOR1 is a cytosolic enzyme
that controls GSNO levels and, in this way, the nitrosation of proteins. GSNOR1 seems to be inhibited
by NO, in which case, the scavenging of GSNO is prevented. Thus, NO controls its production and
scavenging by regulating both nitrate assimilation efficiency and GSNOR1 activity [87,88] (Figure 1). Other posttranslational modifications of proteins mediated by NO are the nitration of tyrosine
and amine groups and the oxidation of thiols and tyrosine [89]. NO can react with superoxide (O2−)
to form peroxinitrite (ONOO−), which is a powerful oxidant contributing to the nitration of protein
tyrosine residues to form 3-nitrotyrosine, which results in regulatory responses. Nitration seems to be
a reversible process that might occur at specific tyrosine residues depending on the local environment
and the secondary and tertiary structure of the protein. A putative specific denitrase removes the nitro
group without degrading the protein, as has been shown in animal systems [89,90]. 6. NO Levels in the Cells Are Regulated Reactive oxygen species (ROS), such as oxygen, singlet oxygen, hydroxyl radical, hydrogen
peroxide, and superoxide anion, all of which are important signaling compounds produced under
several environmental conditions, interact with NO and other reactive nitrogen species (RNS). They
mediate the responses to different environmental situations, even promoting the systemic adaptation
of plants to stress situations [2,91,92]. 7. The NO Synthesis Systems Are Coordinated with Nitrogen Metabolism Under circumstances in which NR is not present or active, another NO-forming system accounts
for NO production and NO effects. Analysis of this topic and possible future directions are
presented here. With an ammonium medium as the sole N-source or in null NR-deficient mutants, NR is absent,
and thus, the dual system NR–NOFNiR is not functional. Nevertheless, NO is being synthesized,
probably due to the existence of a NOS-like activity in algae and plants. This activity can be inhibited
by some compounds, which are primarily arginine analogs acting on the animal-type NOS [6,17]. Plant peroxisomes are single-membrane-bound organelles with an oxidative metabolism and a
simple morphology, but they also have a complex composition of enzymes involved in the metabolism
of oxygen free-radicals. Peroxisomes can generate ROS and nitric oxide and thus important signal
molecules with implications for cellular metabolism in plants [91,93]. Many studies of different plant species have shown the presence of L-Arg-dependent NO
synthase-like enzyme activity, which has biochemical requirements similar to animal NOS (L-Arg,
NADPH, FMN, FAD, Calmodulin, and Ca2+) [94,95]. Corpas and Barroso (2017) postulated that the
L-Arg-dependent NO synthesis that occurs in plants could correspond to cooperation among discrete
proteins, resulting in the formation of a protein complex with requirements for enzyme activity that are
similar to animal NOS [96]. This would explain the lack of success in finding canonical NOS proteins
at the molecular level. On the other hand, coordination between nitrogen assimilation and the nutritional status
within plant metabolism is a critical issue for plant viability. A very abundant family of N- and
C-signaling proteins, widely distributed in nature, is the PII protein family from bacteria, archaea,
and plants [97]. In plants, these nuclear-encoded PII proteins localize in the chloroplast and are
not subject to the covalent modification reported for bacterial PII [98]. PII senses and integrates the
signals of the C–N balance in the cells using 2-ketoglutarate as an indicator, together with the energy
status by competitive ADP/ATP binding [99,100]. Plant PII proteins are conserved throughout the
evolutionary history of the Chloroplastida—green algae and land plants [97]—and participate in a
complex signal-transduction network that mediates nitrogen regulation [101]. In Arabidopsis, the PII
protein controls arginine biosynthesis [102], and PII mutants show an enhanced nitrite uptake by the 7 of 13 Plants 2019, 8, 56 chloroplast [103]. 7. The NO Synthesis Systems Are Coordinated with Nitrogen Metabolism By binding effector molecules, PII interacts with and signals to other chloroplast
proteins, such as N-acetyl-L-glutamate kinase (NAGK), which catalyzes the committed step in the
arginine biosynthesis pathway [102] (Figure 1). Among the effectors binding plant PII protein is
glutamine, which was shown to bind to the termed Q-loop of the C-terminus crystalized plant PII
protein [104]. The binding of glutamine to PII changes its conformation to one that interacts and
activates NAGK for the synthesis of arginine and polyamines. PII–glutamine sensing is a widespread
mechanism in the plant kingdom [104]. In Chlamydomonas, arginine is a component of the same
repressive pathway as ammonium and CYG56, which is a guanylate cyclase activated by NO and
needed to repress nitrogen assimilation genes [105]. Interestingly, plant PII seems to be connected to the regulation of nitrite metabolism, so PII
knockout mutants show an increased sensitivity to nitrite and a decrease in total amino acids, especially
glutamine. Lack of PII seems to increase the C/N balance, as previously shown in cyanobacteria [30]. Recently, Chlamydomonas PII levels have been shown to be tightly controlled by the nitrogen source
and the physiological status of the cells [106]. In fact, PII expression is subject to positive (nitrate and
nitrite) signaling and is downregulated by ammonium via an NO-mediated process that involves an
NO-dependent guanylate cyclase, similar to the negative effect of ammonium on NR expression [6]. PII expression is very similar to that of nitrate assimilation genes [25], so an interaction/coordination
between PII and nitrate assimilation pathways has been suggested [106]. Under standard phototropic conditions, nitrate assimilation takes place under the positive control
of nitrate, mediated by the regulatory gene NIT2 [25]. When both nitrate and ammonium are present,
there is a balance between the positive and negative signals, and NR expression follows that balance;
the NR transcript is detectable even in the presence of ammonium, provided that nitrate is also
present [107]. The negative signal of ammonium on Chlamydomonas NIA1 gene expression depends
on NO and its mediation by an NO-dependent guanylate cyclase (CYG56). So, NO concentrations in
the cells increase with ammonium concentration, leading to complete NR repression [107] by means of
a mechanism that is dependent on a possible NOS, which is inhibited by L-NAME. In different plants, NO production is also sensitive to the mammalian NO synthase inhibitor
L-NAME [2,5]. 7. The NO Synthesis Systems Are Coordinated with Nitrogen Metabolism L-NAME was also shown to affect NO production by interfering with NR activity [108],
which seems to question the mechanisms of action of this compound. However, it has to be considered
that in the double nia1/nia2 Arabidopsis mutant commonly used to study NO effects [54], the content
of nitrite, as expected from the NR deficiency, and of free amino acids, particularly L-arginine, are much
lower than in wild-type plants [109]. Thus, both substrates for NO synthesis would be compromised. yp p
y
p
In conclusion, PII proteins mediate the signaling of the N-source with respect to the carbon status
(2-ketoglutarate). This N can be either oxidized (nitrate, nitrite) with a generally high C/N balance, or
it can be reduced (ammonium) with a generally low C/N balance. With a high C/N balance, the nitrate
assimilation pathway is operative, and NR expression would favor the increase in N capture and its
incorporation into C-skeletons (2-ketoglutarate), which would be abundant. When the C/N balance is
low, NR would be repressed, and the biosynthesis of arginine would be stimulated. So, under these
two extreme conditions, the substrates to produce NO would change. In conditions of high C/N, nitrite
would be efficiently produced; however, with low C/N, arginine biosynthesis would predominate
with low nitrite production. So, it is proposed that the dual NR:NOFNiR system will preponderate at
high C/N, whereas the NOS-like system will be mostly operative at low C/N. PII protein expression
will follow a pattern similar to that of NR [110] to enhance N acquisition and balance the C/N ratio. These two extreme situations might be changed to intermediate ones, depending on the C/N balance
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tightly regulated. Some of the biosynthetic mechanisms are starting to be disentangled, while others
still require additional useful information for their elucidation. Part of the confusion regarding the Plants 2019, 8, 56 8 of 13 primary source of NO might come from the fact that several pathways might function simultaneously
to different extents, depending on nutritional and environmental conditions. Many points still have to
be clarified for plants. primary source of NO might come from the fact that several pathways might function simultaneously
to different extents, depending on nutritional and environmental conditions. Many points still have to
be clarified for plants. Author Contributions: Conceptualization, E.F.; writing-original draft preparation, E.F.; writing-review and
editing M.T.-J., A.L., A.G. and E.F.; funding acquisition, E.F. Funding: This work was funded by MINECO (Grant BFU2015-70649-P), the European FEDER program,
Junta de Andalucía (BIO-502), the Plan Propio de la Universidad de Córdoba, and the U.E.INTERREG VA
POCTEP-055_ALGARED_PLUS5_E. Funding: This work was funded by MINECO (Grant BFU2015-70649-P), the European FEDER program,
Junta de Andalucía (BIO-502), the Plan Propio de la Universidad de Córdoba, and the U.E.INTERREG VA
POCTEP-055_ALGARED_PLUS5_E. Acknowledgments: We thank María Isabel Macías and Aitor Gómez for technical assistance. Acknowledgments: We thank María Isabel Macías and Aitor Gómez for technical assistance. Conflicts of Interest: The authors declare no conflict of interest. References Delledonne, M.; Xia, Y.; Dixon, R.A.; Lamb, C. Nitric oxide functions as a signal in plant disease resistance. Nature 1998, 394, 585–588. [PubMed] 13. Durner, J.; Wendehenne, D.; Klessig, D.F. Defense gene induction in tobacco by nitric oxide, cyclic GMP, and
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article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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Challenges and improvement needs in the care of patients with central diabetes insipidus
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Orphanet journal of rare diseases
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Abstract Central diabetes insipidus (CDI) is a rare condition, with significant impact on patient health and well-being. It is a
chronic condition which usually requires meticulous long-term care. It can affect both children and adults. There is
limited literature considering the needs and challenges inherent in providing high quality care to patients with CDI,
across the care pathway. This paper seeks to address this gap by providing a unique and well-rounded understand‑
ing of clinical and healthcare systems-related challenges. It draws on insights from the literature, from direct clinical
experience contributed by five clinicians as co-authors (providing insights from France, Ireland, Italy, Spain and the
United Kingdom), and from patient perspectives provided through interviews with patient representatives from
three patient organisations. We identify clinical challenges related to the diagnosis of CDI, including differentiating
between other similar conditions and determining the underlying aetiology. Treatment is challenging, given the need
to tailor medication to each patient’s needs and ongoing management is required to ensure that patients continue
to respond adequately to treatment. Ongoing support is required when patients switch between formulations. We
also identify healthcare systems challenges related to limited awareness of CDI amongst primary care physicians and
general paediatricians, and the need for highly skilled specialist care and appropriate workforce capacity. There is also
a significant need for raising awareness and for the education of both healthcare professionals and patients about
different aspects of CDI, with the aim of supporting improved care and effective patient engagement with healthcare
professionals. We reflect on this information and highlight improvement opportunities. These relate to developing
guidance to support patients, carers, primary care physicians and general paediatricians to identify clinical features
earlier, and to consider CDI as a possible diagnosis when a patient presents with suggestive symptoms. Keywords: Central diabetes insipidus, Disease burden, Healthcare improvement, Healthcare systems, Rare disease, Keywords: Central diabetes insipidus, Disease burden, Healthcare improvement, Healthcare systems, Rare disease,
DDAVP Keywords: Central diabetes insipidus, Disease burden, Healthcare improvement, Healthcare systems, Rare disease,
DDAVP Central diabetes insipidus: a rare disease
with significant impacts on patient health
and well‑being and a limited evidence base
on how to improve patient care
Central diabetes insipidus (CDI) is a rare disease affecting approximately 1 in 25,000 individuals [4]. It has signifi-
cant impact on patient health and well-being and usu-
ally requires long term care. The evidence base on how
to care for patients with this condition is fragmented. © 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. Challenges and improvement needs
in the care of patients with central diabetes
insipidus H. Teare1, J. Argente2,3,4, M. Dattani5,6, J. Leger7, M. Maghnie8,9, M. Sherlock10, G.‑C. Ali1, J. Francombe1 and
S. Marjanovic1* S. Marjanovic1* Abstract There is a need to advance knowledge on the diversity
of challenges to patient care and to identify scope for
improvement. *Correspondence: smarjano@randeurope.org
1 RAND Europe, Westbrook Centre Milton Road, Cambridge CB4 1YG, UK
Full list of author information is available at the end of the article In this paper, we provide an overview of the diagno-
sis, treatment and management of patients with CDI to Teare et al. Orphanet Journal of Rare Diseases (2022) 17:58
https://doi.org/10.1186/s13023-022-02191-2 Teare et al. Orphanet Journal of Rare Diseases (2022) 17:58
https://doi.org/10.1186/s13023-022-02191-2 (2022) 17:58 Open Access © 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. Teare et al. Orphanet Journal of Rare Diseases (2022) 17:58 Page 2 of 12 consider the diversity of factors that play a role in deter-
mining the quality of patient care. We identify challenges
and improvement needs in both adult and paediatric
populations. We draw on insights from a narrative litera-
ture review, complemented with the experiential knowl-
edge of clinical experts and the views of representatives
from patient associations. treatment over time, including when managing CDI
alongside other concurrent conditions, particularly
adrenocorticotropic hormone (ACTH) deficiency. Fur-
thermore, the lifelong treatment that patients with CDI
often require [7] places significant demand on healthcare
systems for ongoing management and monitoring. The aim of this review is to expand on the key features
of the care pathway and the associated factors and chal-
lenges that influence patient care, and to reflect on areas
in need of improvement. CDI is characterised by hypotonic polyuria and poly-
dipsia resulting from decreased concentrations of argi-
nine vasopressin (AVP), also known as antidiuretic
hormone (ADH) [4, 11]. Other symptoms can include
fatigue, dizziness, hypotension and tachycardia related to
dehydration, and hypernatremia which is a direct conse-
quence of CDI [15, 24]. Severe dehydration is a particular
challenge in young children, who may not as yet be able
to self-regulate thirst. Children can also experience vom-
iting, constipation, fever, irritability, sleep disturbance,
retardation of growth, failure to thrive and potentially
developmental disability caused by repeated dehydration
and hypernatremia [9, 15, 18, 20]. Methodsh The paper combines findings from a narrative literature
review, insights based on experiential knowledge from
five leading clinical experts from the United Kingdom
(UK), Ireland, Italy, Spain and France who are also co-
authors of the paper, and the views of patient representa-
tives from three associations active in the CDI area (the
Pituitary Foundation, UK; the Pituitary Foundation, Ire-
land; and the Italian Family Association of Septo-optic
Dysplasia and Pituitary Abnormalities, Italy). The disease is caused by damage to AVP-producing
magnocellular neurons in the hypothalamus. The degree
of deficiency of AVP is the main factor that determines
the severity of CDI symptoms. Damage to this region
of the brain can occur either due to traumatic injury
[3, 11] or non-traumatically, for example in response
to a tumour, infiltration, infection or inflammation, or
less frequently as a result of genetic mutations [1, 21]. Hereditary forms of CDI present early in life, whereas
acquired forms can occur at any age. CDI has a similar
prevalence in men and women. A patient’s quality of life
is often impeded as a result of the condition, for exam-
ple from sleep being disturbed by nocturia [25]. There is
also a risk of fatality in the most severe cases, which can
happen because of chronic or severe dehydration, hyper-
natremia, fever and cardiovascular failure due to a com-
promised ability to regulate blood pressure [5, 7]. Adipsia
can increase risk of death [12]. In patients with CDI, an
intact thirst mechanism is critical for protecting against
hypernatremia and dehydration. Narrative review
Th
i
li The narrative literature review was conducted using a
systematic search strategy, with the search conducted
in October 2020. It included papers published between
October 2015 and October 2020, with specified crite-
ria for prioritising papers which were then subjected to
thematic analysis. It focused on understanding the care
pathway for patients with CDI, challenges and wider
influences on patient care, and improvement needs. A
total of 25 papers were included in the review; further
detail of their selection is included in the Additional
file 1. Interviews with patient representatives Perspectives were gathered from three patient repre-
sentatives from associations which support patients
with CDI in Ireland, Italy and the UK (unfortunately
this was not feasible in Spain or France). Their per-
spectives helped to better understand how patients
experience CDI and their views on future priorities to
improve the provision of care. Patient representative
views are referenced in brackets with PR-INT X, with X
being the code number for an individual interviewee. In
some instances, where there is a risk of identity disclo-
sure or linkage of information to a country context, or
in light of informed consent, we withhold a reference to
preserve anonymity. Both adults and children follow a similar diagnostic
pathway. A water deprivation test (WDT), during which
the patient is not allowed to drink, has been the gold
standard for diagnosing CDI [20]. It involves meticulous
and regular measurements of urine production, blood
electrolyte concentrations, plasma and urine osmolal-
ity and weight for approximately 7 h (and occasionally
for shorter durations in children, if cessation of the test
is necessary due to weight loss or hypernatremia) [20]. This usually takes place in a specialist hospital setting
supported by an endocrinologist and specialist nurses,
as water deprivation requires careful monitoring [24]. Patients can find the WDT test extremely unpleasant and
challenging [PR-INT2, PR-INT3]. The water depriva-
tion test is sometimes done in modified versions and for
shorter periods of water deprivation in some paediatric
patients and may be avoided in infancy if hypernatremia
with low urine osmolality is present. Parents of children
with CDI symptoms may also be asked to measure their
child’s fluid balance [PR-INT3]. A WDT may not always
be necessary for pregnant women, for whom such a test
could be overly cumbersome [22]. There is potential for
increased use of alternative diagnostic tests in the future
[PR-INT2], for example measurement of copeptin and
the use of dynamic tests such as arginine or hypertonic
saline infusions. However, the current use of such tests
across different countries remains unclear. We have thematically cross-analysed and triangulated
insights from these various sources to develop a multi-
faceted understanding of care pathways, associated chal-
lenges and improvement needs. There are some caveats to consider when interpreting
our findings. The literature review covered high income
countries and additional insights from low- and middle-
income countries would help to enrich these findings. Focused discussion with clinical experts
fi
d b
ld To refine, nuance and build on insights from the lit-
erature, the paper co-authors—representing health-
care services research experts from the not-for-profit
research institute RAND Europe (SM, HT, JF, DRR,
GCA) and clinicians working with patients with CDI
in France (JL), Ireland (MS), Italy (MM), Spain (JA)
and UK (MD)—engaged in focused discussion. This
included individual discussions between a RAND
Europe health services researcher (either SM, HT,
DRR, or JF) and each individual clinician, a total of five
one-hour discussions. It also included group dialogue
through two online workshops bringing together the
clinical experts and RAND Europe researchers (work-
shops were held on 23 and 27 November 2020). The
workshops were opportunities for collective discus-
sion about care-related improvement needs. They also
served to explore healthcare system related influences CDI is one of four conditions associated with polyuria
and polydipsia, along with nephrogenic diabetes insipi-
dus (NDI), primary polydipsia and gestational diabetes
insipidus [7]. Once diagnosed, treatment of CDI is gen-
erally effective, with desmopressin (DDAVP, D-amino
D-arginine vasopressin- an AVP analogue) used as the
mainstream treatment to help regulate fluid in the body. However, it can be challenging to identify and accurately
diagnose patients and to get them onto appropriate treat-
ment [7, 8, 14, 15, 20, 23, 24]. Optimising dosages and
treatment formulations and ensuring personalised care
is also not straightforward [1, 22, 23]. In addition, it can
be challenging to ensure that patients remain stable on Page 3 of 12 Teare et al. Orphanet Journal of Rare Diseases (2022) 17:58 Teare et al. Orphanet Journal of Rare Diseases (2022) 17:58 Teare et al. Orphanet Journal of Rare Diseases (2022) 17:58 The care pathway and associated challenges
related to diagnosis, treatment and ongoing
management
Diagnosing CDI on quality of care and access to care in more depth,
given that these aspects are under-explored in the lit-
erature. Clinical co-authors from four countries also
gathered estimations of the costs of care for a patient
in their local healthcare setting (i.e. hospital) to provide
a high-level indication of the estimated costs related
to hospital care that are incurred during the patient
pathway. Although these are just estimates, and do not
cover primary care costs for example, they provide ini-
tial insights on the resource implications of caring for
patients with CDI which can be built on in future stud-
ies, and that extend beyond medicine costs alone. on quality of care and access to care in more depth,
given that these aspects are under-explored in the lit-
erature. Clinical co-authors from four countries also
gathered estimations of the costs of care for a patient
in their local healthcare setting (i.e. hospital) to provide
a high-level indication of the estimated costs related
to hospital care that are incurred during the patient
pathway. Although these are just estimates, and do not
cover primary care costs for example, they provide ini-
tial insights on the resource implications of caring for
patients with CDI which can be built on in future stud-
ies, and that extend beyond medicine costs alone. In children and adults, CDI is diagnosed using a series
of tests to confirm polyuria and polydipsia and to try to
understand the underlying cause. Investigations tend to
follow a logical progression of clinical history and exami-
nation followed by biochemical and endocrinological
assessment, followed by radiology. The approach to diag-
nosis is similar across the different healthcare settings
considered in this paper [workshop insights]. In coun-
tries where primary care is generally the first point of
access to healthcare, patients with CDI will usually first
present to a primary care physician (or general practi-
tioner) having experienced symptoms such as dehydra-
tion, thirst and frequent urination [PR-INT1, PR-INT2,
PR-INT3]. Following basic tests by a primary care physi-
cian, in most cases patients will be referred to a specialist
endocrinologist for further diagnostic testing [PR-INT1,
PR-INT2, PR-INT3]. Interviews with patient representatives Absent neurohypophysis can be diagnosed by MRI if the
posterior pituitary bright spot is missing.f Differentiating between the various conditions which
share symptoms of polyuria and polydipsia can be chal-
lenging and can take considerable time [7, 8, 12]. Exist-
ing diagnostic tests can risk false diagnoses, both positive
and negative, particularly in patients with mild or partial
forms of CDI [23 PR-INT2]. Understanding the potential
cause of symptoms can help in the diagnostic process, as
some events, such as traumatic brain injury are associ-
ated with increased likelihood of CDI and can, together
with other tests, help to confirm whether a patient has
CDI or another condition. Therefore, a full patient his-
tory is an important part of the diagnostic process [11]. Timely diagnosis is impeded if symptoms are not
noticed promptly [10], or if other aetiologies for polyu-
ria and polydipsia are investigated first [13]. A lack of
awareness among primary care physicians regarding the
symptoms of CDI [PR-INT1, PR-INT2] can also impede
timely diagnosis and referral. The time to diagnosis can
vary across countries. Differentiating between the various conditions which
share symptoms of polyuria and polydipsia can be chal-
lenging and can take considerable time [7, 8, 12]. Exist-
ing diagnostic tests can risk false diagnoses, both positive
and negative, particularly in patients with mild or partial
forms of CDI [23 PR-INT2]. Understanding the potential
cause of symptoms can help in the diagnostic process, as
some events, such as traumatic brain injury are associ-
ated with increased likelihood of CDI and can, together
with other tests, help to confirm whether a patient has
CDI or another condition. Therefore, a full patient his-
tory is an important part of the diagnostic process [11]. Timely diagnosis is impeded if symptoms are not
noticed promptly [10], or if other aetiologies for polyu-
ria and polydipsia are investigated first [13]. A lack of
awareness among primary care physicians regarding the
symptoms of CDI [PR-INT1, PR-INT2] can also impede
timely diagnosis and referral. The time to diagnosis can
vary across countries. Given desmopressin’s mode of action, ongoing patient
monitoring during treatment dose optimisation is impor-
tant. This is because of the risk of developing hypona-
tremia if too little water is excreted or too much fluid
ingested [13], or hypernatremia [8, 12, 16, 17] if too
much water is lost or too little fluid ingested. Interviews with patient representatives It is impor-
tant to allow water offload in this process, to prevent
hyponatraemia. Optimisation therefore involves a spe-
cialist endocrinology clinic to determine patient response
to treatment and whether the dose needs to be further
adjusted. It usually takes 2–3 days, or up to a week as
an inpatient, but can take longer for infants [workshop
insights]. Treatment optimisation occurs during the same
admission as the diagnostic stage. Costs associated with diagnosis can vary a great deal
depending on the speed of diagnosis and the complexity
of the underlying aetiology, and if additional support is
needed, for example intensive care support in instances
of traumatic brain injury, or genetic testing to confirm
cause (see the cost section below). Interviews with patient representatives The workshops and one-to-one discussions with clinical
experts are limited to the countries they work in and are
based on their personal experience—however these are
all leading experts in their country contexts. The inter-
views are with patient representatives of a limited num-
ber of patient organisations who could engage with the
study, in part related to the small number of existing
patient organisations active in CDI. The cost estimations
in particular are limited by the availability of data and
detail that could be provided, and thus can only provide
an initial indication of cost rather than a comprehensive
overview. Despite these caveats, the insights from the
literature review and complementary views from clini-
cal experts and patient representatives provide a valu-
able contribution to enriching our understanding of how
care for CDI is provided and what the challenges and
improvement needs are. f
Magnetic Resonance Imaging (MRI) is essential to
determine potential causes of CDI—for example it can
show thickening of the pituitary stalk which might indi-
cate inflammation/autoimmune conditions, tumours
or other disorders, or a neoplastic process in the sellar Teare et al. Orphanet Journal of Rare Diseases (2022) 17:58 Teare et al. Orphanet Journal of Rare Diseases (2022) 17:58 Page 4 of 12 Infants and young children will require much lower
doses than adults, which will need to be adjusted over
time, as the children grow and develop. Young infants are
also dependent on fluid such as breast milk or formula
for nutrition, and this can present additional challenges
to fluid regulation and dose optimisation. This is because
the primary therapeutic goals of treating CDI in young
children are to reduce polyuria and decrease excessive
thirst to support appropriate levels of fluid uptake, and
to ensure appropriate growth. Children require adequate
fluid intake and various doses of DDAVP ranging from
a low dose of 10 µg/day for infants in cases of neonatal
DI to 60 µg/day three times a day during later childhood,
although higher doses could be necessary for individu-
alised patients. Careful exact dosing by cutting the
available oral disintegrating tablet DDAVP formulation
should be performed, particularly for young paediatric
patients. Starting with low doses and titration of the dose
is important. region [7]. However, there are challenges associated with
using MRI to identify etiological markers of CDI [20]. Treating CDI: treatment initiation and optimisation
d
h
b
h d Such clinic appointments are the
main healthcare service-related cost associated with the
longer-term management of CDI, as outlined in the cost
section below. should only be administered in intensive care or post-
operatively. According to one patient representative and
in their country context, paediatric patients are gener-
ally initiated on a nasal spray due to ease of administra-
tion, but the formulation may be changed depending on
the patient’s response [interview reference withheld to
preserve anonymity]. In mild cases of CDI in some coun-
tries, clinicians may consider not prescribing DDAVP and
instead suggest strategies for managing fluid balance and
using fluid replacement [interview reference withheld to
preserve anonymity]. Different formulations also take dif-
ferent times to act and hence some patients may prefer
formulations which provide relief quicker [PR-INT2]. As part of patient management, it is sometimes neces-
sary to not only adapt dosage but also to switch between
different formulations of desmopressin. This can happen
if patients find a specific formulation easier to adminis-
ter [15] or due to potential supply issues, or differences
in the effectiveness of a formulation [two interviewees,
interview references withheld to preserve anonymity]. Switching between formulations requires dose titration
to optimise the dose for each patient, as it is not possible
to predict how patients will respond to new formulations
based on their previous treatment protocols [14, 15]. This
is related to the absence of conversion factors between
different formulations. Switching between formulations
will require further in-clinic support, which can add to
the cost of treatment. Establishing conversion factors
between different formulations is challenged by the fact
that different formulations have different bioavailabil-
ity and that there is diversity in how individuals respond
to any specific formulation and dose. The UK electronic
medicines compendium1 provides a correlation table for
oral tablet and melt formulations for adults, but this is
based on bioavailability correlation research which tends
to take place in healthy volunteers, rather than patients
with CDI. Whereas such information may help orient
clinicians, it cannot guide clinical decision-making given
the highly personalised nature of dose optimisation for
CDI, and the frequent need for starting with lower doses
as part of the dose optimisation process. Treating CDI with desmopressin is generally safe and
effective and shown to improve patient quality of life [11],
but can come with side effects. Side effects, while rare,
vary slightly between formulation. Treating CDI: treatment initiation and optimisation
d
h
b
h d Once a diagnosis has been reached, it is necessary to tai-
lor treatment to the individual; given the marked varia-
bility in patient response to treatment, each patient needs
a personalised protocol [2, 9, 20]. Desmopressin is available in several different formu-
lations. Decisions relating to which formulation is most
appropriate depend on the patient being treated. It can be
administered orally as a tablet or as an oral disintegrating
tablet (ODT), as a buccal preparation, as a nasal spray, or
parenterally (usually intramuscularly or intravenously). The latter may be required in patients that are required to
fast pre-operatively or for patients needing intensive care
[1, 6, 8, 10, 24, PR-INT1, PR-INT2]. As introduced previously, CDI is most often treated
by replacing AVP with a synthetic vasopressin analogue
called desmopressin, in both adult and paediatric popula-
tions [1, 6, 8, 10 PR-INT1, PR-INT2, PR-INT3, workshop
insights]. In all patients with chronic CDI, a common
starting point for treatment is to first address nocturia, by
administering the first dose of desmopressin before bed
[22]. The patient’s response and dose will be measured
and adjusted over time until their symptoms are effec-
tively controlled. Initial treatment optimisation generally
involves starting with a low dose and gradually increas-
ing if needed [10]. Some adult patients may only need a
single dose per day to control their symptoms overnight
(although the strength of the single dose can vary signifi-
cantly between patients), while severe cases might need
up to 200 μg twice or three times a day (occasionally up
to 1 mg in total) [11]. Desmopressin lyophilizate sublingual tablets are a
valuable option for treating CDI in infants and young
children, with evidence of more stable absorption than
intranasal formulations and oral tablets [9, PR-INT1
and PR-INT2]. However, it can be difficult to split these
sublingual tablets into small enough doses for infants. Subcutaneous DDAVP administration enables the
administration of small doses to infants but this can lead
to more variable sodium concentrations than other for-
mulations [9, and the experience of an expert involved
with this research suggests that subcutaneous DDAVP Teare et al. Orphanet Journal of Rare Diseases (2022) 17:58 Teare et al. Orphanet Journal of Rare Diseases (2022) 17:58 Page 5 of 12 impact fluid requirements [workshop insights]. Annual
or biannual clinic appointments with an endocrinologist
are often required to ensure treatment doses and formu-
lations remain effective. 1 Electronic Medicines Compendium. 2012. ‘DDVAP Melt 60mcg oral lyoph-
ilisate’. Emc. As of 22 December 2021: https://www.medicines.org.uk/emc/
product/170/smpc#gref Treating CDI: treatment initiation and optimisation
d
h
b
h d The intranasal spray,
for example, may lead to dizziness, eye irritation, head-
ache, flushing, nausea, vomiting, rhinitis or epistaxis and
tachycardia. Evidence suggests that oral DDAVP is bet-
ter tolerated by some patients [20]. Dose adjustment can
help to mitigate side effects [workshop insights]. f
Although desmopressin is the main treatment for CDI
across different types of patient groups at present, some
other treatments are used in some countries (even if not
specific for use in CDI) with slightly different modes of
action and side effects. These include thiazide diuretics,
carbamazepine, chlorpropamide, clofibrate and indapa-
mide [9, 10, 13, 15]. However, desmopressin remains by
far the most commonly used form of treatment [10].h There is a lack of data on the costs associated with
treatment optimisation and these costs are likely, at least
in part, to depend on the complexity and length of the
optimisation process. Managing patients with CDI over time the dose for paediatric patients without discussion with a
specialist. In some countries, repeat prescriptions tend to
be organised by primary care without specialist involve-
ment, although the specialist centre would still direct
dosage and monitor the patient as needed. Long term management decisions other than those
related to medication will also be dependent on whether
patients are able to regulate thirst themselves, or whether
they are adipsic. Patients who are able to experience thirst
are generally encouraged to avoid excess fluid intake, and
drink to thirst rather than habit. For adipsic patients, it
is more difficult to keep track of how much water intake
is needed [11]. Therefore, a routine approach to drinking
with a daily fluid regimen based on strictly regulated fluid
intake with fixed amounts of water, is important to help
mitigate the risk of hyper- or hyponatremia [9]. This can
be particularly challenging if patients contract other ill-
nesses, for example if they are vomiting or eating less or
have diminished consciousness [11]. Fasting for a surgi-
cal procedure can also introduce risk, and must be super-
vised by a specialist, particularly if it requires a change
in treatment formulation, for example if the patient usu-
ally administers ODT [11]. According to experts con-
sulted for this research, physicians should be aware of
associated, treatable hypothalamic abnormalities such
as obesity, sleep apnoea, seizures and thermoregulatory
disorders when managing patients with adipsic diabetes
insipidus. From a fluid balance perspective, these patients
require regular DDAVP to treat their CDI but also need a
daily fluid prescription in order to maintain euvolaemia
(an appropriate blood fluid volume within the body) and
eunatraemia (an appropriate concentration of sodium in
blood plasma). Adjusting the desmopressin dosage and
fluid intake for adipsic patients is generally done in a hos-
pital setting and regular weighing and checking of serum
sodium levels is necessary. Both for adults and children, management of the con-
dition at home is also an important part of the patient
and/or carer journey [PR-INT1, PR-INT3]. Although
patients will tend to have regular six-monthly or annual
follow-up appointments, ensuring appropriate medica-
tion adherence and administration at home is key [PR-
INT2]. Lifestyle issues also require patients to engage
with ongoing management or monitoring through their
own behaviours. Managing patients with CDI over time Specialists—generally
endocrinologists—determine
which brand (or generic) and formulation to use, depend-
ing on what they think is best for the patient or what
they are most familiar with [workshop insights]. In the
countries included in this research, the use of branded
versions appears more common at the point of prescrip-
tion, possibly because generic versions are not available
or because of clinician preference [workshop insights]. Whether there is therapeutic equivalence between
branded or generic products for CDI has not been
researched. If patients are followed up in primary care,
primary care physicians can make decisions to switch
between branded or generic versions, if generic versions
are available. However, they generally would not change Given that CDI is usually a chronic condition, lifelong
management and ongoing monitoring are necessary to
ensure that patients respond well to their specific treat-
ment over time. Ongoing care is also necessary to ensure
that any changes to treatment are carefully managed
[7]. It is difficult to predict how a patient will respond to
treatment. Patient response can be influenced by their
ability to adhere to treatment regimens, for example to
control fluid intake or regularly take medication. It can
also be influenced by other factors such as other ill-
nesses—for example a cold which might affect how well
a patient absorbs desmopressin nasal spray, or vomiting
which will influence the intake of an oral tablet [2, 11, 15,
16]. Patient response to a specific treatment dose and for-
mulation can also be influenced by lifestyle factors such
as foreign travel to a warm country due to changing water
intake behaviours or participating in sport which might Teare et al. Orphanet Journal of Rare Diseases (2022) 17:58 Page 6 of 12 Page 6 of 12 dose adjustment mean that it is important to involve
the relevant experts early in this process to support any
required changes to dose [10]. In paediatric inpatients
who are unable to tolerate oral desmopressin (for exam-
ple while they are unwell with other conditions such as
gastro-intestinal issues) a switch to parenteral therapy
may be required, although such cases are rare. This
would typically be delivered intramuscularly, but intra-
venous pitressin infusion (vasopressin injection) can
also be used. If this is different from the patient’s usual
desmopressin formulation, particularly close monitoring
is required as it is not possible to predict how the patient
will respond [10]. Managing patients with CDI over time For example, stress, menstruation or
unusual consumption of a lot of salty/sweet foods may
require desmopressin to be taken at specific times in the
day [PR-INT2]. Influences on patient care and the care pathway
within healthcare systemsh The diagnosis of patients with CDI, decisions about
treatment dosage and formulation, and ongoing patient
monitoring and management are strongly influenced by
features of the wider healthcare system in which patients
are treated, as discussed below [4, 5, 7–11, 13, 16, 20–23,
25]. Influences related to skills and workforce capacity Awareness raising amongst other professions such as
teachers, university lecturers and air stewards may also
be needed so that patients can manage their condition
with as much ease as possible [PR-INT2].h f
Patient education can help equip patients with knowl-
edge about how to manage their disease, what to look
out for and side effects [8, 12, 23], which can in turn sup-
port effective communications with primary care physi-
cians about treatment and monitoring needs [workshop
insights]. In the UK, the Pituitary Foundation provides
resources that patients can share with general practition-
ers if they suspect they have CDI. There are also efforts in
some countries to rename the condition so as to remove
the term ‘diabetes’ to avoid confusion with diabetes melli-
tus [interview reference withheld to preserve anonymity]. Adjusting treatment formulations and dosages over
time also requires significant skills to make appropri-
ate decisions and to support personalised care, given
that different formulations have significantly differ-
ent bioavailability and that patient response differs [16,
19]. Any potential changes in relation to bioavailability
between branded and generic products require further
research (as there are gaps in evidence on this issue), to
inform policies on generic substitution and automatic
dispensing.hi [
p
y
y]
While there is a need for information and awareness
raising regarding the symptoms of CDI [PR-INT1], there
is also a need for more education on how families can
support treatment and management. This includes a need
for improved access to information about water intake,
food, salt levels and measuring water balance [PR-INT3]. Beyond patients, interviewees also highlighted the need
for better public understanding of CDI [PR-INT1, PR-
INT2], for example to avoid patients being refused access
to toilet facilities in some settings like shops [PR-INT2]. Awareness raising amongst other professions such as
teachers, university lecturers and air stewards may also
be needed so that patients can manage their condition
with as much ease as possible [PR-INT2]. The COVID-19 pandemic has also created specific
challenges for patients with CDI. These include limited
access to primary care physicians to get a referral to an
endocrinologist [interview reference withheld to pre-
serve anonymity], challenges accessing and undertaking
diagnostic testing [PR-INT1], and lack of sufficient follow
up with clinicians related to ongoing management needs. Influences related to skills and workforce capacity l
p
y
Supporting patients with CDI requires specialist skills
involving endocrinologists and including paediatricians
and nurses to support infants and children. Patient sup-
port will also draw on other specialties depending on the
cause of CDI and specific patient needs [9]. For example,
patients with CDI as a result of a tumour, or as a result
of tumour surgery, will require oncologist and neurosur-
geon involvement in care. Patients with CDI as a result
of traumatic brain injury may need intensive care teams,
and patients with CDI in pregnancy will need additional
support, for example from obstetrics [workshop insights]. Appropriate support requires the healthcare system to
have sufficient workforce capacity and a cadre of highly
skilled staff across specialties [2, 11, 15, 16]. Similarly, patients with CDI diagnosis prior to preg-
nancy who are already receiving doses of exogenous vas-
opressin or desmopressin may require increased doses
during pregnancy and additional monitoring to ensure
that there are no complications due to increased dosages
[1]. Given the impact of CDI on growth and development
in infants, more frequent monitoring is also particularly
important in this vulnerable patient population. This will
include monitoring serum sodium, weight and hydra-
tion to allow doses to be modified, and home-monitor-
ing including weighing wet ‘diapers’ [25]. According to
an expert involved with this research, daily weighing of
an infant/young child patient should make it possible to
detect abnormal weight gain, and plasma sodium con-
centration should be monitored frequently to reduce
the risk of hyponatremia. The challenges associated with f
Several of the healthcare systems included in this
paper have specialist centres that support patients across
a large geographic region. This allows expertise to be Teare et al. Orphanet Journal of Rare Diseases (2022) 17:58 Page 7 of 12 engaging with healthcare professionals about opti-
mising treatment [17]. For example, patients may
have preferences for specific formulations [workshop
insights] in light of their absorption, the types of com-
plications experienced or how straightforward the for-
mulation is to administer. Patient engagement is also
essential in the context of monitoring treatment effec-
tiveness and reporting any changes in effectiveness to
healthcare professionals [6, 17], as well as in the con-
text of ensuring dose adherence and compliance with
treatment [8, 20]. For some formulations, patient skills
in administering the treatment, such as dexterity in
administering intranasal formulations, play a role in
treatment effectiveness [16]. Influences related to skills and workforce capacity According to one patient representative, some patient
support groups and helplines saw an increase in enquir-
ies as a result [interview reference withheld to preserve
anonymity]. There is a need to consider how patients can
best be cared for in the context of any future pandem-
ics, both in terms of remote care and any essential face to
face contact with clinicians. Those patients who are on both glucocorticoid (for
example in the case of panhypopituitarism) and DDAVP
replacements may need educational support about spe-
cific sick day management. Glucocorticoids are essen-
tial for the excretion of water. Some patients who have
central diabetes insipidus also have anterior pituitary
dysfunction and as such require glucocorticoid replace-
ment. When a child taking both regular glucocorticoid
and DDAVP becomes unwell, the parents may need to
ensure that the glucocorticoid is doubled or trebled,
and that the child is passing urine before giving further
doses of DDAVP. In the event of inadequate glucocorti-
coid administration, water will not be effectively excreted
and the administration of DDAVP can lead to water Influences related to skills and workforce capacity concentrated but can also require patients to travel long
distances to seek specialist advice and support [PR-INT1,
PR-INT3]. This places significant emphasis on the first
stages of patient support, often through primary care
or other departments such as emergency medicine or
oncology, to recognise specific patient needs [workshop
insights]. In some countries, the limited number of hos-
pitals with relevant clinical expertise can also create long
waiting times for treatment [PR-INT1] or necessitate
remote management. Healthcare professionals in primary care (both pri-
mary care physicians and nurses) can sometimes lack
sufficient awareness about the symptoms of CDI which
can complicate timely diagnosis and referrals [PR-INT1,
PR-INT2, PR-INT3]. This can sometimes lead to inap-
propriate treatment [PR- INT1, PR-INT2]. Primary care
physicians are also sometimes not appropriately trained
on the importance of carefully managing and monitoring
appropriate treatment dosing and formulations as patient
conditions change [workshop insights]. In a hospital care
setting, not all physicians always understand how desmo-
pressin acts, which can complicate the inpatient care of
patients with CDI who are admitted for other conditions
[8, 25]. f
Patient education can help equip patients with knowl-
edge about how to manage their disease, what to look
out for and side effects [8, 12, 23], which can in turn sup-
port effective communications with primary care physi-
cians about treatment and monitoring needs [workshop
insights]. In the UK, the Pituitary Foundation provides
resources that patients can share with general practition-
ers if they suspect they have CDI. There are also efforts in
some countries to rename the condition so as to remove
the term ‘diabetes’ to avoid confusion with diabetes melli-
tus [interview reference withheld to preserve anonymity]. While there is a need for information and awareness
raising regarding the symptoms of CDI [PR-INT1], there
is also a need for more education on how families can
support treatment and management. This includes a need
for improved access to information about water intake,
food, salt levels and measuring water balance [PR-INT3]. Beyond patients, interviewees also highlighted the need
for better public understanding of CDI [PR-INT1, PR-
INT2], for example to avoid patients being refused access
to toilet facilities in some settings like shops [PR-INT2]. Availability and use of appropriate treatment formulations
and medicines management and the role of regulation The availability of appropriate formulations that are safe
and effective as well as convenient to administer plays
a role in treatment decisions and their appropriateness
for users [12, 16, 20, 25]. Communication between cli-
nicians and patients in relation to why some formula-
tions may be available and why there may be shortages
of others (or lack of availability) matters in terms of
patient-centred care and good patient-physician com-
munication [workshop insights]. Patient representatives
raised issues regarding treatment availability and supply
[interview reference withheld to preserve anonymity]. In one country, an interviewee commented that intrana-
sal solution and nasal spray forms of desmopressin have
been unavailable since the autumn of 2020, with patients
and support groups not knowing when the supply will
return [interview reference withheld to preserve ano-
nymity]. Many patients using nasal formulations have
subsequently transferred to sheets/melts, which created
further supply issues. f
Clinical experts from four countries shared cost esti-
mations for their own hospital settings (see Table 1). In
all four countries, the costs of healthcare service provi-
sion (i.e. consultations and diagnostic tests) are covered
by the public healthcare system. However, the clinical
experts also noted that given the personalised nature of
CDI treatment and management, costs per patient can
vary substantially within any one given context [work-
shop insights]. This means that healthcare services costs
are not directly comparable between countries as well as
between different hospital settings. In terms of medicine
costs, in health systems with a mixture of public and pri-
vate funding, desmopressin is usually covered as part of a
long-term illness scheme, and therefore publicly funded
[workshop insights]. Regulation plays an important role in ensuring the
availability of appropriate treatments and in promot-
ing good practice. For example, the intranasal spray for-
mulation of desmopressin is no longer approved by the
United States Food and Drug Administration, and this
has led to a switch to the tablet or melt formulation in
many countries [15]. Similarly, it is important that regula-
tory officials understand reasons for certain decisions, for
example in relation to medicine switching and reasons
for product recalls, to support national decisions and
guidance that can impact patients directly [15]. While there is literature discussing good practice for
specific patient groups (e.g. Influences related to patient engagement with care related
decisions and behaviours Patients and/or caregivers have an important role
to play in the ongoing management of CDI and in Teare et al. Orphanet Journal of Rare Diseases (2022) 17:58 Page 8 of 12 Page 8 of 12 20162) [25]. This can have significant cost implications
over a patient’s lifetime. Data from the UK3 provides
costs for desmopressin acetate, with 90 oral tablets of
100 μg individual doses reported to cost £44 (based on
2016 data, equivalent to 46.62 euros in 2016) and a pack
of 100 sublingual disintegrating tables of 60 μg indi-
vidual doses reported to cost £51 (based on 2016 data,
equivalent to 54.03 euros in 2016). A pharmacoeconomic
analysis of vazomirin spray (a treatment used in Russia
with desmopressin as the active ingredient) compared
with other forms of administration concluded that the
cost of vazomirin spray, compared to vazomirin tablets
is reduced by 45–48% in patients after resection of chi-
asmo-sellar region (CSR) tumours [3]. In general, cost
considerations are an important factor in making treat-
ment decisions. Although this research study did not
focus on gathering specific medicines cost data in indi-
vidual countries as part of its scope, the costs of CDI
treatment are likely to vary across different geographies,
as is the case with many medicines. Therefore, costs in
a US context cannot be used to make inferences about
costs of medicines in different European countries. intoxication and hyponatraemia with related complica-
tions such as seizures. Availability and use of appropriate treatment formulations
and medicines management and the role of regulation children and infants) [9],
and guidance on inpatient management of CDI [4], we
did not identify national level guidelines in the sample
of papers reviewed relating to the countries of interest,
namely: France, Ireland, Italy, Spain or the UK. Given the limitations set out above, the information in
the table below is shared purely for illustrative purposes
and cannot be used to infer costs for an entire system
nor for other settings. It seeks only to provide some ini-
tial information on what healthcare costs are like in some
settings, given the paucity of any such insights in the
existing literature. The data in the table can also not be
aggregated into higher level categories to make inferences 2 OECD Data. 2021. ‘Purchasing power parities (PPP)’. OECD. As of 22
December 2021: https://data.oecd.org/conversion/purchasing-power-parities-
ppp.htm#indicator-chart
3 Horizon Scanning Research & Intelligence Centre. 2016. ‘Low dose
desmopressin lyophilisate (Noqdirna) for nocturia in adults. NIHR Inno-
vation Observatory. As of 22 December 2021: https://www.io.nihr.ac.uk/
wp-content/uploads/migrated/Low-dose-desmopressin-lyophilisate-Noqdi
rna-June16.pdf 3 Horizon Scanning Research & Intelligence Centre. 2016. ‘Low dose
desmopressin lyophilisate (Noqdirna) for nocturia in adults. NIHR Inno-
vation Observatory. As of 22 December 2021: https://www.io.nihr.ac.uk/
wp-content/uploads/migrated/Low-dose-desmopressin-lyophilisate-Noqdi
rna-June16.pdf 2 OECD Data. 2021. ‘Purchasing power parities (PPP)’. OECD. As of 22
December 2021: https://data.oecd.org/conversion/purchasing-power-parities-
ppp.htm#indicator-chart Costs and effective resourcing to ensure appropriate
capacity Economic considerations will vary across healthcare
systems, and economic burden may play a role for some
patients [3, 25]. For example, a US-based study reported
a one-month supply of intranasal DDAVP to cost USD
245.80 (based on 2016 data, equivalent to 179.43 euros
in 2016), while a one-month supply of subcutane-
ous DDAVP cost USD 565.25 for treating infants in the
USA (based on 2016 data, equivalent to 412.63 euros in Page 9 of 12 Teare et al. Orphanet Journal of Rare Diseases (2022) 17:58 Table 1. Examples of costs for treating a patient with CDI—estimates from a sample of hospital settings (Country names withheld to preserve anonymity, all costs expressed in
Euro equivalents)
Stage for each country
A (adults)
B (paediatric)
C (paediatric)
D (paediatric)
Diagnosis
The costs presented relate to the
diagnosis and management of CDI as a
result of acute CDI. This will differ from
adults presenting with polydipsia and
polyuria, and can take over six months
longer to diagnose
Initial visit including blood tests:
EUR 750–800
Cost of hospitalisation and first diag‑
nosis:
EUR 1500–4500
Including admission in the paediatric
endocrine unit for initial evaluation,
diagnosis and treatment initiation (four
days), and for repeated evaluation every
six months during the first three years
after diagnosis (two days for clinical,
biological and cerebral MRI assess‑
ments and treatment optimisation):
EUR 50,000
Cost estimates for paediatric patients
including admission into the paediatric
endocrine unit for initial evaluation,
diagnosis and treatment initiation (three
days), and for repeated evaluation every
six months during the first three years
after diagnosis: EUR 37,600 (costs of
DDAVP not included)
One day of testing including water
deprivation test:
EUR 2300–2500
In patients who need cerebrospinal
fluid analysis and/or pituitary stalk
biopsy, additional costs: EUR 2000–3000
MRI:
EUR 400
Supporting patients (based on the
imaging work-up) every six months for
the first two years of treatment:
EUR 400–1000
Neurosurgery including hospital stay if
required:
EUR 10,000–15,000 (approximately EUR
1500 per day)
Idiopathic CDI may have additional
costs a long time after diagnosis as a
result of complications developing –
specific cost data not available. Costs and effective resourcing to ensure appropriate
capacity ICU stay if required:
EUR 10,000 per day
Genetic diagnosis may be needed in
rare cases: EUR 500
Long term follow-up
Outpatient visits: EUR 300 for two visits
per year
One clinical and biological examination
every six months:
EUR 3000 per year (for following seven
years post diagnosis)
Approximately EUR 2350 per year. Data on costs of treatments (therapeu‑
tics) is not available
Desmopressin:
EUR 6000 for 10 years
Desmopressin treatment (on average
60 ug x 3/day):
EUR 6500 for 10 years
Data on costs of treatments (therapeu‑
tics) is not available
If other hormonal treatment is required,
such as growth hormone treatment
(with about 1 mg per day), an addi‑
tional cost of up to EUR 90,000 for 10
years can be incurred
Approximate total for 10 years
EUR 8450–34,200
(more if longer than one day in ICU, and
not including medication costs)
EUR 12,100–28,500
Approximately EUR 60,000 (depending
on patient complexity)
(if growth hormone treatment if
needed, additional costs are incurred)
Approximately EUR 54,000 (depending
on patient complexity) al settings (Country names withheld to preserve anonymity, all costs expressed in Long term follow-up Teare et al. Orphanet Journal of Rare Diseases (2022) 17:58 Page 10 of 12 about overall diagnosis and care costs, given that differ-
ent settings collect different types of cost-related infor-
mation. However, the table begins to shed light on the
potential cost items implied in the care of patients with
CDI and indicates that the healthcare service costs are
not negligible. professionals to identify symptoms, determine optimal
treatment approaches and to effectively manage changes
in treatment needs over time.h The cost data drawn from four hospital settings, despite
the caveats associated with the data, provides an indica-
tion of the impact that time-consuming and challenging
diagnostic testing and personalisation of treatment can
have on healthcare system resource demands, with costs
concentrated in the early stages of the patient pathway. Further research is needed to understand healthcare ser-
vice-related costs across a broader range of settings. g g
The data provided for each country refer to a specific
patient population (paediatric or adult CDI patients),
with particular mechanisms for data collection and pro-
vision based on information clinicians could gather. Other caveats to bear in mind are that the figures pro-
vided could be broken down by initial diagnosis, long
term care and estimates of total costs for a 10-year
timeframe and indicate highly variable ranges, in part
dependent on patient complexity. For example, cost can
be influenced by whether additional complex testing is
needed beyond standard tests for CDI, such as additional
cerebrospinal fluid analysis or biopsy to determine the
cause of CDI, and by whether patients with CDI caused
by traumatic brain injury need to be supported by neu-
rosurgery. It can also be influenced by how long a patient
needs to stay in hospital and whether they then need
intensive care. If other hormonal treatment is required in
addition to desmopressin, such as growth hormone treat-
ment, there will be an additional cost. Therefore, costs of
care are highly individualised depending on the specific
circumstances of a patient. However, despite these fig-
ures being only indicative and context-specific and illus-
trating diverse cost-ranges across contexts, they indicate
that the costs of caring for patients with CDI are not neg-
ligible and reinforce key insights we have gained about
the complexity of the process. Long term follow-up We have also identified key areas for future explora-
tion, as part of a commitment to improving the diag-
nosis, treatment and management of patients with CDI
and supporting patient quality of life and outcomes. Our
research suggests a need to focus efforts on some key
areas. We acknowledge that the extent to which these
apply more widely—i.e. beyond the countries represented
by the clinical co-authors (France, Ireland, Italy, Spain,
and the UK)—merits further research. However, these
insights are also supported by the challenges highlighted
in the wider global literature. More specifically, in reflecting on the challenges we
have identified in this paper, there is scope to consider
actions which could lead to improved guidance for pri-
mary care physicians on how to identify symptoms
and manage patients with CDI. There is also a need for
more information, education and awareness raising for
patients, carers and families on how to better manage
the condition, including in light of comorbidities and
changes in patient circumstances. Working with patient
associations may be important in this regard. Develop-
ing education and outreach for patients may also help
patients engage with primary care and with specialists in
secondary care. It is worth noting that there can also be additional out
of pocket costs for patients, such as the cost of items nec-
essary for coping with the condition, for example ice cold
water, nappies and toilet rolls [PR-INT2]. Given the complexities of managing patients with CDI,
further research is needed to understand whether there is
potential for greater international consensus or guidance
on best practice in the treatment and management of spe-
cific types of patients, not dismissing the importance of
personalised care. This may require research that would
gather retrospective and real-world data to identify how
specific practices differ across a broad range of contexts
and how they relate to patient outcomes for patients with
specific clinical and behavioural parameters. References 1. Ananthakrishnan S. Diabetes insipidus during pregnancy. Best Pract Res
Clin Endocrinol Metab. 2016;30(2):305–15. 1. Ananthakrishnan S. Diabetes insipidus during pregnancy. Best Pract Res
Clin Endocrinol Metab. 2016;30(2):305–15. 2. Arima H, Oiso Y, Juul KV, Nørgaard JP. Efficacy and safety of desmopressin
orally disintegrating tablet in patients with central diabetes insipidus:
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orally disintegrating tablet in patients with central diabetes insipidus:
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pitalised children. Arch Dis Childh Educ Pract Ed 2017;102:edpract-2016. 10. Elder C, Dimitri P. Diabetes insipidus and the use of desmopressin in hos‑
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Pract Res Clin Endocrinol Metab. 2020:101385. 13. Hunter JD, Calikoglu AS. Etiological and clinical characteristics of central
diabetes insipidus in children: a single center experience. Int J Pediatr
Endocrinol. 2016;2016:3. Discussion and conclusion: towards improved care
pathways Within this paper we have considered current practice
in the diagnosis, treatment and management of CDI,
and discussed the associated challenges relating to dif-
ferential diagnosis and highly personalised treatment and
management. We have shown that CDI requires complex patient
support. This means care delivery needs to be flexible
to meet individual patient needs. The fact that patient
response to the mainstream treatment desmopressin can
change over time makes long term management needs
difficult to predict. This highlights the need for patients
to be well-informed in order to effectively engage with
healthcare professionals in primary and specialist care. It also highlights the need for highly skilled healthcare i
Finally, the COVID-19 pandemic introduced further
challenges for patients with CDI, given high needs for
inpatient care during diagnosis and treatment optimi-
sation and there is scope for the clinical community to
jointly consider how to approach patient care in the con-
text of future pandemic preparedness. We hope that the insights and reflections we have
shared in this research help to raise awareness of the Teare et al. Orphanet Journal of Rare Diseases (2022) 17:58 Page 11 of 12 Page 11 of 12 complexity of managing patients with CDI and support
future efforts of clinicians, patient associations, policy-
makers and the community of patients and carers com-
mitted to improving the care of patients with this rare
but life-impacting condition. Universitario Niño Jesús, Instituto de Investigación La Princesa, Universidad
Autónoma de Madrid, Madrid, Spain. 3 Centro de Investigación Biomédica en
Red (CIBER) de Fisiopatología de la Obesidad y Nutriciόn (CIBEROBN), Instituto
de Salud Carlos III, Madrid, Spain. 4 IMDEA, Food Institute, CEIUAM+CSI, Madrid,
Spain. 5 UCL Great Ormond Street (GOS) Institute of Child Health, University
College London, London, UK. 6 Great Ormond Street Hospital for Children,
London, UK. 7 Assistance Publique‑Hôptaux de Paris, Pediatric Endocrinol‑
ogy‑Diabetology Department, Reference Center for Growth and Development
Endocrine Diseases, Robert Debré University Hospital, Université de Paris,
NeuroDiderot INSERM UMR 1141, 75019 Paris, France. 8 Department of Pedi‑
atrics, IRCCS Istituto Giannina Gaslini, Genoa, Italy. 9 Department of Neurosci‑
ence, Rehabilitation, Ophthalmology, Genetics, Maternal and Child Health,
University of Genova, Genoa, Italy. 10 Department of Endocrinology, Beaumont
Hospital and Royal College of Surgeons in Ireland, Dublin, Ireland. Acknowledgements We would like to thank Pat MacBride from the Pituitary Foundation, UK, Robert
Brady from the Pituitary Foundation, Ireland, and Diana Vitali from the Italian
Family Association of Septo-optic Dysplasia and Pituitary Abnormalities, Italy,
for their insightful contributions to this study, providing an overview of the
patient experience across different health systems. We would also like to
thank Dr. Camilla D’Angelo and Giulia Maistrello, analysts at RAND Europe,
for their help in translation, as part of the recruitment of patient representa‑
tives, and Dr. Daniela Rodriguez Rincon for her involvement in early stages
of the research. This research study was funded by Ferring Pharmaceuticals,
and we thank Teodor Burtea, MD and Lena Maislisch, M.Sc., both from Global
Medical Affairs at Ferring, for their contributions to the concept for this study
at ideation stages. The research was conducted and data analysed entirely
independently, in line with RAND Europe’s role as an impartial, not-for-profit
research organisation. Competing interests The authors have no competing interests to declare. 18. Mavinkurve M, McGrath N, Johnston N, Moloney S, Murphy NP, Hawkes
CP. Oral administration of diluted nasal desmopressin in manag‑
ing neonatal central diabetes insipidus. J Pediatr Endocrinol Metab. 2017;30(6):623–8. Authors’ contributions 5. Barbot M, Ceccato F, Zilio M, Albiger N, Sigon R, Rolma G, et al. Cardio‑
vascular autonomic dysfunction in patients with idiopathic diabetes
insipidus. Pituitary. 2018;21(1):50–5. G-CA and JF were responsible for conducting interviews with patient rep‑
resentatives, all authors were involved in the workshop discussions. SM and
HT contributed to the design of the study. All authors (HT, JA, MD, JL, MM,
MS, G-CA, JF, SM) contributed to drafting and developing the manuscript. All
authors read and approved the final manuscript. 6. Chanson P, Salenave S. Diabetes insipidus and pregnancy. Ann Endocrino
(Paris). 2016;77(2):135–8. 7. Christ-Crain M, Bichet DG, Fenske WK, Goldman MB, Rittig S, Verbalis JG,
et al. Diabetes insipidus. Nat Rev Dis Primers. 2019;5(1):54. 7. Christ-Crain M, Bichet DG, Fenske WK, Goldman MB, Rittig S, Verbalis JG,
et al. Diabetes insipidus. Nat Rev Dis Primers. 2019;5(1):54. Ethics approval and consent to participate 14. Ito A, Nozaki A, Horie I, Ando T, Kawakami A. Relation between change in
treatment for central diabetes insipidus and body weight loss. Minerva
Endocrinol. 2019;44(1):85–90. This research took the form of a literature review, supplemented with a small
number of interviews and workshops with clinical experts and representatives
from patient organisations. It was judged to pose minimal risks to participants
and ethical approval was not sought. The research was conducted in accord‑
ance with the Declaration of Helsinki. All participants gave informed consent
and were provided with participant information sheets as part of this process. 15. Kalra S, Zargar AH, Jain SM, Sethi B, Chowdhury S, Singh AK, et al. Diabetes insipidus: the other diabetes. Indian J Endocrinol Metab. 2016;20(1):9–21. 16. Kataoka Y, Nishida S, Hirakawa A, Oiso Y, Arima H. Comparison of inci‑
dence of hyponatremia between intranasal and oral desmopressin in
patients with central diabetes insipidus. Endocr J. 2015;62(2):195–200. Supplementary Information The online version contains supplementary material available at https://doi.
org/10.1186/s13023-022-02191-2. Consent for publication p
All authors have consented to the publication of this manuscript. 17. Masri-Iraqi H, Hirsch D, Herzberg D, Lifshitz A, Tsvetov G, Benbassat C,
et al. Central diabetes insipidus: clinical characteristics and long-term
course in a large cohort of adults. Endocr Pract. 2017;23(5):600–4. Author details
1 1 RAND Europe, Westbrook Centre Milton Road, Cambridge CB4 1YG, UK. 2 Department of Pediatrics and Pediatric Endocrinology, Hospital Infantil Page 12 of 12 Teare et al. Orphanet Journal of Rare Diseases (2022) 17:58 Teare et al. Orphanet Journal of Rare Diseases (2022) 17:58 19. Nozaki A, Ando T, Akazawa S, Satoh T, Sagara I, Horie I, et al. Quality of
life in the patients with central diabetes insipidus assessed by Nagasaki
Diabetes Insipidus Questionnaire. Endocrine. 2016;51(1):140–7. 20. Patti G, Ibba A, Morana G, Napoli F, Fava D, di Iorgi N, et al. Central diabe‑
tes insipidus in children: Diagnosis and management. Best Pract Res Clin
Endocrinol Metab. 2020:101440. 20. Patti G, Ibba A, Morana G, Napoli F, Fava D, di Iorgi N, et al. Central diabe‑
tes insipidus in children: Diagnosis and management. Best Pract Res Clin
Endocrinol Metab. 2020:101440. 21. Patti G, Scianguetta S, Roberti D, Di Mascio A, Balsamo A, Brugnara
M, et al. Familial neurohypophyseal diabetes insipidus in 13 kindreds
and 2 novel mutations in the vasopressin gene. Eur J Endocrinol. 2019;181(3):233–44. 22. Refardt J, Christ-Crain M. Diabetes insipidus in pregnancy: how to advice
the patient? Minerva Endocrinol. 2018;43(4):458–64. 22. Refardt J, Christ-Crain M. Diabetes insipidus in pregnancy: how to advice
the patient? Minerva Endocrinol. 2018;43(4):458–64. 3. Refardt J, Winzeler B, Christ-Crain M. Diabetes Insipidus: An Upda 23. Refardt J, Winzeler B, Christ-Crain M. Diabetes Insipidus:
Endocrinol Metab Clin North Am. 2020;49(3):517–31. 23. Refardt J, Winzeler B, Christ-Crain M. Diabetes Insipidu
Endocrinol Metab Clin North Am. 2020;49(3):517–31. 24. Sbardella E, Pozza C, Isidori AM, Grossman AB. Endocrinology and adoles‑
cence: dealing with transition in young patients with pituitary disorders. Eur J Endocrinol. 2019;181(4):R155–71. 25. Smego AR, Backeljauw P, Gutmark-Little I. Buccally administered intra‑
nasal desmopressin acetate for the treatment of neurogenic diabetes
insipidus in infancy. J Clin Endocrinol Metab. 2016;101(5):2084–8. Teare et al. Orphanet Journal of Rare Diseases (2022) 17:58 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub‑
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https://www.emerald.com/insight/content/doi/10.1108/IJDI-12-2023-0287/full/pdf?title=efficiency-of-brics-countries-in-sustainable-development-a-comparative-data-envelopment-analysis
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Efficiency of BRICS countries in sustainable development: a comparative data envelopment analysis
|
International journal of development issues
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The current issue and full text archive of this journal is available on Emerald Insight at:
https://www.emerald.com/insight/1446-8956.htm
Efficiency of BRICS countries in
sustainable development: a
comparative data envelopment
analysis
Isabella Melissa Gebert
Iscte-Instituto Universitário de Lisboa, Lisbon, Portugal, and
Efficiency of
BRICS
countries
Received 12 December 2023
Revised 27 February 2024
Accepted 8 March 2024
Felipa de Mello-Sampayo
Iscte-Instituto Universitário de Lisboa and Business Research Unit (BRU-IUL),
Lisbon, Portugal
Abstract
Purpose – This study aims to assess the efficiency of Brazil, Russia, India, China, South Africa (BRICS)
countries in achieving sustainable development by analyzing their ability to convert resources and
technological innovations into sustainable outcomes.
Design/methodology/approach – Using data envelopment analysis (DEA), the study evaluates the
economic, environmental and social efficiency of BRICS countries over the period 2010–2018. It ranks these
countries based on their sustainable development performance and compares them to the period 2000–2007.
Findings – The study reveals varied efficiency levels among BRICS countries. Russia and South Africa lead
in certain sustainable development aspects. South Africa excels in environmental sustainability, whereas
Brazil is efficient in resource utilization for sustainable growth. China and India, despite economic growth,
face challenges such as pollution and lower quality of life.
Research limitations/implications – The study’s findings are constrained by the DEA methodology
and the selection of variables. It highlights the need for more nuanced research incorporating recent global
events such as the COVID-19 pandemic and geopolitical shifts.
Practical implications – Insights from this study can inform targeted and effective sustainability
strategies in BRICS nations, focusing on areas such as industrial quality improvement, employment
conditions and environmental policies.
Social implications – The study underscores the importance of balancing economic growth with social
and environmental considerations, highlighting the need for policies addressing inequality, poverty and
environmental degradation.
Originality/value – This research provides a unique comparative analysis of BRICS countries’ sustainable
development efficiency, challenging conventional perceptions and offering a new perspective on their progress.
Keywords Sustainable development, Data envelopment analysis, Efficiency, BRICS
Paper type Research paper
© Isabella Melissa Gebert and Felipa de Mello-Sampayo. Published by Emerald Publishing Limited.
This article is published under the Creative Commons Attribution (CC BY 4.0) licence. Anyone may
reproduce, distribute, translate and create derivative works of this article (for both commercial and
non-commercial purposes), subject to full attribution to the original publication and authors. The full
terms of this licence may be seen at http://creativecommons.org/licences/by/4.0/legalcode
Fundação para a Ciência e Tecnologia under UIDB/00315/2020 grant is gratefully acknowledged.
The funding body had no influence in the design of the study, collection, analysis or interpretation of
data or in writing the manuscript.
International Journal of
Development Issues
Emerald Publishing Limited
1446-8956
DOI 10.1108/IJDI-12-2023-0287
IJDI
1. Introduction
Economic growth theories have evolved from emphasizing capital accumulation, as in
classical and neoclassical theories, to highlighting the importance of human capital in more
contemporary frameworks such as the Endogenous Growth theory (Choudhry et al., 2020).
Dore and Teixeira (2023) synthesized these factors into seven categories: human capital,
labor and demographic conditions, technology and innovation, macroeconomic conditions,
international trade and FDI, natural resources and institutional conditions. However, the
relative influence of these factors varies significantly between and within countries, as noted
by Chirwa and Odhiambo (2016). In developed countries, key factors include physical
capital, fiscal policy and human capital, among others, while developing countries focus
more on external elements such as FDI and foreign aid. The complex interplay of these
factors is further complicated by issues such as income inequality, labor exploitation and
environmental degradation, underscoring the need for quality growth that reflects true wellbeing (Lopez et al., 2008; Ranis et al., 2000). This recognition has led to the development of
new indicators such as the Human Development Index, the Gini Coefficient and life
expectancy at birth, which include aspects of life quality, income distribution and health, but
often overlook the environmental costs of economic growth (Santana et al., 2014; Chen et al.,
2021; Hasell, 2023).
The concept of Sustainable Development emerged as a response to these challenges,
aiming to harmonize economic growth with environmental conservation (Almeida et al.,
2017). Its roots can be traced back to classical economists such as Malthus, Smith, Ricardo
and Mill, who expressed concerns about the sustainability of economic growth and its
implications for future generations (Purvis et al., 2019). The concept gained prominence after
the 1972 Stockholm Conference, which led to the formation of the United Nations
Environmental Program. However, the conference highlighted the tension between
environmental protection and the development needs of different countries (Chasek, 1994;
Prizzia, 2017). Sustainable development was formally defined as meeting current needs
without compromising future generations’ ability to meet their own (Brundtland, 1987). The
term “sustainability” has since been interpreted in various ways, often encompassing social,
economic and environmental dimensions. This three-pillar model, while not explicitly
outlined in foundational documents such as the Brundtland Report or Agenda 21, has
become a widely accepted framework for understanding sustainability (Purvis et al., 2019;
Moldan et al., 2012). Brown et al. (1987) and Pope et al. (2004) further explored these
dimensions, emphasizing the integration of social needs, environmental protection and
economic growth as key to implementing sustainable development.
Brazil, Russia, India, China and South Africa (BRICS) countries account for approximately
40% of the global population, 30% of the land area, 18% of international trade and a quarter of
the world’s GDP, surpassing the economies of both the USA and the European Union, as
highlighted by O’Neill (2021). Moreover, their substantial role in fossil fuel production and
consumption ranks them among the largest emitters of greenhouse gases (GHGs), significantly
impacting global climate governance, a challenge discussed by Downie and Williams (2018).
The BRICS face the complex task of maintaining rapid economic growth while controlling
carbon emissions. Exploring their historical growth determinants, environmental impacts and
population welfare offers crucial insights for their future development paths.
Following Santana et al. (2014), this study examines how well the BRICS countries are doing
in terms of sustainable development, using the data envelopment analysis (DEA) method that
allows to rank the BRICS countries based on their economic, environmental and social
efficiency. We specifically look at how efficiently these countries have been using their
productive resources and technological innovations to achieve sustainable development over
an eight-year period (2010–2018). The motivation for extending the data for Santana et al.’s
(2014) paper stems from the dynamic nature of economic, social and environmental
development across the BRICS. Santana et al.’s (2014) study provided critical insights into the
efficiency of these countries in converting inputs into sustainable development outcomes over
the period 2001–2007. However, the global economic landscape, environmental challenges and
social dynamics have undergone significant changes since then. Extending the data set beyond
2007 allows for an updated analysis that reflects recent developments, including technological
advancements, policy reforms and shifts in global economic power dynamics. This extension
aims to capture the evolving nature of sustainability efforts within the BRICS, offering a more
current understanding of their progress and challenges in achieving economic growth,
environmental protection and social welfare. It acknowledges the importance of continuous
monitoring and evaluation in the context of sustainable development, providing stakeholders
with relevant data to inform policymaking and strategic planning in an ever-changing global
environment.
The results of this study show the different challenges the BRICS countries have in
achieving sustainable development. Brazil is efficient in using its resources and foreign
investment for sustainable growth but needs to work on issues such as deforestation, social
inequality and political instability. Russia is the most efficient of the group but relies heavily
on oil and gas, which affects its sustainability. It could improve by investing more in human
resources. India and China have grown economically but this has led to more pollution and a
lower quality of life. They need to focus on cleaner industrial growth and better working
conditions and use foreign investment and research to reduce pollution and improve living
standards. South Africa has low pollution levels and is doing well in addressing social and
health issues, but it needs to keep working on these areas to improve people’s lives.
This paper is organized as follows. Section 2 starts discussing BRICS countries’
economic development. The methodology section given in Section 3 explains the research
design and analysis techniques used. The findings and analysis along with the results are
presented in Section 4. The discussion is presented in Section 5. The paper ends with a
conclusion given in Section 6 which summarizes the main points and suggests areas for
future research.
2. Economic growth and sustainable development of the Brazil, Russia, India,
China, South Africa
Since their formal recognition as a cohesive group in 2009, although they did not initially
collaborate closely, as noted by Lowe (2016), BRICS leaders have convened regularly to
formulate cooperative policies and strategies aimed at bolstering their joint economic
growth. Despite attracting global attention for their economic prowess, growth within the
BRICS has seen a period of stagnation and inconsistency since 2011, as observed by Fisher
(2022).
The BRICS consist of a diverse set of countries, each with unique political and economic
frameworks (Chatterjee and Naka, 2022). Brazil and Russia, for instance, are primarily
exporters of raw materials, dependent on their rich mineral reserves. In contrast, China and
India have leveraged their vast labor forces, with China emerging as a major manufacturer
and India as a significant service provider (Streltsov et al., 2021). South Africa stands out as
the most industrialized nation in Africa, with substantial mineral wealth and a reputation as
a stable investment destination (Lowe, 2016). Despite their differences, these countries share
certain common economic traits (Basu et al., 2013). For instance, a large proportion of trade
in intermediates occurs among BRICS countries (De Mello-Sampayo, 2017a, 2017b).
Efficiency of
BRICS
countries
IJDI
Key macroeconomic factors shaping the BRICS’ economic growth include fiscal policy,
exchange rates, trade openness, FDI inflows and inflation rates (Bezerra and Silva, 2021). The
role of FDI in economic development is debated; some studies, such as those of Khalid and
Marasco (2019), suggest it is beneficial, while others present a more nuanced view. Choudhry
et al. (2020) and Saini and Singhania (2018) indicate that the impact of FDI can be significant,
especially when coupled with technology transfer and capital accumulation. Borensztein et al.
(1998) found FDI’s impact to be positive but contingent on the host country’s absorptive
capacity. Further, Khan and Nawaz (2019) observed a positive correlation between trade
openness, FDI and income distribution. Long-term benefits of FDI in BRICS, aligned with
technical cooperation, were noted by Prabhakar et al. (2015) and Agrawal (2015). Joshua et al.
(2020) identified FDI as a crucial element for South Africa’s economic growth, noting that in
2017, BRICS attracted 19% of global FDI inflows (UNCTAD, 2018).
Human capital is another pivotal factor for economic development in these countries. It
enhances trade openness and fosters knowledge transfer, as noted by Nakabashi and
Figueiredo (2005). Barro (1991) posited that human capital facilitates growth by disseminating
technology from more developed countries, allowing less wealthy nations to accelerate their
economic expansion. Fisher (2022) confirmed a strong positive relationship between labor force
participation and GDP per capita growth rate in BRICS from 2009 to 2019. Hartman and Kwon
(2005) found that human capital significantly reduces environmental pollution in China.
Nakabashi and Figueiredo (2005) further argued that human capital indirectly bolsters income
growth per worker through the acceleration of technological diffusion, intensified by imports
and FDI. Hu (2021) echoed these findings, emphasizing the role of human capital in enhancing
the benefits of imported technology and innovation.
Infrastructure investment, encompassing transportation, energy, telecommunications and
other critical facilities, also plays a crucial role in economic performance. Calderon and Serven
(2015) and Kodongo and Ojah (2016) identified a strong link between infrastructure investment
and economic growth. Meidayati (2017) highlighted the impact of telecommunications
infrastructure and market size on developing countries’ economies. However, Apurv and Uzma
(2020) found mixed results regarding the significance of infrastructure investment in economic
growth, particularly in Brazil and South Africa compared to Russia, India and China.
Technological advancements, R&D expenditure and knowledge spillovers are key
contributors to the BRICS’ productivity and competitiveness, as outlined by Franco and
Oliveira (2017) and Hu (2021). Gyedu et al. (2021) analyzed the influence of R&D, trademarks
and patents on economic growth, concluding that innovation investments significantly
bolster growth. Ndlovu and Inglesi-Lotz (2020) explored variations in the impact of R&D
expenditure on economic growth across BRICS countries.
The BRICS’ rapid economic expansion has raised concerns about their seemingly limitless
exploitation of natural resources, as discussed by Gomes and Silva (2017). Their significant
environmental footprint and GHG emissions underscore the urgency of implementing
strategies to mitigate global pollution. While the BRICS are active participants in international
environmental conventions, they lack a unified approach to sustainable policy transition,
operating under the principle of “common but different responsibilities”. Brazil and South
Africa emphasize sustainable development, whereas China and India prioritize economic
growth, as noted by Cavalcanti (2018).
Regarding climate change, Brütsch and Papa (2013) argued that the BRICS lack sufficient
common interests to form a lasting coalition. Although the group has convened environment
and energy minister meetings (BRICS, 2015), they have yet to establish specific cooperation
mechanisms. Each nation faces unique challenges related to climate change. Russia continues
to rely on traditional energy sources, addressing issues such as inequality and poverty. Brazil,
India and China are moving towards renewable energy through legislative changes (Basile and
Cecchi, 2019). South Africa, while transitioning to a low-carbon society, has implemented
strategies to promote social inclusion and address climate change (Cavalcanti, 2018). Each
BRICS country provides detailed reports on their commitments to the UN’s Sustainable
Development Goals and Millennium Development Goals. However, Basile and Cecchi (2019)
noted inconsistencies in their engagement with these goals and the Paris Agreement, with a
continued dependence on traditional energy sources in some countries.
Efficiency of
BRICS
countries
3. Methodology
In this study, we aim to evaluate the efficiency of BRICS countries in channeling their productive
capacities and innovative capabilities into sustainable development. The DEA model is used to
measure and compare the efficiency of BRICS countries over the period from 2010 to 2018. We
conducted three separate DEA analyses, each focusing on one of the sustainable development
pillars: economic, social and environmental sustainability. The source of the data is the World
Data Bank.
The selection of input variables was grounded in existing literature. The variable entitled
Gross Fixed Capital Formation (GFCF) was chosen to represent the capital variable, reflecting
its established correlation with economic growth as discussed in the studies by Meyer and
Sanusi (2019) and Topcu et al. (2020). Following Santana et al. (2014) and Bekun et al. (2019), the
unemployed population was used as a proxy for human capital. Research and development
(R&D) expenditure was used to assess the effect of technological innovation on sustainable
development (Bayarçelik and Tas
el, 2012; Costantini et al., 2023). The variable foreign direct
investment (FDI) inflows are included in our analysis acknowledging its significant role in
innovation and sustainable development in developing countries, as evidenced in studies by
Lee et al. (2021), Chai et al. (2021), Sarkodie and Strezov (2019) and Sunde (2016).
For output variables, gross domestic product (GDP) was selected to represent economic
growth (Apergis and Payne, 2011; Sanz-Díaz et al., 2017). The variable life expectancy was
chosen to reflect the social dimension, aligning with its use as a general health indicator in
studies by Luy et al. (2020), Mariano and Rebelatto (2014) and Magombeyi and Odhiambo (2018).
To proxy environmental sustainability, CO2 emissions were used as a standard metric for
assessing environmental impact (Maryam et al., 2017; Shikwambana et al., 2021; Lee et al., 2021).
The stepwise method was applied to validate the chosen variables for DEA applications.
This process starts with selecting the most statistically significant variable, followed by a
sequential addition or removal of variables based on set criteria. The variables used in the
three DEA applications are detailed in Table 1, categorized by the type of efficiency they
represent (economic, environmental, social), their inputs and outputs.
For the first and second applications, the economic and environmental efficiency was
tested, using GDP and CO2 as the outputs, respectively, and GFCF, R&D, FDI and
unemployed population as inputs for both applications. The third application concerns the
Application
Type of efficiency
Inputs
Output
1
2
3
Economic
Environmental
Social
GFCF, R&D, FDI, unemployed population
GFCF, R&D, FDI, unemployed population
GFCF, R&D, FDI
GDP
CO2 emissions
Life expectancy
Source: Table by authors
Table 1.
Variables used
IJDI
social pillar. Its output is the life expectancy, and the analysis was made considering the
inputs GFCF, R&D and FDI.
The DEA is a non-parametric linear programming method used to measure the efficiency of
decision making units (DMUs) when facing multiple inputs and multiple outputs (Charnes
et al., 1978). One of the most commonly used models is the BCC model (Banker et al., 1984). The
BCC model allows for variable returns to scale (VRS), meaning that as inputs are increased,
outputs do not necessarily increase in a fixed proportion. As the BRICS countries aim to
increase the outputs, i.e. aim for their sustainable development, the BCC–output-oriented model
is used to analyze the economic, social and environmental applications. The output-oriented
DEA-BCC model is characterized by its objective function and constraints, with a focus on
maximizing output efficiency given a set of inputs and outputs, defined as follows:
n
X
Min
vj xj0 – w;
j¼1
Subject to
m
X
ui yi0 ¼ 1;
i ¼ 1
m
X
i ¼ 1
ui yik
n
X
vj xjk þ w # 0; for k ¼ 1; 2; . . . ; h:
j¼1
Where n and m are the number of inputs and outputs analyzed, respectively; h is the number
of decision-making units (DMUs) analyzed; w is the scale factor; vj is the weight of input j for
the DMU; ui is the weight of output i for the DMU; xj0 is the amount of input j of the DMU; yi0
is the amount of output i of the DMU; xjk is the amount of input j of DMU k; and yik is the
amount of input i of DMU k. The nature of the output CO2 emissions, which is undesirable
was transformed to fit the DEA framework. This transformation involved multiplying the
emissions data by “1” and adding a translation vector to ensure the transformed values
remained positive without altering their relational dynamics.
To enhance discrimination between DMU efficiency scores, we used the inverted frontier
method (Angulo Meza et al., 2003). This approach involves reversing the roles of inputs and
outputs in the DEA model and then creating a composite index to rank the units. This index
is computed by averaging the classic frontier efficiency score and the inverted efficiency
score, normalized against the highest value obtained (Leta et al., 2005).
Finally, the study includes an analysis of efficiency trends over the years using the “window
analysis” technique as described by Cooper et al. (2007). This approach treats each unit per year
as a distinct DMU, with a moving average calculated as new units are added and old ones
removed. The number of windows and their respective amplitudes are determined based on the
total number of years analyzed, in this case, resulting in five windows with a five-year amplitude,
as follows:
w¼kpþ1
p¼
kþ1
2
In which, w represents the number of windows; p represents the window amplitude and k
represents the number of years. As the years taken into consideration for this analysis are 9,
the number of windows corresponded to 5 and the amplitude corresponded to 5 years.
4. Results
The results of the DEA across three sustainability dimensions: economic, social and
environmental are presented in Tables 2, 3 and 4, respectively. Efficiency scores for each
country were computed for every analysis window, leading to a comprehensive average
index for each sustainability aspect. Table 5 ranks the countries according to the average
index. Finally, Table 6 provides data on gross domestic product (GDP), CO2 emissions, life
expectancy, GFCF, research and development (R&D) expenditure, foreign direct investment
(FDI) inflows and the unemployed population of BRICS countries, offering insights into their
development trajectories and policy focuses.
The economic dimension results are outlined in Table 2, showing efficiency trends across
five-year windows from 2010 to 2018 for each BRICS country, alongside their average total
Country
Brazil
Russia
India
China
South Africa
1 (2010/14)
(%)
2 (2011–15)
(%)
Window
3 (2012–16)
(%)
4 (2013–17)
(%)
5 (2014–18)
(%)
Mean
total (%)
79.62
90.18
68.26
72.42
74.03
78.16
92.16
68.45
70.57
72.52
77.80
91.69
70.76
70.57
71.77
78.60
91.79
71.68
70.75
71.14
72.58
92.14
70.03
68.27
68.23
77.35
91.60
69.84
70.52
71.54
Source: Table by authors
Country
Brazil
Russia
India
China
South Africa
1 (2010/2014)
(%)
2 (2011–2015)
(%)
Window
3 (2012–2016)
(%)
4 (2013–2017)
(%)
5 (2014–2018)
(%)
Mean total
(%)
90.36
93.49
89.63
52.91
89.54
89.09
93.50
88.14
52.63
97.81
89.31
93.95
88.44
53.87
98.49
89.53
94.13
87.98
53.18
98.40
89.21
95.70
87.76
53.23
99.03
89.50
94.16
88.39
53.16
96.65
1 (2010/2014)
(%)
2 (2011–2015)
(%)
Window
3 (2012–2016)
(%)
4 (2013–2017)
(%)
5 (2014–2018)
(%)
Mean total
(%)
94.54
95.76
47.58
16.45
99.65
94.20
96.57
47.46
15.21
99.47
93.33
97.48
47.51
14.77
99.40
92.11
98.19
47.75
14.20
99.41
89.04
97.69
47.13
13.51
97.63
92.65
97.14
47.49
14.83
99.11
Source: Table by authors
Country
Brazil
Russia
India
China
South Africa
Source: Table by authors
Efficiency of
BRICS
countries
Table 2.
Economic application
Table 3.
Social application
Table 4.
Environmental
application
IJDI
efficiency. Russia emerges as the leader in this category, with Brazil, South Africa and China
following and India trailing. Notably, India and China displayed increasing efficiency scores
in the initial windows, but most countries except Russia experienced a decline in the
subsequent window.
Table 3 details the social sustainability findings, positioning South Africa at the
forefront, closely followed by Russia. Both these countries improved their efficiency scores
over the study period. Brazil, occupying the third rank, showed a declining trend, with India
and China at the lower end, China recording the least efficiency at 53.16%.
Environmental efficiency, as per Table 4, places South Africa at the top with near-perfect
efficiency, closely followed by Russia. Brazil, India and China round out the rankings, with
China significantly lagging behind others with a mere 14.83% efficiency.
To synthesize these results, Table 5 was constructed, offering a consolidated view of the
efficiency rankings across all three applications. Russia and South Africa consistently rank
high, while India and China are positioned at the bottom of the efficiency scale.
Table 6 provides a breakdown of the input and output values for the variables used in the
DEA, averaged over the period 2010–2018. Table 6 serves as a quantitative foundation,
underpinning the DEA analysis and offering deeper insights into the variables influencing
each country’s sustainable development efficiency. Notably, China leads in GDP, CO2
emissions and life expectancy, reflecting its rapid industrial growth and large population.
India, with significantly high CO2 emissions, also has a high unemployed population,
suggesting economic growth may not be equitably translating into job creation. Russia and
Brazil present moderate figures in most categories, though Brazil’s relatively high
Application
Social
Economic
Table 5.
Mean efficiency
rankings of BRICS
from 2010 to 2018
Brazil
Russia
India
China
South Africa
3rd
2nd
4th
5th
1st
3rd
2nd
4th
5th
1st
Source: Table by authors
Country
Brazil
Russia
India
China
South Africa
Table 6.
Descriptive statistics
2nd
1st
5th
4th
3rd
Environmental
GDPa
1,790,640.45
1,359,389.57
2,002,225.282
10,413,780.02
338,802.96
Mean total (2010–2018)
CO2
Life
emissionsb expectancyc
GFCFd
R&De
454540.79
1628548.22
2062884.15
9741657.54
430355.03
74.1
71.9
68.9
76.7
63.0
FDIf
329,921,331.99 21,565.3 65,892,09
291,458,527.51 14,454,58 25,851.68
609,207,470.49 14,053.51 33,945.33
4,483,614.96 208,971.73 249,339.79
59,412.47
2,376.60
3,561.31
Unmployed
populationg
9.21
4.29
37.85
35.36
5.10
Notes: World Data Bank; agross domestic product (constant 2015, million US$); CO2 emissions (kt); clife
expectancy at birth (years); dgross fixed capital formation (constant, million 2015, US$); eR&D expenditure (constant
2015, million US$); fFDI inflows (constant 2015, million US$); gunmployed population (million)
Source: Table by authors
unemployment rate stands out. China’s substantial R&D expenditure and FDI inflows
underline its position as a global economic powerhouse with a strong emphasis on
innovation. In comparison, there are relatively low R&D investments in Russia and South
Africa.
The comparative analysis shown in Table 7 underscores the diverse trajectories of
BRICS nations in balancing economic growth with social welfare and environmental
sustainability.
Brazil has seen a shift in its sustainability landscape, with its economic efficiency slightly
declining from the top position, indicating slight decline in its economic management
effectiveness. Socially, Brazil experienced a significant drop in efficiency, moving from first
to third place, suggesting challenges in sustaining its previously achieved social welfare
gains. Environmentally, Brazil improved its efficiency score but fell in rankings, implying
that while it has intensified its environmental conservation efforts, other BRICS nations
have made more substantial strides in this domain.
Russia has demonstrated remarkable progress, especially in economic terms, where it
leaped from the lowest to the highest efficiency score, showcasing significant improvements
in economic management and output efficiency. Socially, Russia maintained its second-place
ranking with an enhanced efficiency score, indicating steady advances in social welfare. The
country also made notable gains in environmental efficiency, moving up in both score and
ranking, which underscores Russia’s effective strategies in environmental management.
India has remained at the lower end of economic efficiency among the BRICS, with only a
minor improvement in score, highlighting ongoing difficulties in efficiently converting
inputs into economic outputs. In the social domain, despite maintaining a low ranking,
India’s improved score signals progress in social development. However, India faced a sharp
decline in environmental efficiency, suggesting escalating challenges in managing
environmental sustainability amid its economic and demographic growth.
Economically, China saw a slight improvement in its efficiency score but dropped in
ranking, suggesting that while there has been some progress, other BRICS countries have
outpaced China in economic efficiency. Socially, China’s efficiency score and ranking both
Country
Application
Brazil
Economic
Social
Environmental
Economic
Social
Environmental
Economic
Social
Environmental
Economic
Social
Environmental
Economic
Social
Environmental
Russia
India
China
South Africa
Efficiency score
(2001–2007)a (%)
Rank
(2001–2007)a
Efficiency score
(2010–2018)b (%)
Rank
(2010–2018)b
98
99
90
51
89
78
49
49
81
65
56
21
66
76
99
1st
1st
2nd
4th
2nd
4th
5th
5th
3rd
3rd
4th
5th
2nd
3rd
1st
77
90
93
92
94
97
70
88
48
71
53
15
72
97
99
2nd
3rd
3rd
1st
2nd
2nd
5th
4th
4th
4th
5th
5th
3rd
1st
1st
Sources: aSantana et al. (2014) results; bour results; table by authors
Efficiency of
BRICS
countries
Table 7.
Evolution of BRICS
countries’ efficiency
in economic, social
and environmental: a
comparative analysis
IJDI
declined, reflecting significant hurdles in addressing social disparities despite its rapid
economic growth. Environmentally, China experienced the most considerable decline in
efficiency, remaining the least efficient, which highlights the severe environmental
challenges it faces, underscoring the urgent need for sustainable environmental policies.
South Africa experienced a drop in its comparative economic ranking but socially, the
country has made remarkable strides, a testament to its effective strategies and policies
aimed at enhancing social welfare and addressing societal challenges. Environmentally,
South Africa has upheld its premier status, demonstrating a consistent dedication and
effective approach to environmental management and sustainability initiatives.
5. Discussion
In Brazil, median input levels, coupled with the second-highest foreign direct investment
(FDI) inflows, have resulted in a paradox of high-efficiency scores and a mid-range ranking.
This dichotomy points to a potential for Brazil to enhance its output maximization.
Economic growth data from the Brazilian Institute of Geography and Statistics highlight
uneven GDP growth, notably a decline from an average of 3.6% annually between 2001 and
2010 to just 0.7% between 2011 and 2018. This fluctuation reflects the impact of fiscal
policies aimed at stimulating private consumption and demand, which, despite initial
success, led to economic downturns post-2013 (Costa et al., 2017).
Economically, Brazil’s efficient conversion of median inputs into high outputs places it
second in economic efficiency among the BRICS. This suggests that maintaining input
levels while refining fiscal policies could elevate GDP output. Environmentally, Brazil ranks
third with a 92.68% efficiency score, reflecting its substantial renewable energy sector,
primarily hydropower (Udemba and Tosun, 2022). However, high GHG emissions because
of deforestation counter this achievement (Timperley, 2018). The correlation between rising
FDI and decreasing CO2 emissions (Khatoon et al., 2022), amidst increasing deforestation,
underscores the need for focused environmental policies.
Socially, Brazil’s life expectancy of around 75 years (2018) and significant strides in
reducing inequality during the Worker’s Party (PT) governance (Oliveira, 2023) contrast
with its third-place social efficiency ranking. Programs such as Bolsa Família have
substantially improved living conditions (Campoli et al., 2019), yet political shifts and
reduced focus on such initiatives (Costa, 2019) have stalled progress, emphasizing the
imperative of sustained social policies.
Considering the comparative analysis with Santana et al.’s (2014) findings, Brazil’s
trajectory in sustainability shows a moderate downturn in economic efficiency from its
previously top position, a significant drop in social welfare efficiency from first to third
place and an enhancement in its environmental endeavors. However, this progress is
shadowed by more substantial advancements made by other BRICS nations in
environmental management during the same period.
Russia’s median Global Fixed Capital Formation (GFCF) and research and development
(R&D) inputs, combined with the lowest unemployment, have yielded the highest efficiency
rates in the group. Post-USSR, the Russian economy’s transition to a market-based system
saw concentrated economic power and reliance on oil, accounting for 20% of GDP (Orazalin
and Mahmood, 2018; Dabrowski, 2023). Despite a modest GDP, Russia’s efficient use of
inputs, especially in using human capital and infrastructure development (Serbian et al.,
2023), has led to a top economic efficiency ranking.
Environmentally, Russia stands second with a 97.14% efficiency rate, managing low CO2
outputs from median inputs. However, as the world’s fourth-largest GHG emitter
(Zagoruichyk, 2022), optimizing R&D and FDI towards reducing emissions is crucial.
Socially, Russia’s advancements in poverty reduction, despite a lower life expectancy of
72.7 years, reflect a 94.16% efficiency rating, underscoring the role of government initiatives
in social development (Rudenko and Satre, 2018).
In comparison to Santana et al.’s (2014) earlier analysis, Russia demonstrates remarkable
progress, moving from the lowest to the highest in economic efficiency, steadfastly holding
its rank in social welfare and making notable strides in environmental efficiency. This
upward trajectory signifies Russia’s successful implementation of effective management
strategies and policy advancements, distinguishing its comprehensive growth and
sustainability efforts among the BRICS nations.
India’s high unemployment and R&D inputs, juxtaposed with median FDI inflows, have
not translated into proportional outputs, placing it last in economic efficiency. Post-1991
reforms propelled growth, shifting the economic structure towards services over industry
(The World Bank, 2018a, 2018b, 2018c; Anand, 2014). However, this premature
deindustrialization might explain India’s low economic efficiency, as the advantages of
industrialization remain unexploited.
Environmentally, India ranks fourth with a 47.49% score, reflecting increased CO2
emissions alongside economic growth (Zameer et al., 2020; Timperley, 2019). The primary
emission sources, including energy and agriculture, suggest inefficiencies in input
utilization. Socially, India’s rapid growth has not inclusively benefitted its population,
evident in its fourth-place social efficiency ranking and life expectancy of around 69 years.
Initiatives aimed at reducing poverty and promoting non-farm employment (Pattayat et al.,
2022) indicate potential areas for improvement.
Relative to Santana et al.’s (2014) results, India’s journey reflects ongoing challenges in
economic efficiency and environmental sustainability. Despite slight enhancements in
economic performance and social development, India has experienced a discernible
deterioration in environmental efficiency. This contrast highlights the persistent difficulties
India faces in aligning its rapid economic and demographic expansion with effective
environmental governance and sustainability practices.
China, despite its high inputs and outputs, demonstrates suboptimal efficiency scores. As
the world’s second-largest economy, its fourth-place economic efficiency ranking might stem
from its shift to a service-oriented economy and rising unemployment influenced by
technological advancements (Du and Wei, 2022; The World Bank, 2018a, 2018b, 2018c).
In environmental terms, China ranks last with a 14.83% efficiency score, exacerbated by
a vertically managed administrative system that prioritizes economic growth over
environmental standards (Chai et al., 2021). Addressing FDI’s role in pollution (Azam et al.,
2019; Chai et al., 2021) is essential for environmental improvement. Socially, China’s
challenges in poverty, inequality and labor market distortions (Ebenstein et al., 2015)
contribute to its last-place social ranking.
When juxtaposed with Santana et al.’s (2014) findings, China’s path reflects incremental
progress in economic efficiency and a decline in social welfare efficiency, alongside facing
pronounced environmental hurdles. These dynamics underscore the pressing necessity for
China to devise and implement more robust policies that reconcile its swift economic ascent
with the imperatives of environmental protection and social equity.
South Africa, with the lowest inputs and outputs, has attained notable efficiency across
all domains. Its third-place economic ranking, with a 70.52% score, suggests potential for
growth through enhanced FDI and R&D (Makhoba et al., 2019; Sunde, 2016; Quaynor et al.,
2022). Environmental leadership is evident in its top ranking, reflecting the lowest CO2
outputs among BRICS and the impact of optimized inputs on environmental quality (Joshua
et al., 2020). Socially, despite low life expectancy, South Africa’s high efficiency score is
Efficiency of
BRICS
countries
IJDI
attributed to significant improvements in health and a need for policies promoting inclusive
growth (WHO Africa, 2022; Francis and Webster, 2019; The World Bank, 2018a, 2018b,
2018c).
Comparing South Africa’s sustainable development from 2000–2007 to the latest data,
the country shows a nuanced evolution. Economically, there is a slight improvement in
efficiency, but its ranking among BRICS nations has fallen, indicating faster progress by
others. Socially, South Africa has moved to the top in efficiency, reflecting strong efforts in
addressing social issues. Environmentally, it continues to lead, showing consistent
commitment to sustainability. This comparison reveals South Africa’s progress in social
and environmental areas, despite economic challenges relative to other BRICS countries.
6. Conclusions
This study delves beyond traditional gross domestic product metrics to understand
economic development, emphasizing the importance of sustainable growth that harmonizes
economic expansion with quality-of-life improvements. Emerging countries, particularly the
BRICS countries, have gathered global attention because of their rapid economic growth,
substantial populations and rich natural resources. Hence, analyzing their growth bases and
impacts on future generations is crucial, especially regarding how endogenous growth
drivers can bolster sustainable development.
Focusing on the BRICS countries’ resource conversion efficiency into sustainable
development, this research used DEA across economic, social and environmental facets
from 2010 to 2018. Compared to the previous study that used the 2000–2007 period, Russia
and South Africa emerge as leaders, with Brazil also performing well. Russia’s success
highlights the impact of strategic investments in human capital and green technologies.
South Africa’s leading environmental efforts and top social ranking demonstrate progress in
overcoming social challenges. Brazil shows effective resource and FDI conversion but faces
challenges such as deforestation and social inequality. India needs to address CO2 emissions
and improve working conditions, leveraging FDI and R&D for environmental and social
gains. China’s low efficiency underscores the need for better industrial practices,
employment conditions and targeted technological investments. Despite differing time
frames, the studies collectively illustrate the ongoing efforts of BRICS nations to balance
economic advancement with environmental protection and social inclusion, highlighting
both achievements and areas requiring further attention.
While insightful, this study misses recent global events, notably the COVID-19 pandemic
and the Russia–Ukraine conflict, that have likely impacted BRICS’ sustainable development
trajectories. Thus, future research must incorporate these developments, expanding
variables and methodologies to offer a more complete understanding of the BRICS’
sustainable development paths.
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Corresponding author
Felipa de Mello-Sampayo can be contacted at: fdmso@iscte-iul.pt
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Efficiency of
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Deep-learning-based Automatic Detection and Classification of Traffic Signs Using Images Collected by Mobile Mapping Systems
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*Corresponding author: e-mail: kemyoung@nsu.ac.kr
https://doi.org/10.18494/SAM3956 Keywords: high-definition maps, traffic sign, mask R-CNN, Inception-v3, autonomous driving Keywords: high-definition maps, traffic sign, mask R-CNN, Inception-v3, autonomous driving As interest in autonomous driving has increased in recent years, various sensors have been
developed for use in vehicles to detect and classify traffic signs. When road traffic facilities are
not recognized owing to the malfunction of sensors, point cloud data and images collected by
mobile mapping systems (MMSs) are used to construct high-definition maps containing road
traffic facility information. However, when traffic signs, among the targets of high-definition
map construction, are constructed using point cloud data, it becomes difficult to detect and
classify traffic signs because they are highly reflective. In this study, we detected and sub-
classified traffic signs by combining Mask Regions with Convolutional Neuron Network (Mask
R-CNN) and Inception-v3 models based on image data obtained using MMSs. Image data
obtained by various types of MMS were used to detect traffic signs and classification results
were verified. The detection accuracy of traffic signs was 87.6% and the classification accuracy
was 77.5%; thus, the method proposed in this study can be used to automatically construct
traffic signs for high-definition maps. 4801 4801 Sensors and Materials, Vol. 34, No. 12 (2022) 4801–4812
MYU Tokyo Sensors and Materials, Vol. 34, No. 12 (2022) 4801–4812
MYU Tokyo
S & M 3144 Sensors and Materials, Vol. 34, No. 12 (2022) 4801–4812
MYU Tokyo Deep-learning-based Automatic Detection and Classification of
Traffic Signs Using Images Collected by Mobile Mapping Systems Hyeong-Yoon So and Eui-Myoung Kim* Graduate School of Spatial Information Engineering, Namseoul University,
91, Daehak-ro, Seonghwan-eup, Seobuk-gu, Cheonan-si, Chungcheongnam-do, 31020 Republic of Korea (Received April 27, 2022; accepted August 3, 2022; online published August 15, 2022) 1.
Introduction As interest in autonomous driving has increased, studies are actively underway to detect and
classify road traffic facilities on roads using various sensors, such as LiDAR, RADAR, and
cameras. However, when sensors are used, there are cases when a malfunction occurs or objects
of interest are not detected under adverse circumstances, including long distance, blind spots, or
bad weather conditions.(1) Accordingly, the need to construct high-definition maps is imperative
so that this information can compensate for the malfunction of the sensors. In South Korea, mobile mapping systems (MMSs) are currently used to collect various data,
such as horizontal images, panoramic images, point clouds, and positional information to
construct high-definition maps.(2) The process of producing high-definition maps is as follows:
the operator uses the collected point cloud to detect and classify objects of interest manually with
the naked eye, and then digitizes them to construct maps. However, in the process of detecting
and classifying objects of interest, those that are highly reflective to laser beams, such as traffic ISSN 0914-4935 © MYU K.K. https://myukk.org/ ISSN 0914-4935 © MYU K.K. https://myukk.org/ ISSN 0914-4935 © MYU K.K. https://myukk.org/ 4802 Sensors and Materials, Vol. 34, No. 12 (2022) signs, have an insufficient point density in the point cloud, as shown in Figs. 1(b) and 1(d),
making it difficult to classify and locate traffic signs with the naked eye. In contrast, classifying
traffic signs and determining their locations are improved using images from MMS cameras, as
shown in Figs. 1(a) and 1(c). In the case of traffic signs, using images from MMS cameras, rather
than using point clouds, is effective when detecting and classifying objects of interest. In studies on the detection of traffic signs using point clouds collected by MMSs, after
classifying the ground and non-ground surfaces, methods such as Euclidean distance clustering,
filtering and clustering using reflection intensity, and bag of visual phrases (BoVP) can be used
to detect traffic signs.(3,4) However, there are limitations, namely, the reflection intensity must be
normalized for traffic sign detection, the process of preprocessing the point clouds consumes
much time, and the results of traffic signs vary depending on the threshold setting determined by
the user experience. For the efficient construction of high-definition maps, research is required
to detect and classify traffic sign objects using images. 1.
Introduction Studies on the detection of traffic signs using images have used image processing algorithms
and convolutional neural network (CNN)-based deep learning models. Studies using image
processing algorithms include a study using a FAST feature point extraction method and a study
using the Viola-Jones algorithm to determine the edge of a traffic sign and then detect the traffic
sign region using support vector machine (SVM).(5,6) These methods can be used to detect the
edge of the traffic sign, but their limitation is that the region cannot be detected if no edge is
determined for the traffic sign. Another study used color, shape, and both color and shape to detect traffic signs.(7) In the
case of the color-based method, the process was fast and simple, but it was sensitive to light. The
shape-based method was limited in that the detection result varied depending on the type of edge
detector used. When color and shape were used simultaneously, traffic signs could be detected
effectively using a maximally stable extremal region (MSER) and high-contrast region extraction
(HCRE), but the limitation in using color and shape together is that the detection accuracy of the
traffic signs varied depending on the color enhancement method. In a study that detected objects
using a machine learning or CNN-based deep learning algorithm, the You Only Look Once
(YOLO) V4-Tiny model was trained using approximately 9000 images to detect and classify 45 (a)
(b)
(c)
(d)
Fig. 1. (Color online) (a), (c) Traffic signs in images from MMS. (b), (d) Traffic signs in point cloud from MMS. (a) (c) (d) (b) (d) (a) (b) (c) Fig. 1. (Color online) (a), (c) Traffic signs in images from MMS. (b), (d) Traffic signs in point cloud from MMS. Sensors and Materials, Vol. 34, No. 12 (2022) 4803 types of traffic sign, but it showed a low accuracy of approximately 52%.(8) After training a
Mask R-CNN model using 6000 images, a high accuracy of 95.2% was obtained after detecting
and classifying ten types of traffic sign.(9,10) Through these research cases, it was confirmed that
the Mask R-CNN model can be used to detect and classify traffic signs, and the outlines of the
traffic signs can be found through segmentation, a characteristic of the Mask R-CNN model. 1.
Introduction The studies on object classification include the use of the MSER method to detect traffic
signs in images and Inception-v3 to classify them into three main categories, namely, warning,
regulatory, and mandatory signs, and the Belgian traffic sign dataset (BTSD) was used to train
an Inception-v3 model and classify 62 traffic signs.(11–14) Through these studies, it can be seen
that in order to classify traffic signs, it is necessary to find the areas of traffic signs to be
extracted from images first. Therefore, the purpose of this study is to propose a methodology for
automatically detecting and classifying traffic signs by combining a Mask R-CNN model and an
Inception-v3 model in images acquired using MMSs. 2.1
Methodology In this study, we propose a methodology that combines a Mask R-CNN model and an
Inception-v3 model to detect and sub-classify traffic signs in the images obtained using MMSs
(Fig. 5). The first step in automatically constructing traffic signs on high-definition maps is to
detect the region of the traffic sign in each image and define its main classification as a warning,
regulatory, or mandatory sign. The Mask R-CNN model was applied to the traffic sign regions in the original images
collected using MMSs in order to define their respective main classifications. Then, the image
coordinates (x, y) for the edge of the traffic sign region were obtained and recorded in a
JavaScript object notation (JSON) format file. The JSON format file includes records of
information, such as the main classification code, main classification name, and upper left and
lower right image coordinates of the object region. After automatically detecting the region of the traffic sign in the image and providing a main
classification as either a warning, regulatory, or mandatory sign, the Inception-v3 model was
required to sub-classify the traffic sign type in the second step. To this end, the Inception-v3
model was applied to read the JSON format file (the output of the first step), and after creating
cropped images using the upper left and lower right image coordinates of the object region, the
sub-classification class was assigned, attendant to the main classification. 2.
Materials and Methods The traffic signs defined to construct high-definition maps in South Korea are classified into
three main categories (Fig. 2): warning signs (B1_1), regulatory signs (B1_2), and mandatory
signs (B1_3), which consist of 47, 27, and 35 classes, respectively, comprising a total of 103
classes as sub-classifications.(2) The warning signs have a triangular shape with a red edge and
the regulatory signs have a circular shape with a red edge. The mandatory signs are circular,
triangular, or square with a blue edge. Unlike Korean traffic signs, U.S. warning signs have a yellow diamond shape as shown in
Fig. 3(a), and regulatory and mandatory signs include squares and letters on a white background
as shown in Figs. 3(b) and 3(c).(15) Unlike the Korean warning signs shown in Fig. 4(a), the Czech warning signs in Europe are
triangular signs with white backgrounds as shown in Figs. 4(b) and 4(c). In addition, comparing
the Korean and Austrian regulatory signs shows that the shapes are similar, but the background
color or symbols are different.(16) (a)
(b)
(c)
Fig. 2. (Color online) (a) Warning signs defined in high-definition maps, (b) regulatory sings, and (c) mandatory
signs. (a) (c) (b) (c) (a) Fig. 2. (Color online) (a) Warning signs defined in high-definition maps, (b) regulatory sings, and (c) mandatory
signs. Sensors and Materials, Vol. 34, No. 12 (2022) 4804 Fig. 3. (Color online) (a) Warning, (b) regulatory, and (c) mandatory signs in the United States. (a)
(b)
(c) (b) (c) (a) (a) (a) (b) (c) Fig. 3. (Color online) (a) Warning, (b) regulatory, and (c) mandatory signs in the United States. Fig. 4. (Color online) (a) Korean warning sign, (b) Czech warning sign, (c) French warning sign, (d) Korean no
overtaking regulatory sign, (e) Austrian no overtaking regulatory sign, (f) Korean one-way mandatory sign, and (g)
Austrian one-way mandatory sign. (a)
(b)
(c)
(d)
(e)
(f)
(g) (e)
(f)
(g) (d) (d) (b) (e) Fig. 4. (Color online) (a) Korean warning sign, (b) Czech warning sign, (c) French warning sign, (d) Korean no
overtaking regulatory sign, (e) Austrian no overtaking regulatory sign, (f) Korean one-way mandatory sign, and (g)
Austrian one-way mandatory sign. 2.2 Construction of training data In this study, the AI-Hub data managed by the National Information Society Agency (NIA)
was used to train the Mask R-CNN and Inception-v3 models.(17) The “Panorama Image Set of Sensors and Materials, Vol. 34, No. 12 (2022) 4805 Fig. 5. (Color online) Methodology for detecting and classifying traffic signs. Fig. 5. (Color online) Methodology for detecting and classifying traffic signs. Road Environments (v. 1.0)” provided by AI-Hub is a dataset that provides eight-direction
images (.jpg) captured in the daytime using a horizontal camera mounted on an MMS and the
label information (.json), such as traffic lights, traffic signs, and road surface marking (Table 1). The label information has records of the image name, class code and name, and upper left and
lower right image coordinates. AI-Hub provides approximately 330000 images of training data, but in some cases, the label
was missing as shown in Fig. 6(a), or the labeling standard was not met as shown in Fig. 6(b). Therefore, we manually labeled 10000 images to develop the training data for traffic signs. To proceed with the training of the Mask R-CNN model, we divided the 10000 manually
constructed images in a ratio of 8:2 to create training and validation data. Table 2 details the
construction of the training and validation data. To proceed with the training of the Inception-v3 model, we used 86400 images provided by
the AI-Hub and the object region information in the label file (.json) to create cropped images as
shown in Fig. 7. The created cropped images were separated into three folders [warning (B1_1),
regulatory (B1_2), and mandatory (B1_3)] through the information provided in the label file
(.json). Then, three training datasets were constructed so that each folder would have sub-folders
consisting of sub-classification codes. 3.1
Training and validation of Mask R-CNN model The training was performed for 600 epochs using the constructed training data, and 100
steps were performed in one epoch. Among the 600 weighted models created through the
epochs, a weighted model that had a low loss rate indicating the difference between the values
predicted using the model and the true values of the problem and a high mean average precision
(mAP) using the validation data was used to detect the traffic sign. The mAP represents an index
that can consider both detection rate (recall) and accuracy (precision) at the same time. Table 3 shows the loss and mAP for 200, 400, 568, and 600 epochs performed to detect the
traffic signs, and Fig. 8 shows a graph of the loss rate for the validation data. Using Table 3 and 4806 Sensors and Materials, Vol. 34, No. 12 (2022) Table 1
(Color online) Sample images and label information provided by AI-Hub. Category
Description
Format
Example
Planar split
images
8 split images
.jpg
Annotation
information
Object location
and type
information
.json
{
"interface": {
"id": "028770",
"filename": "028770_Panorama_crop_2.jpg",
"path": "02/crop_image/",
"resolution": [1000, 1000],
"location": "Gangnam-gu",
"datetime": "2020-01-09 11:28:18.0",
"annotations": [
{
"annotation_id": 161305,
"annotation_type": "bbox",
"class_code": "0201022600000",
"class_name": "Traffic sign/Regulatory sign/Slow",
"memo": "",
"coord_xy": [[863, 940], [373, 443]]
},
{
"annotation_id": 161314,
"annotation_type": "polygon",
"class_code": "0204001300000",
"class_name": "Road sign/Crosswalk warning",
"memo": "",
"coord_xy": [[613, 660, 700, 646], [562, 555, 591, 599] ]
}
]
}
} Table 1
(Color online) Sample images and label information provided by AI-Hub. Category
Description
Format
Example
Planar split
images
8 split images
.jpg
Annotation
information
Object location
and type
information
.json
{
"interface": {
"id": "028770",
"filename": "028770_Panorama_crop_2.jpg",
"path": "02/crop_image/",
"resolution": [1000, 1000],
"location": "Gangnam-gu",
"datetime": "2020-01-09 11:28:18.0",
"annotations": [
{
"annotation_id": 161305,
"annotation_type": "bbox",
"class_code": "0201022600000",
"class_name": "Traffic sign/Regulatory sign/Slow",
"memo": "",
"coord_xy": [[863, 940], [373, 443]]
},
{
"annotation_id": 161314,
"annotation_type": "polygon",
"class_code": "0204001300000",
"class_name": "Road sign/Crosswalk warning",
"memo": "",
"coord_xy": [[613, 660, 700, 646], [562, 555, 591, 599] ]
}
]
}
} { (a)
(b)
Fig. 6. (Color online) (a) Missing label contained in AI-hub dataset and (b) non-standard label. (a) (b) (a) (b) Fig. 6. (Color online) (a) Missing label contained in AI-hub dataset and (b) non-standard label. Sensors and Materials, Vol. 34, No. 12 (2022) 4807 (a)
(b)
Fig. 7. (Color online) (a) AI-Hub original image (.jpg), (b) AI-Hub labeling data (.json), and (c) cropped images. 3.2
Assessment of Mask R-CNN model Three types of MMS test set were used to objectively assess the performance of the training
results of the Mask R-CNN model, which was intended to detect and classify traffic signs into
either warning, regulatory, or mandatory signs. In test set 1, we used the images not used in the
training of the Mask R-CNN model from the data provided by AI-Hub. For test set 2, we used
‘Pegasus: Two Ultimate’, Leica’s MMS. Lastly, for test set 3, we used R1, an MMS of NaverLabs. Table 4 provides detailed information and shows the mAP results for the test sets. In assessing the accuracy of detecting the traffic signs and determining their main
classification, high accuracies of 91.3, 81.2, and 90.2% were obtained for the test datasets of test
sets 1, 2, and 3, respectively. Figure 9 shows sample results of the detection of the traffic signs
and their main classification in the three test sets using the weighted Mast R-CNN model. 3.1
Training and validation of Mask R-CNN model Table 2
Detailed information of training and validation data. Training set (8000 images)
Validation set (2000 images)
ID
CODE
Description
Number of labels
Ratio (%)
Number of labels
Ratio (%)
1
B1_1
Warning
446
9.7
116
10.0
2
B1_2
Regulatory
2162
46.9
535
46.2
3
B1_3
Mandatory
2000
43.4
508
43.8
Total
4608
1159
(c) Table 2
Detailed information of training and validation data. Table 2
Detailed information of training and validation data. (a) (b) (c) (c) (b) (a) Fig. 7. (Color online) (a) AI-Hub original image (.jpg), (b) AI-Hub labeling data (.json), and (c) cropped images Fig. 8. (Color online) Loss graph for validation data. Table 3
Mean average precision and loss by number of epochs observed during training. Epoch
200
400
568
600
Train_mAP (%)
72.5
93.8
95.5
95.4
Train_loss (%)
30.3
19.8
12.7
13.3
Val_mAP (%)
88.6
89.9
90.7
90.1
Val_loss (%)
47.8
47.3
37.9
46.0 Fig. 8. (Color online) Loss graph for validation data. 4808 Sensors and Materials, Vol. 34, No. 12 (2022) Fig. 8, we found that in the case of 568 epochs, the training accuracy (95.5%) and the validation
accuracy (90.7%) were the highest, and the loss was the lowest. On the basis of these results, a
weighted model of 568 epochs was used for the detection of traffic signs to classify the traffic
signs into three main categories, namely, warning, regulatory, and mandatory signs. Table 5 Dataset
AI-Hub Dataset
IMAGE
86400
Epoch
100000
Learning rate
0.001
Batch size
100
Warning
Regulatory
Mandatory
Original
Using_IDG
Original
Using_IDG
Original
Using_IDG
mAP (%)
83.1
90.0
88.9
95.3
86.2
91.5
Loss (%)
84.9
21.1
38.1
20.4
56.3
17.5 3.3 Training and validation of Inception-v3 model The Inception-v3 model was used to sub-classify the traffic signs, which were already
classified as warning, regulatory, or mandatory signs. We created sub-classifiers based on the
main categories to train the Inception-v3 model. The three sub-classifiers were trained by setting
the epochs to 100000 times, the learning rate to 0.001, and the batch size to 100. As a result of
the training, we found that the training accuracy was 83.1% for the warning sign sub-classifier,
88.9% for the regulatory sign sub-classifier, and 86.2% for the mandatory sign sub-classifier. That the training accuracy was not higher than 90% was determined to be due to the
imbalance in the sub-classifier data used for the training. Therefore, the amount of the training
data was increased by repeating the processes of rotating the image, changing the image
brightness, and changing the image size using the Image Data Generate (IDG) function provided
by TensorFlow version 2.4.(18,19) As a result, the training accuracy improved to 90.0% for the
warning signs, 95.3% for the regulatory signs, and 91.5% for the mandatory signs (Table 5). Table 4
Detailed information on the test sets and the accuracy of their classification using Mask R-CNN model. Test set 1
(1000 images)
Test set 2
(1000 images)
Test set 3
(175 images)
ID
Description
Number
of labels
Ratio
(%)
Average
precision
AP (%)
Number
of labels
Ratio
(%)
Average
precision
AP (%)
Number
of labels
Ratio
(%)
Average
precision
AP (%)
1
Warning
74
12.1
95.0
156
24.6
80.6
3
1.4
100.0
2
Regulatory
330
54.0
92.3
358
56.4
86.5
134
63.2
94.7
3
Mandatory
207
33.9
86.7
121
19.1
76.3
75
35.4
76.0
Total
611
mAP
91.3
635
mAP
81.2
212
mAP
90.2 Detailed information on the test sets and the accuracy of their classification using Mask R-CNN model. Sensors and Materials, Vol. 34, No. 12 (2022) 4809 Fig. 9. (Color online) (a) Sample results of detection and classification using Mask R-CNN model in test sets 1, (b)
2, and (c) 3. (a)
(b)
(c) (c) (a) (b) (a) (b) (c) Fig. 9. (Color online) (a) Sample results of detection and classification using Mask R-CNN model in test sets 1, (b)
2, and (c) 3. 3.4
Assessment of Inception-v3 model To assess the performance of the trained warning, regulatory, and mandatory sign classifier
models, we created and used cropped images based on the label data of the test datasets used for
the performance assessment of the Mask R-CNN model. According to the performance assessment results of the three sub-classifiers, the accuracies
(mAP) were 77.3, 78.6, and 76.7% shown in Table 6. All three datasets were able to partially
increase the evaluation accuracy by increasing the diversity of training data using IDG, but the Sensors and Materials, Vol. 34, No. 12 (2022) 4810 Table 6
(Color online) Classification results of symmetric traffic signs. (Color online) Classification results of symmetric traffic signs. Images
Code
Keep Left
(313)
Keep Right
(314)
Classification
result
Code
Amount
Ratio (%)
Code
Amount
Ratio (%)
313
232
96.27
314
34
75.56
309
1
0.41
313
11
24.44
306
1
0.41
308
1
0.41
307
1
0.41
314
4
1.66
311
1
0.41
Total
241
Total
45 high evaluation accuracy was not recorded. This means that the classification weight model
calculated from the training data constructed using IDG has not been generalized. To solve this
problem, it can be seen that it is necessary to increase the diversity of forms for training traffic
signs by additionally constructing new data in training data. Table 6 shows the classification results of symmetrical traffic signs. It was found that left-side
traffic was misclassified as right-side traffic and vice versa. For this reason, it can be seen that
the accuracy of classification is rather low in the case of symmetrical traffic signs. 4.
Conclusions In this study, a methodology that combines Mask R-CNN and Inception-v3 models to detect
and sub-classify 103 traffic signs to produce high-definition maps of South Korea using images
obtained by MMSs is described. The Mask R-CNN model was used to determine the regions of
the objects, and the main categories were defined as warning, regulatory, or mandatory signs. These categories were then used in the Inception-v3 model to sub-classify the traffic signs into
103 classes. The weighted Mask R-CNN model was trained to detect the traffic signs defined in the high-
definition maps using the images captured by MMSs and to classify them into three main
categories. The trained and weighted model was assessed using the test datasets obtained from
various MMSs, and an average accuracy of 87.6% was obtained. The weighted model was trained using Inception-v3 to create sub-classification categories for
the traffic signs. The imbalance of the data between the classification targets in the training
process of the weighted model could be partially solved by using the IDG function. Combining
these models enabled the classification of 103 types of traffic sign in high-definition maps with
an average accuracy of 77.5%. Sensors and Materials, Vol. 34, No. 12 (2022) 4811 The methodology proposed in this study can be used to detect and classify traffic signs
systematically to produce the high-definition maps required for autonomous driving. It will be
necessary to obtain additional training data to improve the accuracy of the models. References Korean Soc. Surv. Geod. 37 (2019) 119. https://doi.org/10.7848/ksgpc.2019.37. and I. Y. Hong: J. Korean Soc. Surv. Geod. 37 (2019) 119. 19 Image Data Generator of Tensorflow Core v2.0: https://www.tensorflow.org/api_docs/python/tf/keras/
preprocessing/image/ImageDataGenerator (accessed June 2022). References 1 Y. S. Na, S. K. Kim, Y. S. Kim, J. Y. Park, J. M. Jeong, K. C. Jo, S. J. Lee, S. J. Cho, M. H. Sunwoo, and J. M. 1 Y. S. Na, S. K. Kim, Y. S. Kim, J. Y. Park, J. M. Jeong, K. C. Jo, S. J. Lee, S. J. Cho, M. H. Sunwoo, and J. M. Oh: J. Korean Soc. Automot. Eng. 28 (2020) 797. https://doi.org/10.7467/KSAE.2020.28.11.797 2 National Geographic Information: HD Map Quality Crafting Manual, https://www.ngii.go.kr/kor/contents/
view.do?sq=1241&board_code=contents_data (accessed June 2022). 3 Y. Yu, J. Li, C. Wen, H. Guan, H. Luo, and C. Wang: J. Photogramm. Remote Sens. 113 (2016) 106. https://doi. org/10.1016/j.isprsjprs.2016.01.005 4 A. Alvaro, S. Mario, A. Juan, A. Garcia, and R. Belen: Expert Syst. Appl. 89 (2017) 286. https://doi. org/10.1016/j.eswa.2017.07.042 5 M. J. Choi, J. K. Suhr, K. Choi, and H. G. Jung: IEEE 7 (2019) 149846. https://doi.org/10.1109/
ACCESS.2019.2947287 6 P. Viola and M. J. Jones: Int. J. Comput. Vis. 57 (2004) 137. https://doi.org/10.1023/B:VISI.0000013087.49260. fb 7 C. Liu, S. Li, F. Chang, and Y. Wang: IEEE 7 (2019) 86578. https://doi.org/10.1109/ACCESS.2019.2924947 7 C. Liu, S. Li, F. Chang, and Y. Wang: IEEE 7 (2019) 86578. https://doi.org/10.1109/ACCESS.2019.2924947
8 L. Wang, K. Zhou, A. Chu, G. Wang, and L. Wang: IEEE 9 (2021) 124963. https://doi.org/10.1109/
ACCESS.2021.3109798 8 L. Wang, K. Zhou, A. Chu, G. Wang, and L. Wang: IEEE 9 (2021) 124963. https://doi.org/10.1109/
ACCESS.2021.3109798 9 D. Tabernik and D. Skočaj: IEEE Trans. Intell. Transp. Syst. 21 (2020) 1427. https://doi.org/10.1109/
TITS.2019.2913588 10 K. He, G. Gkioxari, P. Dollar, and R. Girshick: Proc. IEEE Int. Conf. Comput. Vis. (2017) 2980. https://doi. org/10.48550/arXiv.1703.06870 g
11 Y. Yang, H. Luo, H. Xu, and F. Wu: IEEE Trans. Intell. Transp. Syst. 17 (2016) 2022. https://doi.org/10.1109/
TITS.2015.2482461 12 C. Lin, L. Li, W. Luo, K. Wang, and J. Guo: Period. Polytech. Transp. Eng. 47 (2018) 242. https://do
org/10.3311/PPtr.11480f 13 C. Szegedy, V. Vanhoucke, S. Ioffe, J. Shlens, and Z. Wojna: Proc. IEEE Comput. Soc. Conf. Comput. Vis. Pattern Recognit. (2016) 2818. https://doi.org/10.1109/CVPR.2016.308 14 D. G. Kim, Y. G. Kang, Y. R. Park, N. Y. Kim, and J. Y. Lee: Spat. Inf. Res. 28 (2020) 241. https://do
org/10.1007/s41324-019-00285-x 15 United States Road Symbol Signs: https://mutcd.fhwa.dot.gov/services/publications/fhwaop02084/index.htm
(accessed June 2022). 16 Comparison of European Road Signs: https://en.wikipedia.org/wiki/Comparison_of_European_road_sig
(accessed June 2022). 17 The Ministry of Science and ICT in Korea: https://aihub.or.kr (accessed June 2022). 18 J. S. Kim and I. Y. Hong: J. About the Authors Hyeong-Yoon So received his B.S. degree from Namseoul University, Korea,
in 2021. He has been enrolled in the master’s program in spatial information
engineering at Namseoul University, Korea, since 2021. His research interests
are in photogrammetry, artificial intelligence, and GIS. (ssoss95@naver.com) Sensors and Materials, Vol. 34, No. 12 (2022) 4812 Eui-Myoung Kim received his B.S. and M.S. degrees from Gyeongsang
National University, Korea, in 1994 and 1996, respectively, and his Ph.D. degree from Yonsei University, Korea, in 2000. From 2000 to 2002, he was a
senior researcher at the Korea Institute of Civil Engineering and Building
Technology (KICT). He worked as a postdoctoral fellow at the University of
Calgary, Canada, from 2003 to 2005. He was with the Korea Geospatial
Information and Communication (KSIC) from 2005 to 2006. Since 2007, he
has been a professor at Namseoul University, Korea. His research interests are
in photogrammetry and GIS. (kemyoung@nsu.ac.kr) Eui-Myoung Kim received his B.S. and M.S. degrees from Gyeongsang
National University, Korea, in 1994 and 1996, respectively, and his Ph.D. degree from Yonsei University, Korea, in 2000. From 2000 to 2002, he was a
senior researcher at the Korea Institute of Civil Engineering and Building
Technology (KICT). He worked as a postdoctoral fellow at the University of
Calgary, Canada, from 2003 to 2005. He was with the Korea Geospatial
Information and Communication (KSIC) from 2005 to 2006. Since 2007, he
has been a professor at Namseoul University, Korea. His research interests are
in photogrammetry and GIS. (kemyoung@nsu.ac.kr) Eui-Myoung Kim received his B.S. and M.S. degrees from Gyeongsang
National University, Korea, in 1994 and 1996, respectively, and his Ph.D. degree from Yonsei University, Korea, in 2000. From 2000 to 2002, he was a
senior researcher at the Korea Institute of Civil Engineering and Building
Technology (KICT). He worked as a postdoctoral fellow at the University of
Calgary, Canada, from 2003 to 2005. He was with the Korea Geospatial
Information and Communication (KSIC) from 2005 to 2006. Since 2007, he
has been a professor at Namseoul University, Korea. His research interests are
in photogrammetry and GIS. (kemyoung@nsu.ac.kr)
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Study on the mechanical properties of clayey slip zone soil considering montmorillonite content
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Research Square (Research Square)
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cc-by
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Research Article Keywords: cohesive slipband soils, expansion characteristics, hydrophilic minerals, montmorillonite,
shear strength parameters
Posted Date: January 4th, 2023
DOI: https://doi.org/10.21203/rs.3.rs-2178907/v1
License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read Full License Posted Date: January 4th, 2023 DOI: https://doi.org/10.21203/rs.3.rs-2178907/v1 License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read Full License Page 1/21 Page 1/21 Page 1/21 Abstract The strength property of slip zone soil determines the evolution mechanism and deformation damage of
landslides. When the slip zone soil contains a certain amount of hydrophilic minerals, it, therefore, has
swelling properties. These swelling properties cause the mechanical property of cohesive slip zone soils
to differ from those of conventional-type of slip zone soils. Thus, four cases of typical landslide cohesive
slip zone soils in the Shanxi Province of the Yellow River Basin were used as research objects to study the
mechanism of hydrophilic minerals on the mechanical properties of cohesive slip zone soils. The mineral
fractions of the cohesive slip zone soils were analyzed using X-ray diffraction tests, followed by direct
shear tests to analyze the pattern of the shear strength parameters of cohesive slip zone soils under
different hydrophilic mineral contents with water content. Finally, the free swelling rate test was used to
verify that montmorillonite minerals are the main hydrophilic minerals affecting the swelling
characteristics of cohesive slip zone soils. The results were as follows. The cohesion and internal friction
angle of clayey slipband soils exhibited two patterns (negative exponential/negative correlation) with the
variation of water content, and the critical values of the pattern of the variation of the shear strength
parameters with water content were affected by montmorillonite content ranging from 17.65% to 34.52%. The shear strength attenuation of clayey slip zone soils showed a positive correlation with the change in
montmorillonite content, and the swelling property caused by montmorillonite minerals was the intrinsic
reason for the shear strength attenuation of clayey slip zone soils under different water content
conditions. The research results explain the shear strength decay law of cohesive slip zone soils from the
perspective of mineral composition and reveal the unique strength properties of these slip zone soils. 1 Introduction Domestic and foreign research res
contain a certain amount of clay minerals(Yan C J et al,200
B L et al,2021) but are different from general clay soils, and
these slip soils swelling characteristics(Chen J et al,2014;K
of hydrophilic minerals in different areas of cohesive slip so
mineral contents cause different swelling results. These giv
characteristics, which cause different degrees of weakening
attenuation(Baldermann A et al,2021). The experimental study on the strength characteristics of slipband soils containing clay particles by Chen
Xiaoping (Chen X P et al,2011)showed that the shear stress–shear strain relationship was of a hardening
type; with increased shear displacement, the shear stress exerted itself to the peak relatively quickly and
thereafter basically stabilized. Meanwhile, Han Lei et al.(Han L et al,2018) found that gravelly clayey
slipband soils also exhibit strain-hardening characteristics through triaxial tests. The study of Bo Wu et
al.(Wu B et al,2018) showed that under any normal stress, loess-type slipband soils exhibit strain-
softening shear properties. In another study, Yiying Zhang et al.(Zhang Y Y et al,2021) conducted ring
shear tests on tuff rubble slipband soils, which also exhibited strain-softening shear characteristics. This
further illustrated that the mechanical properties of cohesive slipband soils are different from those of
other types of slipband soils. In addition, Wen BP et al. (Wen B P et al,2017)found that slipband soils
containing clay minerals underwent two different creep patterns under residual conditions. Meanwhile, Li
Jiang et al.(Li J et al,2015) studied the changes in microstructure and mechanical properties of softened
interbedded slip zone soils after softening, where microstructural damage was the intrinsic cause of the
decrease in mechanical strength of slip zone soils. Scaringi G et al.(Scaringi G et al,2018) found the time
course pattern of the mechanical properties of cohesive slip zone soils with time. In summary, cohesive
slip zone soils have mechanical properties that distinguish them from other types of slip zone soils, and
their in-depth study should broaden the research field of slip zone soils. Domestic and foreign scholars have conducted studies on the physical properties and mechanical
properties of slip zone soils. However, there is a lack of research on the correlation between the mineral
components and mechanical properties of clayey slip zone soils, not to mention the explanation of the
decay of their strength properties with water content from a mineral perspective. 1 Introduction Therefore, in this paper,
four cases of clayey slip zone soils in the Shanxi Province of the Yellow River Basin, where weak layer-
controlled landslides typically occur, were used as the research objects. The mineral components and
mechanical properties of these slip zone soils were systematically studied using X-ray diffraction (XRD)
analysis, direct shear test, and free expansion rate test. The research results can further reveal the
characteristics of the strength properties of such slip zone soils and contribute to the scientific prevention
and control of landslide hazards. 1 Introduction Slippery zone soil is an important research topic for determining landslide geological disaster genesis
mechanisms and evolution processes. In this respect, scholars have studied the microscopic
characteristics(Cheng Q G,1999;Wu B et al,2018;Sun J X et al,2021), mineral fraction(Yan C J et
al,2002;Cui D S et al,2018), grain size gradation(Wang L et al,2017), resistivity properties(Long J H et
al,2007), and mechanical properties(Yagi N et al,2003;Long J H et al,2010;Chen X L et al,2015;Jiang QQ et
al,2019;Zhou JJ et al,2020) of slipband soils. One type of slip zone soil is controlled and gradually
evolved by mudstone, shale, and clayey rock. These slip zone soils are called clayey slip zone soils
because of the large amount of clay minerals in the mineral fraction. Moreover, they often have scale-like
microstructure characteristics(Dimaio C et al,2010), as well as low permeability, high plasticity, and low
friction angle. According to the practical experience of the subject group in landslide engineering for
many years, clayey slipband soil landslides are widely distributed in the Yellow River basin in the loess,
coal, and bauxite strata and in slopes controlled by soft-type strata with mainly expansive minerals,
posing a great threat to the safety of people and property in the Yellow River basin every year. From the summary of the research progress on slip zone soils by Li et al(Li X et al,2010)., the
classification of slip zone soils still must be further enhanced and the understanding of the essential
factors of slip zone soil strength further deepened. Therefore, systematic research on cohesive slip zone Page 2/21 soils must be conducted. Domestic and foreign research results have shown that cohesive slip soils
contain a certain amount of clay minerals(Yan C J et al,2001;Dai S B et al,2005;Zheng G D et al,2006;Zhu
B L et al,2021) but are different from general clay soils, and the presence of hydrophilic minerals gives
these slip soils swelling characteristics(Chen J et al,2014;Khan M S et al,2018). The types and contents
of hydrophilic minerals in different areas of cohesive slip soils vary, and different types of hydrophilic
mineral contents cause different swelling results. These give cohesive slip soils different swelling
characteristics, which cause different degrees of weakening effects on the degree of shear strength
attenuation(Baldermann A et al,2021). soils must be conducted. 2 Test Sample
2.1 Sampling process Page 3/21 Page 3/21 Through field investigations and engineering practice project data collection, experimental samples were
obtained from boreholes, exploratory wells, and probing troughs, among others, for typical weak layer
control landslides in the Shanxi Province, as shown in Fig. 1, with a total of four typical landslide cases. The weight of the experimental samples obtained for each landslide viscous slip zone soil was 10 kg. Then, the soil samples were immediately packed into containers and sealed using cling films and brought
back to the laboratory. g
g
g
g p
p
j
,
p
p
obtained from boreholes, exploratory wells, and probing troughs, among others, for typical weak layer
control landslides in the Shanxi Province, as shown in Fig. 1, with a total of four typical landslide cases. The weight of the experimental samples obtained for each landslide viscous slip zone soil was 10 kg. Then, the soil samples were immediately packed into containers and sealed using cling films and brought
back to the laboratory. 3.1 Test procedure XRD tests were conducted using a Smartlab SE03030502 model X-ray diffractometer. The soil samples
were first taken from each typical landslide cohesive slip zone soil, pounded into blocks, and ground into
powder using an agate mortar. The powder was then passed through a 200-mesh sieve (no granularity
and flour texture to the touch), dried at a temperature of 105°C for 12 h, and then loaded into IMEC-type
sample tubes for backup. The X-ray diffractometer was calibrated according to the instrument manual
before use and was ready for small-angle (0.6°–5°) and wide-angle (5°–85°) diffraction test start-up with
a scan step of 0.01°. The test target was a copper target (radiation source ray λ = 0.154056 nm), and the
test was started using an external calculation book control system. 2.2 Determination of the saturated water content The landslide cases in Dongshan, Taiyuan; Linxian, Lvliang; Xiangning, Linfen; and Zezhou, Jincheng,
were labeled as DS, LX, XN, and ZZ, respectively. The saturated water content of each sample was
measured in an indoor laboratory, and the test results are shown in Table 1. Table 1
Saturated water content of the soil samples
Soil sample
number
Protostratigraphic
era
Original stratigraphic
lithology
Saturated water content
(%)
DS
C3t
Carbonaceous mudstone
39.64
LX
T2t
Mudstone
30.32
XN
N2b
Clay
27.99
ZZ
C3t
Shale
31.12 Table 1 3.2 Analysis of mineral components The raw XRD data were processed using the Jade 6.5 software. The diffraction pattern was determined
by taking the diffraction angle (2θ) as the horizontal coordinate and the intensity of the diffraction peaks
as the vertical coordinate. Using the experimentally obtained diffraction pattern, the physical phase Page 4/21 Page 4/21 species categories of the mineral components contained in the clayey slip zone soil were determined by
comparing the standard diffraction characteristics of various minerals provided by the ICDD and the
JCPDS. species categories of the mineral components contained in the clayey slip zone soil were determined by
comparing the standard diffraction characteristics of various minerals provided by the ICDD and the
JCPDS. Mineral peaks did not appear in the small-angle test diffraction patterns, and the minerals present in the
small-angle diffraction did not appear in the experimental samples. From the diffraction pattern curve of
the wide-angle test, as shown in Fig. 2, the identification and analysis concluded that clayey slipband soil
samples contained minerals such as montmorillonite, kaolinite, illite, chlorite, gypsum, chlorite, talc, black
mica, quartz, and white mica. Moreover, there were some differences in the mineral analogs and contents
among the samples consistent with the research results of Shaobin Dai et al(Dai S B et al,2005). In
addition, the hydrophilic minerals in the mineral fraction of the clayey slickensides were mainly
montmorillonite, kaolinite, illite, and chlorite. The mineral composition of the clayey slip zone soil was
different from that of the general slip zone soil, where the usual soil does not contain a certain amount of
feldspar(Yan C J et al,2002;Dai S B et al,2005), calcite(Cui D S et al,2018), and other minerals. Another
explanation is that the feldspars in slip soils are rapidly transformed into montmorillonite and kaolinite
when they undergo this process(Baldermann A et al,2021), which is also the reason for the high content
of hydrophilic minerals in clayey slip soils. After the qualitative analysis of the phases, the exact content of each mineral was further determined
using the K-value method and the adiabatic method, as shown in Fig. 4. The percentages of hydrophilic
minerals contained in the clayey slip zone soils of the typical landslides were 60.10%, 42.40%, 54.14%,
and 35.58%, respectively, and their common hydrophilic minerals were montmorillonite and kaolinite. 3.2 Analysis of mineral components In
addition, the landslides in Dongshan, Taiyuan; Xiangning, Linfen; and Zezhou, Jincheng, also contained
illite minerals, whereas the landslide in Linxian, Lvliang, contained chlorite minerals, of which
montmorillonite minerals had the highest percentage of all four hydrophilic minerals. The presence of a
large number of hydrophilic mineral components in the mineral fraction of clayey slip zone soils is an
important basis for distinguishing them from other types of slip zone soils. 4.1 Straight shear test The slow shear test was conducted using the ZJ strain-controlled straight shear instrument produced by
Nanjing Soil Instrument Factory Co., Ltd. The test was performed in strict accordance with the Standard
for Geotechnical Test Methods GB/T50123-2019. Appropriate amounts of cohesive slip zone soil
samples were obtained and ground through a 0.5-mm soil sieve to study the effect of different moisture
contents on the shear strength of cohesive slip zone soils. As the rock and soil bodies in the Yellow River
basin and other arid regions often contain crystalline salts, changes in environmental factors such as
acidity and alkalinity brought about by their dissolution in aqueous solutions were avoided. Pyrolysis
(500°C) and elution (deionized water)(Bing H et al,2011;Zhao Z W et al,2021) were thus adopted in
sequence to wash the salts to eliminate alkaline solution ions (e.g., Na+ and Ca2+), acid solution ions Page 5/21 (e.g., SO4
2− and NO3−), and organic impurities. Then, the cohesive slip zone soil after salt washing was
dried and processed, each time weighing 50 g of dry soil samples and setting the slow shear test
moisture content ratio according to the saturated moisture content steps of each typical landslide
cohesive slip zone soil in Table 1. Each case test sample was prepared in four groups with different water
contents; 5, 7.5, 10, 15, and 20 ml of distilled water were drawn using a medical syringe, and the soil
samples were evenly sprayed with 10%, 15%, 20%, 30%, and 40% of the water contents. The containers
were then sealed using a cling film and left for 24 h. Straight shear specimens (61.8 mm in diameter and
20 mm in height) of the remodeled cohesive slip zone soils were prepared using the pressed sample
method and then placed on the straight shear table with an impermeable film separating the upper and
lower ends of the specimens from the permeable stone to avoid the effect of matrix suction during the
shear test. Then, vertical pressures were applied at 100, 200, 300, and 400 kPa, respectively, with a shear
volume of 6 mm and a shear rate of 0.02 mm/min. The experimental data were recorded according to the
shear displacement. The variation patterns of shear stress with shear displacement for cohesive slipband soils were obtained,
as shown in Fig. 3. The unified law of change was divided into three stages. 4.1 Straight shear test The first stage shows that
the shear stress increases rapidly with increasing shear displacement. The second stage shows that the
rate of increase of shear stress gradually slows down and the curve shows a curved arc section. Finally,
the third stage shows a stable fluctuation period, the shear stress growth rate tends to stabilize, and the
growth rate remains basically the same. The variation patterns of shear stress with shear displacement
did not show a yielding phenomenon but showed hardening-type characteristics, consistent with the
results of Chen Xiaoping(Chen X P et al,2011) and Han Lei et al(Han L et al,2018). on clay-bearing
slipband soils. Because the slipband soil underwent complex processes such as shear misalignment and
grinding and crushing, which led to the damage of the clay mineral grain crystal layer, it did not show the
strain-softening phenomenon as what appeared in the ring shear test of slipband soil with artificially
added clay grain content conducted by Cao Shichao et al(Cao S C et al,2019). In addition, from the shear
stress–shear strain test curve, it can be initially seen that the peak shear stress decreases with increasing
water content under the same vertical pressure condition. The shear stress corresponding to a shear displacement of 5 mm was used as the shear strength. At this
time, most of the working conditions have reached the peak shear stress. Then, the shear strength of
each typical landslide cohesive slip zone soil was extracted and its relationship with the vertical pressure
was plotted, as shown in Fig. 4. The unified law showed that the shear strength increased as the vertical
pressure increased, which exhibited the same regular characteristics as those of the general slip zone
soil: that is, with higher vertical pressure, the clay slip zone soil compactness increased, and the mineral
particles were in closer contact. In addition, the shear strength of the cohesive slipband soils decreased
with increasing water content, which verified the preliminary conclusions drawn above. However, the
degree of influence of the water content varied among the test samples. For instance, the shear strength
of the clayey slip zone soil of the landslide in Dongshan, Taiyuan, was greatly influenced by the water
content because hydrophilic minerals accounted for 60.10% of its mineral fraction, where the swelling Page 6/21 Page 6/21 characteristics of the extremely hydrophilic clay minerals under high water content conditions led to an
increase in shear strength attenuation. 4.1 Straight shear test The next landslide cases affected by water content were the LX
and XN clayey slip zone soils, which comprised 42.40% and 54.14% of hydrophilic minerals, respectively. The least affected by water content changes was the landslide clayey slip zone soil in Zechou, Jincheng
City, with only 25.77% of hydrophilic minerals. Using the data on the shear strength and vertical pressure of each viscous slip zone soil above, the
cohesion and internal friction angle can be calculated through linear fitting combined with the Coulomb
strength formula, and the calculation results are shown in Table 2. A scatter plot of the variation of each
shear strength parameter with moisture content was plotted, as shown in Fig. 5. The cohesion and
internal friction angle of the cohesive slip zone soil decreased as the water content increased, which
followed the general law characteristics of unsaturated soil. In addition, the shear strength parameters
showed two patterns with the variation of moisture content. Table 2
Shear strength parameters of the cohesive slip zone soils
Sample number
Water content (%)
Cohesive force (kPa)
Internal friction angle (°)
DS
10
49.11
21.52
20
42.35
17.65
30
35.23
7.49
40
25.16
4.35
LX
10
18.43
26.71
15
13.56
22.27
20
10.33
21.21
30
3.08
17.76
XN
10
22.46
22.59
15
15.63
21.25
20
9.34
19.32
30
5.03
17.96
ZZ
10
18.32
25.19
15
13.86
23.84
20
11.35
23.38
30
10.07
23.09 Linear and exponential functions were used to fit and analyze the relationship between cohesion and
internal friction angle with the variation of moisture content. The formulas are given as follows. Linear and exponential functions were used to fit and analyze the relationship between cohesion and
internal friction angle with the variation of moisture content. The formulas are given as follows. y = Aω + B 1
y = Aω + B
y = C exp(−ω/D) + E 1 1 1
y = C exp(−ω/D) + E y = C exp(−ω/D) + E y = C exp(−ω/D) + E 2 where y is the cohesion or angle of internal friction; ωis the water content; and A, B, C, D, and E are the
model parameters. On the basis of the test data in Table 2 to fit the linear and exponential functions, the
fitted curve is shown in Fig. 5. 4.1 Straight shear test where y is the cohesion or angle of internal friction; ωis the water content; and A, B, C, D, and E are the
model parameters. On the basis of the test data in Table 2 to fit the linear and exponential functions, the
fitted curve is shown in Fig. 5. The results of the fitted function for the variation of cohesion with moisture content are as follows. The results of the fitted function for the variation of cohesion with moisture content are as follows. The results of the fitting function of the variation of the angle of internal friction with the moisture
content are as follows. The cohesion and internal friction angle of the ZZ clay slip soils with low hydrophilic mineral content
showed negative exponential relationships with water content, whereas the cohesion and internal friction
angle of the DS clay slip soils with high clay mineral content showed negative linear relationships with
water content. These indicated that the hydrophilic mineral content affected the shear strength
parameters of the clay slip soils with water content. Meanwhile, XN with high hydrophilic mineral content
and LX with low content of clayey slickensides showed opposite patterns, and the influence of
montmorillonite as the main hydrophilic mineral on the decay pattern of shear strength parameters was
well explained. The critical values of the patterns of shear strength parameters with water content for the
XN clayey slip zone soil with lower montmorillonite content ranged from 17.65–34.52%. Further, the shear strength attenuation degree (water content 10–30%) of the clayey slip zone soils of
each typical landslide was plotted with the variation of montmorillonite content, as shown in Fig. 6. The
degree of cohesion decay showed a certain negative correlation with the decrease in montmorillonite
content, which was because montmorillonite minerals absorb water and swell under low water content
conditions to form a more compact structure between particles, and the degree of cohesion decay
showed a smaller degree. However, Fig. 5 shows that with the continuous increase in water content, the
decreasing cohesion trends of the DS and LX clayey slip zone soils, which had the greatest Further, the shear strength attenuation degree (water content 10–30%) of the clayey slip zone soils of
each typical landslide was plotted with the variation of montmorillonite content, as shown in Fig. 6. 4.1 Straight shear test The
degree of cohesion decay showed a certain negative correlation with the decrease in montmorillonite
content, which was because montmorillonite minerals absorb water and swell under low water content
conditions to form a more compact structure between particles, and the degree of cohesion decay
showed a smaller degree. However, Fig. 5 shows that with the continuous increase in water content, the
decreasing cohesion trends of the DS and LX clayey slip zone soils, which had the greatest
montmorillonite contents, were greater than those of the XN and ZZ samples, and the cohesion showed a
positive correlation with montmorillonite content. This was because montmorillonite minerals continued
to absorb water under high water contents and swell violently, and the resulting swelling force caused the
more fragile particles to break and disintegrate. Moreover, the porosity jump caused a decrease in the
degree of association between particles, resulting in a continuous decrease in cohesion and showing a
positive correlation characteristic trend. The degree of decay of the internal friction angle showed a good
positive correlation in the range of 10–30% water content, and the trend of the degree of decay shown in
Fig. 5 remained unchanged. This indicates that the cohesion of the cohesive slip zone soil has a certain
hysteresis with respect to the internal friction angle. 4.2 Free expansion rate test The free expansion rate test of the cohesive slip zone soils was continued to further verify the shear
strength decay law of cohesive slip zone soils. Using a WX-2000 swelling rate tester, appropriate amounts
of soil samples after washing salt treatment and passing through a 0.5-mm soil sieve and drying were
taken. Then, distilled water was added, and the samples were placed in a loose state where they were free
to swell in air. The free swelling rate of clay slip belt soil was calculated using formula (3): δef =
× 100
Vwe −V0
V0 δef =
× 100
Vwe −V0
V0 3 Equation:
is the free swelling rate;
is the volume of the sample after swelling in water;
is the
initial volume of the sample. δef
Vwe
V0 Page 9/21 Page 9/21 The free swelling rate of each typical landslide cohesive slip zone soil specimen was obtained, and the
swelling time was more than 3 d. At this time, the volume of each sample was stable after water
absorption and swelling. The free expansion rate was up to 62.5%, with a minimum of 13.0%. A line
graph of the free swelling rate as affected by the montmorillonite and kaolinite mineral contents is
plotted in Fig. 7. There was a positive correlation between the free swelling rate of viscous slickensides
and the increase in montmorillonite content, whereas the effect of kaolinite clay minerals on the free
swelling rate showed a local dislocation relationship. The natural swelling rate test results verified the
conclusion that montmorillonite is the main expansive clay mineral in the slow shear test, consistent with
the swelling research results of single clay minerals by Bao-Long Zhu et al(Zhu B L et al,2021). The
spacing before and after swelling of montmorillonite was about 1 nm, whereas the spacings before and
after swelling of the other hydrophilic minerals were only 0.0006–0.008 nm. Thus, montmorillonite was
the main hydrophilic mineral for water absorption and swelling. However, the free swelling rate results did
not increase equiproportionally with the increase in montmorillonite content because the shear
fragmentation experienced by the slipband soils led to the damage and destruction of the crystalline layer
of montmorillonite clay minerals, which eventually showed different proportional degrees. 5 Discussion The microstructure of montmorillonite minerals as a composite aluminum-silicate crystal exhibits scale-
like or lamellar shapes(Dimaio C et al,2010), as shown in Fig. 8. Their mechanism of hydration expansion
is that the cations (Na+/Ca2+) inherent between the crystals of montmorillonite form a hydration film
through the adsorption of water molecules on the surface, which continuously increases the lattice
spacing and manifests as volume expansion in the macroscopic scale. The expansion force generated by
the water absorption and expansion of montmorillonite minerals also makes the contact between the
mineral particles closer, changing the contact mode between the mineral particles from a point–point
contact to a line–line contact or even a face–face contact. The increased degree of particle association
manifests in the shear stress–shear strain curve as a hardening characteristic, which is a unique feature
of the mechanical properties of cohesive slip zone soils. In addition, because the vertical load was
applied to the test sample in the slow shear test, the cohesive slip zone soil cannot achieve free
expansion but instead underwent isovolume expansion under constant volume conditions, which
produced an expansion force on the cover of the straight shear pedestal(Xiong Y F et al,2021;Zhu B L et
al,2021) and caused a reduction effect on the vertical load in the axial direction. Moreover, the actual load
value applied to the sample was less than the theoretical load value, causing the attenuation of cohesion
with the change in moisture content to weaken and the attenuation of the angle of internal friction to
increase, resulting in the correlation characteristics in Fig. 6. This effect becomes more prominent as the
water content of the prepared samples increased and the mineral content of montmorillonite increased. The degree of decay of cohesion and internal friction angle was increased by the combined effect of
water content and montmorillonite content, which led to the fragmentation of fragile particles inside the
microstructure after water absorption and swelling. Meanwhile, the leap in porosity led to a continuous
decrease in the degree of association between particles, which finally caused a continuous decrease in Page 10/21 Page 10/21 cohesion and internal friction angle, showing the two patterns in Fig. 5. Therefore, the reason for the
degree of shear strength attenuation of the clayey slipband soils is the presence of the montmorillonite
mineral in addition to the water content factor. 6 Conclusion In this study, relevant experimental analyses were conducted on four cases of typical landslide cohesive
slip zone soils in the Shanxi Province of the Yellow River Basin to study the mechanism of the influence
of hydrophilic minerals on the mechanical properties of cohesive slip zone soils. The following research
conclusions were obtained: ) The hydrophilic minerals in the mineral fraction of the clay slip zone soils were mainly philic minerals in the mineral fraction of the clay slip zone soils were mainly (1) The hydrophilic minerals in the mineral fraction of the clay slip zone soils were mainly
montmorillonite, kaolinite, illite, and chlorite. The percentages of the hydrophilic minerals in the clayey
slip zone soils of each typical landslide were 60.10% (DS), 42.40% (LX), 54.14% (XN), and 35.58% (ZZ),
and montmorillonite minerals had the highest percentages in hydrophilic minerals. (2) The stress–strain curve of the cohesive slipband soil shows a hardening-type characteristic. The
variations of cohesion and internal friction angle with water content showed two patterns. The samples
with low montmorillonite content showed a negative exponential relationship, whereas the samples with
high montmorillonite content showed a negative correlation. Critical values for the patterns of variation of
shear strength parameters with moisture content as affected by the montmorillonite content ranged from
17.65–34.52%. (3) The shear strength attenuation degree of clayey slipband soils showed a positive correlation with the
change in montmorillonite content. When the range of water content was small, the attenuation degree of
cohesion showed a negative correlation with the change in montmorillonite content; when the range of
water content was large, the attenuation degree of cohesion showed a positive correlation with the
change in montmorillonite content. Moreover, the degree of decay of the internal friction angle with the
variation of montmorillonite content all showed positive correlation characteristics. (3) The shear strength attenuation degree of clayey slipband soils showed a positive correlation with the
change in montmorillonite content. When the range of water content was small, the attenuation degree of
cohesion showed a negative correlation with the change in montmorillonite content; when the range of
water content was large, the attenuation degree of cohesion showed a positive correlation with the
change in montmorillonite content. Moreover, the degree of decay of the internal friction angle with the
variation of montmorillonite content all showed positive correlation characteristics. 5 Discussion When the content of intact montmorillonite clay minerals
increased to a certain degree or with an artificially proportioned hydrophilic mineral content, the
mechanical properties exhibited a strain-softening phenomenon(Zhang Y Y et al,2021), but the softening
amplitude was small and its mechanical properties favored the nature of clay. However, the mechanical
properties of clayey slip soils obtained from the test were not exactly the same as those of clay. Therefore, clayey slip zone soils are an extremely special type of slip zone soil. cohesion and internal friction angle, showing the two patte
degree of shear strength attenuation of the clayey slipband
mineral in addition to the water content factor. When the co
increased to a certain degree or with an artificially proportio
mechanical properties exhibited a strain-softening phenom
amplitude was small and its mechanical properties favored
properties of clayey slip soils obtained from the test were n
Therefore, clayey slip zone soils are an extremely special ty The results of the water absorption and swelling of clayey slip zone soils showed plastic deformation
and sharp decay of shear strength on a macroscopic scale, which verified that natural slopes and
artificial slopes containing weak interlayers are prone to landslides through sliding along the weak layer
under the conditions of natural rainfall and artificial irrigation, among others. Acknowledgements The authors would like to thank the Shanxi Metallurgical Geotechnical Engineering Investigation Ltd for
their cooperation in conducting the experiment tests. Funding This research was partially funded by the Development Fund Project of State Key Laboratory of Deep
Geotechnical Mechanics and Underground Engineering of China(SKLGDUEK2124)and Shanxi Natural
Science Foundation of China(20210302124112). Abbreviations XRD:X-ray diffraction;ICDD:International Centre For Diffraction Data;JCPDS:Joint Committee on Powder
Diffraction Standards 6 Conclusion (4) There was a positive correlation between the free swelling rate of clayey slip zone soils and the
content of montmorillonite minerals. Montmorillonite was the main expansive clay mineral, and its (4) There was a positive correlation between the free swelling rate of clayey slip zone soils and the
content of montmorillonite minerals. Montmorillonite was the main expansive clay mineral, and its Page 11/21 swelling property was the intrinsic reason for the shear strength decay of the clayey slip zone soil under
different water content conditions. swelling property was the intrinsic reason for the shear strength decay of the clayey slip zone soil under
different water content conditions. Author details 1College of Mining Engineering,Taiyuan University of Technology,Taiyuan,030024,China;2Architectural
Design and Research Institute (Group) Co.,Ltd.,Tongji University,Shanghai,200092,China;3Shanxi
Metallurgical Geotechnical Engineering Investigation Ltd,Taiyuan,030000,China Competing interests I have declare that there is no any competing interests. Availability of data and materials All the datasets that have been used and analysed during the current study is available from the
corresponding author on reasonable request. Authors’ contributions Funding acquisition,L.JH.;Methodology,L.JH.,Y.ZQ.and J.YQ.;Project administration,L.JH. and
Y.ZQ.;Experiment,J.YQ.,C.CY.and D.R.;Software,Y.ZQ.and Y.HY.;Supervision,L.JH.;Writing-Original
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15. Li J, Xu Q, Hu ZM et al(2015)Experimental research on softening of undisturbed saturated slip soil in
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undisturbed soil.Journal of Arid Land Resources and Environment35(3):126–
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undisturbed soil.Journal of Arid Land Resources and Environment35(3):126–
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458 21. Wang L, Zhao FS, Cheng XH et al (2017)Physical and mechanical properties and microstructures of
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458 22. Wen BP, Jiang XZ (2017) Effect of gravel content on creep behavior of clayey soil at residual state:
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implication for its role in slow-moving landslides. Landslides 14(2):559–576. http://doi.org/10.1007/s10346-016-0709-3 23. Wu B, Zhao FS, Wang L et al (2018)Physical and mechanical properties of slip zone soil in loess-
bedrock landslides and its evolutionary process.Coal Geology&Exploration46(4):129–134 + 141 23. Wu B, Zhao FS, Wang L et al (2018)Physical and mechanical properties of slip zone soil in loess-
bedrock landslides and its evolutionary process.Coal Geology&Exploration46(4):129–134 + 141 24. Page 12/21 Xiong YF, Lv SH, Xiao X(2021)Study on the Stability of a Landslide Considering the Action of
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zone in reservior district of Three Gorges Profect. Rock and Soil Mechanics 23(Sup 1): 23–
26.http://doi.org/10.16285/j.rsm.2002.s1.007 26. Yan CJ, Tang HM, Chen JY et al (2002)Studies of soil microstructures and compositions of slipping
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26.http://doi.org/10.16285/j.rsm.2002.s1.007 27. Yan CJ, Tang HM, Sun Y Z(2001)Study on the soil of slipping zone in landslides and its significance
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by scanning electron microscope and X-Ray diffractometer.Geological Science and Technology
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776.http://doi.org/10.13544/j.cnki.jeg.2020-610 28. Zhang YY, Guo CB, Yang ZH et al(2021)Study on shear strength of deep-seated sliding zone soil of
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Prefecture,Japan. Geochimica35(2):201–210.http://doi.org/10.19700/j.0379-1726.2006.02.012 31. Zhou JJ, Zhao FS, Yuan XQ et al (2020)Creep process and the microstructural evolution of sliding-
zone soil.Hydrogeology & Engineering Geology47(3):115–121. http://doi.org/10.16030/j.cnki.issn.1000-3665.2019010011 31. Zhou JJ, Zhao FS, Yuan XQ et al (2020)Creep process and the microstructural evolution of sliding-
zone soil.Hydrogeology & Engineering Geology47(3):115–121. http://doi.org/10.16030/j.cnki.issn.1000-3665.2019010011 32. Zhu BL, Li Q, Wang SL et al (2021) Study on time-dependent effect and three-dimensional macro-
micro swelling pressure model of black shale under influence of water. J Railway Sci Eng
18(10):2610–2619. http://doi.org/10.19713/j.cnki.43-1423/u.T20201067 32. Zhu BL, Li Q, Wang SL et al (2021) Study on time-dependent effect and three-dimensional macro-
micro swelling pressure model of black shale under influence of water. J Railway Sci Eng
18(10):2610–2619. http://doi.org/10.19713/j.cnki.43-1423/u.T20201067 Figures Figures Page 15/21
Figure 1
Clay slip zone soil of each typical landslide. (a) Location of the Yellow River Basin and landslide ca
China based on the 1980 Chinese National Geodetic Coordinate System. (b–e) Landslides in (b)
Dongshan, Taiyuan, (c) Linxian, Lvliang, (d) Xiangning, Linfen, and (e) Zezhou, Jincheng Page 15/21
Figure 1
Clay slip zone soil of each typical landslide. (a) Location of the Yellow River Basin and landslide cases in
China based on the 1980 Chinese National Geodetic Coordinate System. (b–e) Landslides in (b)
Dongshan, Taiyuan, (c) Linxian, Lvliang, (d) Xiangning, Linfen, and (e) Zezhou, Jincheng Figure 1 Page 15/21
Clay slip zone soil of each typical landslide. (a) Location of the Yellow River Basin and landslide cases in
China based on the 1980 Chinese National Geodetic Coordinate System. (b–e) Landslides in (b)
Dongshan, Taiyuan, (c) Linxian, Lvliang, (d) Xiangning, Linfen, and (e) Zezhou, Jincheng Figure 2
XRD pattern analysis and mineral composition. (a) DS-XRD, (b) LX-XRD, (c) XN-XRD, and (d) ZZ-XRD Figure 2
XRD pattern analysis and mineral composition. (a) DS-XRD Figure 2 XRD pattern analysis and mineral composition. (a) DS-XRD, (b) LX-XRD, (c) XN-XRD, and (d) ZZ-XRD XRD pattern analysis and mineral composition. (a) DS-XRD, (b) LX-XRD, (c) XN-XRD, and (d) ZZ-XRD XRD pattern analysis and mineral composition. (a) DS-XRD, (b) LX-XRD, (c) XN-XRD, and (d) ZZ-XRD d mineral composition. (a) DS-XRD, (b) LX-XRD, (c) XN-XRD, and (d) ZZ-XRD Page 16/21 Page 16/21 ure 3
ear stress versus shear position curve (vertical pressure: 300 kPa). (a) DS, (b) LX, (c) XN, and (d) ZZ gure 3
hear stress versus shear position curve (vertical pressure: 300 kPa). (a) DS, (b) LX, (c) XN, and (d) ZZ Figure 3 Shear stress versus shear position curve (vertical pressure: 300 kPa). (a) DS, (b) LX, (c) XN, and (d) ZZ Page 17/21 Page 17/21 ure 4
ationship between the shear strength and vertical pressure. (a) DS, (b) LX, (c) XN, and (d) ZZ Figure 5 Relationship between the shear strength parameters and water content. (a) c and (b) φ Figure 6
Relationship between shear strength parameters and montmorillonite content. (a) c and (b) φ Figure 4 Relationship between the shear strength and vertical pressure. (a) DS, (b) LX, (c) XN, and (d) ZZ Relationship between the shear strength and vertical pressure. (a) DS, (b) LX, (c) XN, and (d) ZZ Page 18/21 Figure 5
Relationship between the shear strength parameters and water content. (a) c and (b) φ Figure 6 Relationship between shear strength parameters and montmorillonite content. (a) c and (b) φ Relationship between shear strength parameters and montmorillonite content. (a) c and (b) φ Page 19/21 Page 19/21 Figure 7
Free expansion rate test results Figure 7
Free expansion rate test results Figure 7 Free expansion rate test results Page 20/21 Figure 8 Mechanism of water absorption and expansion of cohesive slip zone soil Mechanism of water absorption and expansion of cohesive slip zone soil Page 21/21 Page 21/21 Page 21/21
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Vertebral cryptococcosis in an immunocompetent patient - a case report
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Abstract We report an unusual case of 70 years old, immunocompetent woman who was diagnosed with vertebral cryptococcosis. The diagnosis was made
on the basis of radiological and histological findings. The outcome was favorable under antifungal treatment. &Corresponding author: Ammouri Wafa, Department of Internal medicine, Avicenne university hospital, Rabat, Morocco &Corresponding author: Ammouri Wafa, Department of Internal medicine, Avicenne university hospital, Rabat, Morocco Key words: Cryptococcosis, vertebral, immunocompetent, C neoformans, Morocco Received: 19/03/2011 - Accepted: 10/04/2011 - Published: 10/04/2011 Received: 19/03/2011 - Accepted: 10/04/2011 - Published: 10/04/2011 Open Access Bachir Houda1, Ammouri Wafa1,&, Tazi Mezalek Zoubida1, Adnaoui Mohamed1, Aouni Mohamed1, Har 1Department of Internal medicine, Avicenne University Hospital, Rabat, Morocco Pan African Medical Journal – ISSN: 1937- 8688 (www.panafrican-med-journal.com)
Published in partnership with the African Field Epidemiology Network (AFENET). (www.afenet.net) Introduction Cryptococcosis is infection with Cryptococcus neoformans fungus. C neoformans is an opportunistic fungal infection that defines
immunosuppression. It can infrequently be seen in immunocompetent patients. Cryptococcosis presents in two forms: pulmonary and
cerebromeningeal. Skeletal cryptococcosis is uncommon. There have been only occasional case reports of thoracic vertebral cryptococcosis
presenting as a cord compression in an immunocompetent patient [1].We report a case of an unusual presentation of cryptococcal infection in an
immunocompetent patient. Conclusion Vertebral cryptococcosis can rarely occur in an immunocompetent person without HIV, where it usually goes undiagnosed. The clinician should
know this rare and severe infectious complication because the treatment is specific and the prognosis depends of the precociousness of the
diagnosis. Discussion Cryptococcus neoformans is encapsuled yeast, found in pigeon and other bird dropping. It can be inhaled by humans. Most infectious occur in
immunocompromised patients, with HIV, transplants patients or patients on long-term corticosteroid therapy [1]. The disease also appears to be
more frequent in diabetes [2]. It can rarely be seen in immunocompetent patients [3]. The overall incidence of crytococcosis in immunocompetent
individuals has been estimated at 0.2 per million per year [4]. The lung is the portal of entry and then the dissemination is haematogenous [5]. Bones involvement in this Infection is not common. The first described case was reported by Busse and Bushke in 1894 – 1895, which
demonstrated that the yeast produced osteomyelitis of the tibia [6]. A variety of bones can be involved by the fungal organism, but vertebras are
the single most likely site [7]. The vertebral involvement can mimic tuberculosis of the spine [7]. Like our patient, radiological findings of cryptococcal bone lesions are non-
specific, consisting of osteolytic destruction of the vertebral bodies with paraspinal abscesses; thus simulating tuberculosis, which is more prevalent
in tropical areas [4]. Diagnosis of bony infection requires biopsy unless C neoformans can be is isolated from another body site; less than half of the patients will have a
positive serum cryptococcal antigen test [8]. The level of antigen titre correlates with the severity of the disease [5]. Diagnosis of bony infection requires biopsy unless C neoformans can be is isolated from another body site; less t
positive serum cryptococcal antigen test [8]. The level of antigen titre correlates with the severity of the disease Cryptococcal vertebral infection causing cord compression can be treated surgically through debridement and bone grafting. The combination of
medical treatment is necessary including, antifungal agents such as amphotericin B, flucytosine and fluconazole [9]. Combination therapy might be
needed even if patients are immunocompetent [9]. Patient and case report A 70 years old woman was admitted for paraplegia. Past medical history of the patient revealed bilateral sciatica evolving since July 2009, not
alleviated by symptomatic treatment. A month prior admission, the sciatica symptoms worsened towards a gradual onset of paraplegia, prompting
the current consultation. No other significant medical history was noted. On admission, body temperature was 37°, heart rate was 75b/mn, and
blood pressure 130/60 mmhg. Neurological examination showed spastic paraparesis, deep tendon reflexes of the lower extremities were lively. Babinski reflex was bilaterally positive. Laboratory findings showed: erythrocyte sedimentation rate 100mm/h, white blood cell count 4800/ mm³,
lymphocytes 1600/mm³ and haemoglobin level 12g/dl HIV serology was negative; complement levels and measurement of CD4/CD8 T-lymphocyte ratio were within the normal range. HIV serology was negative; complement levels and measurement of CD4/CD8 T-lymphocyte ratio were within the A lumbar magnetic resonance imaging was performed showing a lesion process extending from Th8 to Th10, with spinal cord compression. The
lesion was hypo-intense on T1-weighted images and hyper-intense on T2-weighted images (Figure 1, 2). Culture of cerebrospinal fluid established
the presence of C neoformans. Posterior laminectomy was performed, revealing an abnormal tissue lesion. Biopsy showed, on histological examination, numerous fungal
organisms with thick capsules and a granulomatosis inflammatory process without necrosis. There were no malignant cells (Figure 3). Chest X ray
examination and thoracic computed tomography were normal. The diagnosis of spinal cryptococcosis in an immunocompetent patient was established and the patient was treated by intravenous amphotericin B
(0.7 mg/ kg/day) with relay by fluconazol (400mg/d). On follow-up, the patient remains well with no neurological or other sequel. Pan African Medical Journal. 2011; 8:42 This article is available online at: http://www.panafrican-med-journal.com/content/article/8/42/full/ © Bachir Houda et al. The Pan African Medical Journal - ISSN 1937-8688. 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. Page number not for citation purposes
1 Pan African Medical Journal – ISSN: 1937- 8688 (www.panafrican-med-journal.com)
Published in partnership with the African Field Epidemiology Network (AFENET). (www.afenet.net) Page number not for citation purposes Introduction Authors’ contribution All authors have read and agreed to the final version of this manuscript and have equally contributed to its content and to the management of the
case. Figures Figure 1: The lesion process extending to the right posterior hemi-arch of the eighth thoracic vertebrae (TH8), responsible for an important spinal
cord compression at this level. Figure 2: The lesion process extending to the right posterior hemi-arch of the ninth (TH9), responsible for an important spinal cord compression
at this level. Figure 3: Fungal organisms with thick capsules, a granulomatous inflammatory process without necrosis, an abundant eosinophile cytoplasm and
cells with a dark ring in the periphery. Marking cytokeratin was negative (hematoxylin eosin, original x 100) Competing interests The authors declare no competing interests. Page number not for citation purposes Authors’ contribution Page number not for citation purposes
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4 Page number not for citation purposes Page number not for citation purposes
6 References 1. Awashti M, Patankar T, Sah P, Castillo M. Cerebral cryptococcosis: atypical appearances on CT. Br J Radiol. 2001 Jan;74(877):83-5. This
article on PubMed 2. Glynn MJ, Duckworth G, Ridge JA, Grange WJ, Gibbs DD. Cryptococcal spondylitis: solitary infective bone lesions are not always
tuberculosis. Br J Rheumatol. 1994 Nov;33(11):1085-6. This article on PubMed 3. Saigal M G, Donovan Post M Judith, Lolayekar S, Murtaza A. Unusual Presentation of central nervous system cryptococcal infection in an
immunocompetent patient. Am J Neuroradiol. 2005; 26: 2522-2526. This article on PubMed 4. Gupta S K, Chhabra R, Sharma B S, Khosla A. Vertabral cryptococcosis simulating tuberculosis. British Journal of Neurosurgery. Br J
Neurosurg. 2003 Dec;17(6):556-9. This article on PubMed 5. Mitchell TG, Perfect JR. Cryptococcosis in the era of AIDS-100 years after the discovery of Cryptococcus neoformans. Clin Microbiol Rev. 1995; 8: 515-48. This article on PubMed 5. Mitchell TG, Perfect JR. Cryptococcosis in the era of AIDS-100 years after the discovery of Cryptococcus neoformans. Clin Microbiol Rev. 1995; 8: 515-48. This article on PubMed 6. Bushke A. Ueber eine durch coicidien hemorgerufene krankhitedes menschen. Deutsch Med Wochenschr. 1895; 21:14 6. Bushke A. Ueber eine durch coicidien hemorgerufene krankhitedes menschen. Deutsch Med Wochenschr. 1895; 21:14 7. Gurevitz O, Goldschmied-Reuven A, Block C, Kopoloric J, Fecfel Z, Hassin D. Cryptococcus neoformans vertebral osteomyelitis. J Med Vet
Mycol. 1994;32(4):315-8. This article on PubMed 8. Casadevall A, Perfect JR. Cryptococcus neoformans. Washington DC. ASM Press 9. Matsushita T, Suzuki K. Spastic paraparesis due to cryptococcal osteomyelitis :a case report. Clin Orthop Relat Res. 1985 Jun;(196):279-
84. This article on PubMed Page number not for citation purposes
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| ERROR: type should be string, got "https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0.\nl\n.\n0 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 Impact of Hurricane Irma on Coral Reef Sediment Redistribution at \n5 \nLooe Key Reef, Florida, USA \nKimberly K. Yates1, Zachery Fehr2, Selena Johnson1, David Zawada1 \n1U.S. Geological Survey, St. Petersburg, FL, 33701, United States \n2Cherokee Nation System Solutions, Tulsa, OK, 74166, United States, Contractor to the U.S. Geological Survey \nCorrespondence to: Kimberly K. Yates (kyates@usgs.gov) \n10 Rates of elevation \nchange during this post-storm period were one to two orders of magnitude greater than decadal and multi-decadal rates of \nchange in the same location, and changes showed erosion of approximately 50% of sediment deposited during the storm event \nas seafloor sediment distribution began to re-equilibrate to non-storm sea state conditions. Our results suggest higher resolution \n30 \nelevation-change data collected over seasonal and annual time periods could enhance characterization and understanding of \nshort-term and long-term rates and processes of seafloor change and help guide post-storm recovery and restoration of benthic \nhabitats in topographically complex coral reef systems. Abstract. Understanding event-driven sediment transport in coral reef environments is essential to assessing impacts to reef \nspecies, habitats, restoration, and mitigation, yet there remains a global knowledge gap due to limited quantitative studies. Hurricane Irma made landfall in the Lower Florida Keys with sustained 209 km h-1 winds and greater than 8 m waves on 10 \nSeptember 2017, directly impacting the Florida Reef Tract (FRT), and providing an opportunity to perform a unique comprehensive, quantitative assessment of its impact on coral reef structure and sediment redistribution. We used lidar and \n15 \nmultibeam derived digital elevation models (DEMs) collected before and after the passing of Hurricane Irma over a 15.98 km2 \narea along the Lower FRT including Looe Key Reef to quantify changes in seafloor elevation, volume, and structure due to \nstorm impacts. Elevation change was calculated at over 4-million point-locations across 10 habitat types within this study area \nfor two time periods using data collected from 1) approximately one year before the passing of Irma and three to six months 15 comprehensive, quantitative assessment of its impact on coral reef structure and sediment redistribution. We used lidar and \n15 \nmultibeam derived digital elevation models (DEMs) collected before and after the passing of Hurricane Irma over a 15.98 km2 \narea along the Lower FRT including Looe Key Reef to quantify changes in seafloor elevation, volume, and structure due to \nstorm impacts. Elevation change was calculated at over 4-million point-locations across 10 habitat types within this study area \nfor two time periods using data collected from 1) approximately one year before the passing of Irma and three to six months following the storm’s impact, and 2) from three to six months after, and up to 16.5 months after, the storm. Impact of Hurricane Irma on Coral Reef Sediment Redistribution at \n5 \nLooe Key Reef, Florida, USA \nKimberly K. Yates1, Zachery Fehr2, Selena Johnson1, David Zawada1 \n1U.S. Geological Survey, St. Petersburg, FL, 33701, United States \n2Cherokee Nation System Solutions, Tulsa, OK, 74166, United States, Contractor to the U.S. Geological Survey \nCorrespondence to: Kimberly K. Yates (kyates@usgs.gov) \n10 Impact of Hurricane Irma on Coral Reef Sediment Redistribution at \n5 \nLooe Key Reef, Florida, USA \nKimberly K. Yates1, Zachery Fehr2, Selena Johnson1, David Zawada1 \n1U.S. Geological Survey, St. Petersburg, FL, 33701, United States \n2Cherokee Nation System Solutions, Tulsa, OK, 74166, United States, Contractor to the U.S. Geological Survey \nCorrespondence to: Kimberly K. Yates (kyates@usgs.gov) \n10 rly K. Yates1, Zachery Fehr2, Selena Johnson1, David Zawada1 Abstract. Understanding event-driven sediment transport in coral reef environments is essential to assessing impacts to reef \nspecies, habitats, restoration, and mitigation, yet there remains a global knowledge gap due to limited quantitative studies. Hurricane Irma made landfall in the Lower Florida Keys with sustained 209 km h-1 winds and greater than 8 m waves on 10 \nSeptember 2017, directly impacting the Florida Reef Tract (FRT), and providing an opportunity to perform a unique \ncomprehensive, quantitative assessment of its impact on coral reef structure and sediment redistribution. We used lidar and \n15 \nmultibeam derived digital elevation models (DEMs) collected before and after the passing of Hurricane Irma over a 15.98 km2 \narea along the Lower FRT including Looe Key Reef to quantify changes in seafloor elevation, volume, and structure due to \nstorm impacts. Elevation change was calculated at over 4-million point-locations across 10 habitat types within this study area \nfor two time periods using data collected from 1) approximately one year before the passing of Irma and three to six months \nfollowing the storm’s impact, and 2) from three to six months after, and up to 16.5 months after, the storm. Elevation-change \n20 \ndata were then used to generate Triangulated Irregular Network (TIN) models in ArcMap to calculate changes in seafloor \nvolume during each time-period. Our results indicate that Hurricane Irma was primarily a depositional event that increased \nmean seafloor elevation and volume at this study site by 0.34 m and up to 5.4 Mm3, respectively. Sediment was transported \nprimarily west-southwest (WSW) and downslope modifying geomorphic seafloor features including the migration of sand \nwaves and rubble fields, formation of scour marks in shallow seagrass habitat, and burial of seagrass and coral-dominated \n25 \nhabitat. Approximately 16.5 months after Hurricane Irma (during a 13-month period between 2017 and 2019), net erosion was \nobserved across all habitats with mean elevation-change of -0.15 m and net volume change up to -2.46 Mm3. https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 1 Introduction Coral coverage \nhas been reported at less than 7% along the Florida Keys Reef Tract and less than 3% along the northern FRT in recent years \n50 \n(Jackson et al., 2014; Walton et al, 2018; Knowlton, 2020); and many of Florida’s reefs are in a net erosional state (Yates et \nal., 2017; Morris et al, 2022). Additionally, seagrass has been decreasing in coverage since early Thalassia testudinum die-\noffs in 1987 and more contemporary die-offs in 2015 following storm events and water quality variations (Hall et al., 2016). al., 2009; Gorstein et al., 2016, Towle et al., 2020). Benthic communities of the Florida Reef Tract (FRT) have been degrading \n40 \nfor the past several decades. Coral coverage has declined across the Caribbean and Florida reefs by more than 50% since the \n1970’s due to coral disease and bleaching (Porter et al., 2001; Patterson et al., 2002; Williams and Miller, 2012; Joyner et al., \n2015; Walker et al., 2018), pollution and overfishing (Littler et al., 1986; Lapointe & Clark, 1992; and Hughes 1994), and \nmass-mortality of macroalgal grazers (e.g., Lessios et al. 1983). Progression of climate change has increased thermal stress, al., 2009; Gorstein et al., 2016, Towle et al., 2020). Benthic communities of the Florida Reef Tract (FRT) have been degrading \n40 \nfor the past several decades. Coral coverage has declined across the Caribbean and Florida reefs by more than 50% since the \n1970’s due to coral disease and bleaching (Porter et al., 2001; Patterson et al., 2002; Williams and Miller, 2012; Joyner et al., \n2015; Walker et al., 2018), pollution and overfishing (Littler et al., 1986; Lapointe & Clark, 1992; and Hughes 1994), and \nmass-mortality of macroalgal grazers (e.g., Lessios et al. 1983). Progression of climate change has increased thermal stress, coral bleaching and disease, ocean acidification, and corallivory (predation of corals) (Wilkinson 1996; Mumby et al., 2006; \n45 \nBrandt and McManus, 2009; Soto et al., 2011; Kuffner et al., 2015; Randall and van Woesik, 2015; Muehllehner et al., 2016; \nHughes et al., 2017; Rice et al., 2019). These multiple stressors and increased storm occurrences have caused a shift from \nstony-coral-dominated reefs to macroalgae and octocoral dominated reefs (Bohsnack 1983; Hughs, 1994; Knowlton, 1992; \nMiller et al., 2002; Norstrom et al., 2009; Bruno et al., 2009; Ruzicka et al., 2013 and Jackson et al., 2014). Impact of Hurricane Irma on Coral Reef Sediment Redistribution at \n5 \nLooe Key Reef, Florida, USA \nKimberly K. Yates1, Zachery Fehr2, Selena Johnson1, David Zawada1 \n1U.S. Geological Survey, St. Petersburg, FL, 33701, United States \n2Cherokee Nation System Solutions, Tulsa, OK, 74166, United States, Contractor to the U.S. Geological Survey \nCorrespondence to: Kimberly K. Yates (kyates@usgs.gov) \n10 Elevation-change \n20 \ndata were then used to generate Triangulated Irregular Network (TIN) models in ArcMap to calculate changes in seafloor \nvolume during each time-period. Our results indicate that Hurricane Irma was primarily a depositional event that increased \nmean seafloor elevation and volume at this study site by 0.34 m and up to 5.4 Mm3, respectively. Sediment was transported \nprimarily west-southwest (WSW) and downslope modifying geomorphic seafloor features including the migration of sand following the storm’s impact, and 2) from three to six months after, and up to 16.5 months after, the storm. Elevation-change \n20 \ndata were then used to generate Triangulated Irregular Network (TIN) models in ArcMap to calculate changes in seafloor \nvolume during each time-period. Our results indicate that Hurricane Irma was primarily a depositional event that increased \nmean seafloor elevation and volume at this study site by 0.34 m and up to 5.4 Mm3, respectively. Sediment was transported \nprimarily west-southwest (WSW) and downslope modifying geomorphic seafloor features including the migration of sand waves and rubble fields, formation of scour marks in shallow seagrass habitat, and burial of seagrass and coral-dominated \n25 \nhabitat. Approximately 16.5 months after Hurricane Irma (during a 13-month period between 2017 and 2019), net erosion was \nobserved across all habitats with mean elevation-change of -0.15 m and net volume change up to -2.46 Mm3. Rates of elevation \nchange during this post-storm period were one to two orders of magnitude greater than decadal and multi-decadal rates of \nchange in the same location, and changes showed erosion of approximately 50% of sediment deposited during the storm event as seafloor sediment distribution began to re-equilibrate to non-storm sea state conditions. Our results suggest higher resolution \n30 \nelevation-change data collected over seasonal and annual time periods could enhance characterization and understanding of \nshort-term and long-term rates and processes of seafloor change and help guide post-storm recovery and restoration of benthic \nhabitats in topographically complex coral reef systems. as seafloor sediment distribution began to re-equilibrate to non-storm sea state conditions. Our results suggest higher resolution \n30 \nelevation-change data collected over seasonal and annual time periods could enhance characterization and understanding of \nshort-term and long-term rates and processes of seafloor change and help guide post-storm recovery and restoration of benthic \nhabitats in topographically complex coral reef systems. 1 1 Introduction Coral reefs provide a variety of services to coastal communities including protection from coastal hazards such as storms, \n35 \nwaves, and erosion (Ferrario et al., 2014; Storlazzi et al., 2021); socioeconomic benefits such as fisheries, recreation, and \ntourism (Moberg and Folk, 1999; Hall et al. 2020); and they support numerous habitats and diverse marine species (Knowlton, \n2020). Socioeconomic benefits of Florida reefs have an estimated value of over 8 billion dollars a year, supporting 39,000 \nSouth Florida jobs and 70,400 total jobs, with at least 2.9 billion dollars contributing directly to the local economy (Krediet et Coral reefs provide a variety of services to coastal communities including protection from coastal hazards such as storms, \n35 \nwaves, and erosion (Ferrario et al., 2014; Storlazzi et al., 2021); socioeconomic benefits such as fisheries, recreation, and \ntourism (Moberg and Folk, 1999; Hall et al. 2020); and they support numerous habitats and diverse marine species (Knowlton, \n2020). Socioeconomic benefits of Florida reefs have an estimated value of over 8 billion dollars a year, supporting 39,000 \nSouth Florida jobs and 70,400 total jobs, with at least 2.9 billion dollars contributing directly to the local economy (Krediet et \nal., 2009; Gorstein et al., 2016, Towle et al., 2020). Benthic communities of the Florida Reef Tract (FRT) have been degrading \n40 \nfor the past several decades. Coral coverage has declined across the Caribbean and Florida reefs by more than 50% since the \n1970’s due to coral disease and bleaching (Porter et al., 2001; Patterson et al., 2002; Williams and Miller, 2012; Joyner et al., \n2015; Walker et al., 2018), pollution and overfishing (Littler et al., 1986; Lapointe & Clark, 1992; and Hughes 1994), and \nmass-mortality of macroalgal grazers (e.g., Lessios et al. 1983). Progression of climate change has increased thermal stress, \ncoral bleaching and disease, ocean acidification, and corallivory (predation of corals) (Wilkinson 1996; Mumby et al., 2006; \n45 \nBrandt and McManus, 2009; Soto et al., 2011; Kuffner et al., 2015; Randall and van Woesik, 2015; Muehllehner et al., 2016; \nHughes et al., 2017; Rice et al., 2019). These multiple stressors and increased storm occurrences have caused a shift from \nstony-coral-dominated reefs to macroalgae and octocoral dominated reefs (Bohsnack 1983; Hughs, 1994; Knowlton, 1992; \nMiller et al., 2002; Norstrom et al., 2009; Bruno et al., 2009; Ruzicka et al., 2013 and Jackson et al., 2014). 1 Introduction Coral coverage has been reported at less than 7% along the Florida Keys Reef Tract and less than 3% along the northern FRT in recent years \n50 \n(Jackson et al., 2014; Walton et al, 2018; Knowlton, 2020); and many of Florida’s reefs are in a net erosional state (Yates et \nal., 2017; Morris et al, 2022). Additionally, seagrass has been decreasing in coverage since early Thalassia testudinum die-\noffs in 1987 and more contemporary die-offs in 2015 following storm events and water quality variations (Hall et al., 2016). Multi-decadal seafloor elevation-change analyses along the FRT indicate that degradation of coral reefs and surrounding \n55 \nseafloor habitats has led to substantial erosion and loss of elevation from the 1930’s to 2002 and increased water depths to \nlevels not expected until near the year 2100 (Yates et al., 2017). Projected socioeconomic impacts due to continued FRT coral \nreef degradation and loss of seafloor elevation estimate increases of flooding risk from storms and coastal inundation to more \nthan 7,300 people and $823.6 million (2010 U.S. dollars, USD) in direct and indirect damage to housing, buildings, and Multi-decadal seafloor elevation-change analyses along the FRT indicate that degradation of coral reefs and surrounding \n55 \nseafloor habitats has led to substantial erosion and loss of elevation from the 1930’s to 2002 and increased water depths to \nlevels not expected until near the year 2100 (Yates et al., 2017). Projected socioeconomic impacts due to continued FRT coral \nreef degradation and loss of seafloor elevation estimate increases of flooding risk from storms and coastal inundation to more \nthan 7,300 people and $823.6 million (2010 U.S. dollars, USD) in direct and indirect damage to housing, buildings, and businesses, annually (Storlazzi et al., 2021). Storm frequency and strength are projected to increase as sea-surface temperatures \n60 \nand atmospheric energy increase due to climate change and global warming (Elsner et al., 2008, Bhatia et al., 2019; Knutson \net al., 2020). While advances have been made in understanding long-term change in seafloor elevation and structure and its \npotential socioeconomic consequences, understanding the effects of event-driven changes to seafloor geomorphology due to \nstorms remains a major knowledge gap. businesses, annually (Storlazzi et al., 2021). Storm frequency and strength are projected to increase as sea-surface temperatures \n60 \nand atmospheric energy increase due to climate change and global warming (Elsner et al., 2008, Bhatia et al., 2019; Knutson \net al., 2020). https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 Major tropical storms persistently impact the state of Florida with historical hurricane impacts estimated to have caused more \nthan $450 billion dollars of damage across the state from the early 1900’s to 2007 (Malmstadt et al., 2009). The Middle to \nLower Florida Keys (from Islamorada to Key West) has been impacted by 15 major hurricane landfall events (Category 3 \nthrough 5) and numerous tropical storm and Category 1 and 2 hurricanes from the early 1900’s to 2022 (NOAA, 2022a). Hurricane Irma made landfall at Cudjoe Key in the Lower Florida Keys after passing directly over Looe Key Reef on 10 \n70 \nSeptember 2017 as a category 4 hurricane with maximum wind speeds of 213 km h-1 (115 kts) (Cangialosi et al., 2021) and \nsignificant wave heights of approximately 14 m a few kilometers offshore of the Florida Keys (Xian et al., 2018, Fig. 1a, b). Satellite imagery showed extensive sediment plumes throughout South Florida and the FRT caused by sediment resuspension \nand transport during the storm (Fig. 1c, d). The storm damaged up to 75% of buildings near its landfall point and caused Hurricane Irma made landfall at Cudjoe Key in the Lower Florida Keys after passing directly over Looe Key Reef on 10 \n70 \nSeptember 2017 as a category 4 hurricane with maximum wind speeds of 213 km h-1 (115 kts) (Cangialosi et al., 2021) and \nsignificant wave heights of approximately 14 m a few kilometers offshore of the Florida Keys (Xian et al., 2018, Fig. 1a, b). Satellite imagery showed extensive sediment plumes throughout South Florida and the FRT caused by sediment resuspension \nand transport during the storm (Fig. 1c, d). The storm damaged up to 75% of buildings near its landfall point and caused \napproximately 50 billion USD of wind and water damage across the state of Florida (Xian et al., 2018; Cangialosi et al., 2021; \n75 \nNOAA, 2022b). Prior to Hurricane Irma, the most recent, direct impact to Looe Key Reef from a tropical storm was in 2008 \nduring Tropical Storm Fay (NOAA, 2022a). Diver\nbased surveys of coral reefs at 57 locations along the FRT by the National Oceanic and Atmospheric Administration showed \n80 \nhighest levels of damage in the Middle to Lower Keys including dislodged and fractured corals, clogged and damaged sponges, \nheavy sedimentation, burial of corals, displaced rubble and sand, reef erosion, fractured substrate, and marine debris; 14% of \nsites showed severe impact, 33% showed moderate impact, and 53% showed minimal impact (Viehman et al., 2018). Looe \nKey Reef, located near the hurricane landfall location, showed more than 26% prevalence of hurricane-impacted corals (Florida based surveys of coral reefs at 57 locations along the FRT by the National Oceanic and Atmospheric Administration showed \n80 \nhighest levels of damage in the Middle to Lower Keys including dislodged and fractured corals, clogged and damaged sponges, \nheavy sedimentation, burial of corals, displaced rubble and sand, reef erosion, fractured substrate, and marine debris; 14% of \nsites showed severe impact, 33% showed moderate impact, and 53% showed minimal impact (Viehman et al., 2018). Looe \nKey Reef, located near the hurricane landfall location, showed more than 26% prevalence of hurricane-impacted corals (Florida Resilience Program, 2017). Similar surveys along the northern FRT from Key Biscayne north showed from approximately 5% \n85 \nto 17% of 62 sites with impacts to corals including dislodged and buried colonies, and at least one site with slabs of hardbottom \n2 to 5 m in size fractured and displaced several meters (Walker, 2018). Analyses of long-term monitoring-transect data at 40 \nsites throughout the Florida Keys National Marine Sanctuary (FKNMS) showed instantaneous losses in seagrass and \ncalcareous green macroalgae density after the storm passed, particularly in the Lower Florida Keys near where Hurricane Irma Resilience Program, 2017). Similar surveys along the northern FRT from Key Biscayne north showed from approximately 5% \n85 \nto 17% of 62 sites with impacts to corals including dislodged and buried colonies, and at least one site with slabs of hardbottom \n2 to 5 m in size fractured and displaced several meters (Walker, 2018). Analyses of long-term monitoring-transect data at 40 \nsites throughout the Florida Keys National Marine Sanctuary (FKNMS) showed instantaneous losses in seagrass and \ncalcareous green macroalgae density after the storm passed, particularly in the Lower Florida Keys near where Hurricane Irma made landfall (Wilson et al., 2020). 1 Introduction While advances have been made in understanding long-term change in seafloor elevation and structure and its \npotential socioeconomic consequences, understanding the effects of event-driven changes to seafloor geomorphology due to \nstorms remains a major knowledge gap. 65 2 2 Hurricane Irma made landfall at Cudjoe Key in the Lower Florida Keys after passing directly over Looe Key Reef on 10 \n70 \nSeptember 2017 as a category 4 hurricane with maximum wind speeds of 213 km h-1 (115 kts) (Cangialosi et al., 2021) and \nsignificant wave heights of approximately 14 m a few kilometers offshore of the Florida Keys (Xian et al., 2018, Fig. 1a, b). Satellite imagery showed extensive sediment plumes throughout South Florida and the FRT caused by sediment resuspension \nand transport during the storm (Fig. 1c, d). The storm damaged up to 75% of buildings near its landfall point and caused \napproximately 50 billion USD of wind and water damage across the state of Florida (Xian et al., 2018; Cangialosi et al., 2021; \n75 approximately 50 billion USD of wind and water damage across the state of Florida (Xian et al., 2018; Cangialosi et al., 2021; \n75 \nNOAA, 2022b). Prior to Hurricane Irma, the most recent, direct impact to Looe Key Reef from a tropical storm was in 2008 \nduring Tropical Storm Fay (NOAA, 2022a). Numerous rapid assessments of seafloor habitats were conducted along the FRT in the weeks following Hurricane Irma. Diver-\nbased surveys of coral reefs at 57 locations along the FRT by the National Oceanic and Atmospheric Administration showed \n80 \nhighest levels of damage in the Middle to Lower Keys including dislodged and fractured corals, clogged and damaged sponges, \nheavy sedimentation, burial of corals, displaced rubble and sand, reef erosion, fractured substrate, and marine debris; 14% of \nsites showed severe impact, 33% showed moderate impact, and 53% showed minimal impact (Viehman et al., 2018). Looe \nKey Reef, located near the hurricane landfall location, showed more than 26% prevalence of hurricane-impacted corals (Florida \nResilience Program, 2017). Similar surveys along the northern FRT from Key Biscayne north showed from approximately 5% \n85 \nto 17% of 62 sites with impacts to corals including dislodged and buried colonies, and at least one site with slabs of hardbottom \n2 to 5 m in size fractured and displaced several meters (Walker, 2018). Analyses of long-term monitoring-transect data at 40 \nsites throughout the Florida Keys National Marine Sanctuary (FKNMS) showed instantaneous losses in seagrass and \ncalcareous green macroalgae density after the storm passed, particularly in the Lower Florida Keys near where Hurricane Irma \nmade landfall (Wilson et al., 2020). Additionally, several locations showed moderate burial of seagrass with up to 5 to 10 cm \n90 \nof sand, while other locations showed heavy erosion or moderate seagrass canopy thinning (Wilson et al., 2020). Reef Visual \nCensus (RVC) surveys including structure from motion (SfM) habitat photogrammetry at sites in the Lower Florida Keys from \nFebruary 2017 to December 2018 showed a 30% decrease in macroalgae at the Looe Key Sanctuary Preservation Area (SPA) \nand a 30% increase at the Looe Key Special Use Area (SPU) post Irma; while both Looe Key locations showed a 10% decrease \nin live coral cover and a 20% increase in octocoral cover (Simmons et al., 2022). Comparison of restored (outplant) coral \n95 \nsurvival rates at two fore reef and two patch reef sites near Tavernier Key in the Upper Florida Keys showed approximately \n85% outplant survival at all locations prior to the passage of Hurricane Irma; however, no outplants survived at the fore reef \nsites and only 51% of outplants survived at the patch reef sites post-Irma, the difference likely due to protection of the patch \nreefs from dissipation of wave energy by the reef crest (Lohr et al., 2020). Examination of Diadema antillarum sea urchins (a Numerous rapid assessments of seafloor habitats were conducted along the FRT in the weeks following Hurricane Irma. Diver-\nbased surveys of coral reefs at 57 locations along the FRT by the National Oceanic and Atmospheric Administration showed \n80 \nhighest levels of damage in the Middle to Lower Keys including dislodged and fractured corals, clogged and damaged sponges, \nheavy sedimentation, burial of corals, displaced rubble and sand, reef erosion, fractured substrate, and marine debris; 14% of \nsites showed severe impact, 33% showed moderate impact, and 53% showed minimal impact (Viehman et al., 2018). Looe \nKey Reef, located near the hurricane landfall location, showed more than 26% prevalence of hurricane-impacted corals (Florida Numerous rapid assessments of seafloor habitats were conducted along the FRT in the weeks following Hurricane Irma. Additionally, several locations showed moderate burial of seagrass with up to 5 to 10 cm \n90 \nof sand, while other locations showed heavy erosion or moderate seagrass canopy thinning (Wilson et al., 2020). Reef Visual \nCensus (RVC) surveys including structure from motion (SfM) habitat photogrammetry at sites in the Lower Florida Keys from \nFebruary 2017 to December 2018 showed a 30% decrease in macroalgae at the Looe Key Sanctuary Preservation Area (SPA) \nand a 30% increase at the Looe Key Special Use Area (SPU) post Irma; while both Looe Key locations showed a 10% decrease in live coral cover and a 20% increase in octocoral cover (Simmons et al., 2022). Comparison of restored (outplant) coral \n95 \nsurvival rates at two fore reef and two patch reef sites near Tavernier Key in the Upper Florida Keys showed approximately \n85% outplant survival at all locations prior to the passage of Hurricane Irma; however, no outplants survived at the fore reef \nsites and only 51% of outplants survived at the patch reef sites post-Irma, the difference likely due to protection of the patch \nreefs from dissipation of wave energy by the reef crest (Lohr et al., 2020). Examination of Diadema antillarum sea urchins (a 3 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 100 re 1. Location of the Florida Keys Reef Tract, Hurricane Irma trackline and impact. (a) NOAA, National Weather Service WSR-\nradar image (decibels, DBZ) from south Florida on 10 September 2017 at 5:22 am Eastern Daylight Time (EDT) showing approach of\nicane Irma (inset black line = hurricane trackline). (b) Significant wave height (m) from the U.S. Geological Survey (USGS) Coupled\nan, Atmosphere, Wave, Sediment Transport (COAWST) model on 10 September 2017 at 5:00 am EDT (Warner et al., 2010, image\nit: Patricia Dalyander, USGS). (c) Satellite imagery from 30 August 2017, 11 days prior to landfall of Hurricane Irma in the Florida\ns (NASA, 2023, EOSDIS Worldview Imagery). (d) Satellite imagery from 13 September 2017, 3 days after Hurricane Irma landfall in\nFlorida Keys showing extensive resuspended sediment plume (NASA, 2023, EOSDIS Worldview Imagery). Red boxes show the location\nooe Key Reef relative to other reefs along the reef tract Figure 1. Location of the Florida Keys Reef Tract, Hurricane Irma trackline and impact. https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 key reef grazer) density, size structure, and coral reef community structure before and 2.5 months after Irma at 10 locations in \n110 \nthe Middle and Upper Florida Keys showed a significant decrease in D. antillarum density with increased sedimentation, \nsuggesting sediment transport caused mortality through abrasion and burial (Kobelt et al. 2019). key reef grazer) density, size structure, and coral reef community structure before and 2.5 months after Irma at 10 locations in \n110 \nthe Middle and Upper Florida Keys showed a significant decrease in D. antillarum density with increased sedimentation, \nsuggesting sediment transport caused mortality through abrasion and burial (Kobelt et al. 2019). While observational data from several locations indicate seafloor sediments were transporte While observational data from several locations indicate seafloor sediments were transported and likely caused damage to \nbenthic habitats, the direct impact of Hurricane Irma or other tropical storms on seafloor elevation and geomorphologic \n115 \nstructures has not previously been quantified along the FRT. In this study, we used high-resolution light-detection-and-ranging \n(lidar) and multibeam bathymetry data collected before and after the passage of Hurricane Irma to quantify seafloor elevation \nand volume change of benthic habitats and geomorphological structures resulting from the storm’s impact and post-storm re-\nequilibration of seafloor sediments at more than 4-million point-locations at the Looe Key Reef system in the Lower FRT. benthic habitats, the direct impact of Hurricane Irma or other tropical storms on seafloor elevation and geomorphologic \n115 \nstructures has not previously been quantified along the FRT. In this study, we used high-resolution light-detection-and-ranging \n(lidar) and multibeam bathymetry data collected before and after the passage of Hurricane Irma to quantify seafloor elevation \nand volume change of benthic habitats and geomorphological structures resulting from the storm’s impact and post-storm re-\nequilibration of seafloor sediments at more than 4-million point-locations at the Looe Key Reef system in the Lower FRT. 2.1 Looe Key Reef Study Site The FRT is the only living coral barrier reef in the continental United States, and it spans more than 580 km along the east \ncoast of Florida from St. Lucie Inlet to the Dry Tortugas, with total reef area of approximately 1,179 km2 (Finkl and Andrews, \n2008; Jackson et al., 2014; Florida Department of Environmental Protection, 2022). Water depth along the FRT is up to approximately 20 m with discontinuous spur and groove formations and patch reefs separated by tidal passes, and it is \n125 \ncharacterized by both coral-dominated and non-coral dominated seafloor habitat as characterized and mapped by the Florida \nFish and Wildlife Conservation Commission-Fish and Wildlife Research Institute (FWC, 2015). Much of the FRT is protected \nby the FKNMS, Biscayne National Park, and Dry Tortugas National Park, and includes several sanctuary preservation areas \n(SPAs) and special use areas (SPUs) within FKNMS, including the Looe Key SPA and SPU, that together protect over 6000 approximately 20 m with discontinuous spur and groove formations and patch reefs separated by tidal passes, and it is \n125 \ncharacterized by both coral-dominated and non-coral dominated seafloor habitat as characterized and mapped by the Florida \nFish and Wildlife Conservation Commission-Fish and Wildlife Research Institute (FWC, 2015). Much of the FRT is protected \nby the FKNMS, Biscayne National Park, and Dry Tortugas National Park, and includes several sanctuary preservation areas \n(SPAs) and special use areas (SPUs) within FKNMS, including the Looe Key SPA and SPU, that together protect over 6000 marine species (Keller and Donahue, 2006). Looe Key Reef is a barrier bank reef located approximately 10 km offshore in the \n130 \nLower Florida Keys, south of Cudjoe Key, and it is characterized by a prominent, shallow reef crest with two extensive coral \nrubble fields, a fore reef with a spur-and-groove formation, a forereef terrace and deep reef zone, and a back reef area with \nseagrass communities, patch reefs, and individual coral heads (Fig. 2a-d). Seagrass beds and sandflats with intermittent patch \nreefs extend shoreward from Looe Key Reef proper to Hawk Channel, approximately 2 km to the north. Looe Key SPA, marine species (Keller and Donahue, 2006). (a) NOAA, National Weather Service WSR-\n88D radar image (decibels, DBZ) from south Florida on 10 September 2017 at 5:22 am Eastern Daylight Time (EDT) showing approach of \nHurricane Irma (inset black line = hurricane trackline). (b) Significant wave height (m) from the U.S. Geological Survey (USGS) Coupled \nOcean, Atmosphere, Wave, Sediment Transport (COAWST) model on 10 September 2017 at 5:00 am EDT (Warner et al., 2010, image \ncredit: Patricia Dalyander, USGS). (c) Satellite imagery from 30 August 2017, 11 days prior to landfall of Hurricane Irma in the Florida \nKeys (NASA, 2023, EOSDIS Worldview Imagery). (d) Satellite imagery from 13 September 2017, 3 days after Hurricane Irma landfall in \nthe Florida Keys showing extensive resuspended sediment plume (NASA, 2023, EOSDIS Worldview Imagery). Red boxes show the location \nof Looe Key Reef relative to other reefs along the reef tract. 105 4 2.1 Looe Key Reef Study Site Looe Key Reef is a barrier bank reef located approximately 10 km offshore in the \n130 \nLower Florida Keys, south of Cudjoe Key, and it is characterized by a prominent, shallow reef crest with two extensive coral \nrubble fields, a fore reef with a spur-and-groove formation, a forereef terrace and deep reef zone, and a back reef area with \nseagrass communities, patch reefs, and individual coral heads (Fig. 2a-d). Seagrass beds and sandflats with intermittent patch \nreefs extend shoreward from Looe Key Reef proper to Hawk Channel, approximately 2 km to the north. Looe Key SPA, located at approximately 24° 32' N, 81° 24' W, is just over 18 km2 and surrounds LKR proper which is less than 1.7 km2. Looe \n135 \nKey Reef contains a coral nursery and several restoration sites for coral outplants; it is one of seven FKNMS iconic reefs, and \nthe focus of a major collaborative habitat restoration effort known as Mission: Iconic reefs (NOAA Fisheries, 2022). located at approximately 24° 32' N, 81° 24' W, is just over 18 km2 and surrounds LKR proper which is less than 1.7 km2. Looe \n135 \nKey Reef contains a coral nursery and several restoration sites for coral outplants; it is one of seven FKNMS iconic reefs, and \nthe focus of a major collaborative habitat restoration effort known as Mission: Iconic reefs (NOAA Fisheries, 2022). 5\nThe northeastern eyewall of Hurricane Irma passed directly over LKR with the storm’s center passing approximately 9 km \nwest of LKR. However, the storm was approximately 684 km in diameter and covered the entire FRT and much of South \n140 \nFlorida. The National Weather Service’s technical summary of the storm reported tropical storm force winds more than 640 The northeastern eyewall of Hurricane Irma passed directly over LKR with the storm’s center passing approximately 9 km 5\nwest of LKR. However, the storm was approximately 684 km in diameter and covered the entire FRT and much of South \n140 \nFlorida. The National Weather Service’s technical summary of the storm reported tropical storm force winds more than 640 5\nwest of LKR. However, the storm was approximately 684 km in diameter and covered the entire FRT and much of South \n140 \nFlorida. The National Weather Service’s technical summary of the storm reported tropical storm force winds more than 640 5 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 km away from the storm’s center, and hurricane force winds more than 125 km from the storm’s center (NWS, 2022b). Gale \nforce winds (sustained winds above 63 km h-1) were detected by the evening of 9 September 2017 at the National Oceanic and \nAtmospheric Administration - National Ocean Service (NOAA-NOS), Tides and Currents Station at Vaca Key (number \n8723970), 35 km to the northeast of LKR; maximum sustained winds of 213 km h-1 were reported as the storm made landfall, 155 and latent gale force winds were detected after the storm passed on the evening of 10 September 2017 (NOAA-NOS, 2023). 155 \nThe average wind direction for this period was 67.01 degrees indicating winds moved from ENE toward WSW. Wind speeds \nfell sharply below gale force after the storm, shifting north eastward. Wind conditions were relatively quiescent from July \n2016 through January 2019 (except during Hurricane Irma) with wind speeds occasionally ranging up to approximately 56 km \nh-1 during winter storms (NOAA-NOS, 2023). and latent gale force winds were detected after the storm passed on the evening of 10 September 2017 (NOAA-NOS, 2023). 155 \nThe average wind direction for this period was 67.01 degrees indicating winds moved from ENE toward WSW. Wind speeds \nfell sharply below gale force after the storm, shifting north eastward. Wind conditions were relatively quiescent from July \n2016 through January 2019 (except during Hurricane Irma) with wind speeds occasionally ranging up to approximately 56 km \nh-1 during winter storms (NOAA-NOS, 2023). https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 gure 2. Looe Key Reef location, bathymetry, and seafloor habitats. (a) Location of the Florida Keys along the southern coast of Florida, \nckline of Hurricane Irma and the location of its landfall (red box). (b) Proximity of Hurricane Irma’s trackline to the Looe Key Reef study \ne (purple box), location of landfall at Cudjoe Key, and location of Vaca Key where the nearest NOAA-NOS (2023) Tides and Currents \ntion was located. (c) 2016 lidar bathymetric map of the Looe Key Reef study site showing location of Florida Keys National Marine \nnctuary Special Preservation Area (SPA), Special Use Area (SPU), and geomorphic features of focused investigation for this study. (d) \nbitat distribution at the Looe Key Study site from Florida Fish and Wildlife Conservation Commission-Fish and Wildlife Research \ntitute (2015). Figure 2. Looe Key Reef location, bathymetry, and seafloor habitats. (a) Location of the Florida Keys along the southern coast of Florida, \ntrackline of Hurricane Irma and the location of its landfall (red box). (b) Proximity of Hurricane Irma’s trackline to the Looe Key Reef study \nsite (purple box), location of landfall at Cudjoe Key, and location of Vaca Key where the nearest NOAA-NOS (2023) Tides and Currents \n145 \nstation was located. (c) 2016 lidar bathymetric map of the Looe Key Reef study site showing location of Florida Keys National Marine \nSanctuary Special Preservation Area (SPA), Special Use Area (SPU), and geomorphic features of focused investigation for this study. (d) \nHabitat distribution at the Looe Key Study site from Florida Fish and Wildlife Conservation Commission-Fish and Wildlife Research \nInstitute (2015). 150 150 6 The 2016 lidar DEM refers to data that were collected on 23 July 2016 (13.5 months before the passage of Hurricane Irma) by \nthe NOAA Office for Coastal Management, National Geodetic Survey, Topobathy Lidar Dem Block 1 dataset (Office for \nCoastal Management, 2017). The 2017 multibeam DEM refers to multibeam bathymetry data collected by the U.S. Geological \nSurvey in December 2017, and February–March 2018 at Looe Key Reef (between three and six months after the passage of \nHurricane Irma), specifically to examine impacts from the storm (Fredericks et al., 2019). The 2019 lidar DEM refers to data \n170 \ncollected January 8–31, 2019 by NOAA NGS Topobathy Lidar DEM Hurricane Irma: Miami to Marquesas Keys, FL dataset \n(National Geodetic Survey, 2022). The Florida Fish and Wildlife Conservation Commission (FWC) Unified Florida Reef Tract \n(UFRT) Map version 2.0, Level 2 habitat categories (FWC, 2015) were used to delineate geographic boundaries for 10 habitat 2.2 Elevation and Habitat Data \n160 Three Digital Elevation Models (DEMs) derived from lidar or multibeam bathymetric surveys were used for seafloor elevation- \nand volume-change analyses and are referenced in this study as 2016 lidar, 2017 multibeam, and 2019 lidar (Table 1). Table 1: Elevation datasets used in this analysis; collection dates are specific to the geographic extent of this study. Digital Elevation Model \nSource \nCollection Dates \nHorizontal Resolution/ \nVertical RMSE \n(meters/meters) \n2016 NOAA NGS Topobathy \nLidar DEM:Florida Keys \nOuter Reef Block 01 \nOffice for \nCoastal \nManagement, \n2017 \n23 July 2016 \n \n1.0/0.15 \nMultibeam bathymetry data \ncollected in December 2017, \nFebruary and March 2018 at \nLooe Key, the Florida Keys \nFredericks et al, \n2019 \nLeg 1: 12 December 2017 – 16 December 2017 \nLeg 2: 2 February 2018 – 9 February 2018 \nLeg 3: 9 March 2018 –11 March 2018 \n \n1.0/0.14 \n2018-2019 NOAA NGS \nTopobathy Lidar Hurricane \nIrma: Miami to Marquesas \nKeys, FL \nNational \nGeodetic Survey, \n2022 \n8 January 2019– 31 January 2019 \n \n1.0/0.11 \nRMSE = root mean square error. 165 165 The 2016 lidar DEM refers to data that were collected on 23 July 2016 (13.5 months before the passage of Hurricane Irma) by \nthe NOAA Office for Coastal Management, National Geodetic Survey, Topobathy Lidar Dem Block 1 dataset (Office for \nCoastal Management, 2017). The 2017 multibeam DEM refers to multibeam bathymetry data collected by the U.S. Geological \nSurvey in December 2017, and February–March 2018 at Looe Key Reef (between three and six months after the passage of \nHurricane Irma), specifically to examine impacts from the storm (Fredericks et al., 2019). The 2019 lidar DEM refers to data \n170 \ncollected January 8–31, 2019 by NOAA NGS Topobathy Lidar DEM Hurricane Irma: Miami to Marquesas Keys, FL dataset \n(National Geodetic Survey, 2022). The Florida Fish and Wildlife Conservation Commission (FWC) Unified Florida Reef Tract \n(UFRT) Map version 2.0, Level 2 habitat categories (FWC, 2015) were used to delineate geographic boundaries for 10 habitat The 2016 lidar DEM refers to data that were collected on 23 July 2016 (13.5 months before the passage of Hurricane Irma) by \nthe NOAA Office for Coastal Management, National Geodetic Survey, Topobathy Lidar Dem Block 1 dataset (Office for \nCoastal Management, 2017). The 2017 multibeam DEM refers to multibeam bathymetry data collected by the U.S. https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 types within the LKR study site (Fig. 2d). Habitat labelled as ‘not classified’ was indistinguishable during mapping due to \nturbidity, cloud cover, water depth, or other interferences with obtaining an optical signature of the seafloor (Zitello et al., \n175 \n2009). turbidity, cloud cover, water depth, or other interferences with obtaining an optical signature of the seafloor (Zitello et al., \n175 \n2009). turbidity, cloud cover, water depth, or other interferences with obtaining an optical signature of the seafloor (Zitello et al., \n175 \n2009). 2.2 Elevation and Habitat Data \n160 Geological \nSurvey in December 2017, and February–March 2018 at Looe Key Reef (between three and six months after the passage of Hurricane Irma), specifically to examine impacts from the storm (Fredericks et al., 2019). The 2019 lidar DEM refers to data \n170 \ncollected January 8–31, 2019 by NOAA NGS Topobathy Lidar DEM Hurricane Irma: Miami to Marquesas Keys, FL dataset \n(National Geodetic Survey, 2022). The Florida Fish and Wildlife Conservation Commission (FWC) Unified Florida Reef Tract \n(UFRT) Map version 2.0, Level 2 habitat categories (FWC, 2015) were used to delineate geographic boundaries for 10 habitat Hurricane Irma), specifically to examine impacts from the storm (Fredericks et al., 2019). The 2019 lidar DEM refers to data \n170 \ncollected January 8–31, 2019 by NOAA NGS Topobathy Lidar DEM Hurricane Irma: Miami to Marquesas Keys, FL dataset \n(National Geodetic Survey, 2022). The Florida Fish and Wildlife Conservation Commission (FWC) Unified Florida Reef Tract \n(UFRT) Map version 2.0, Level 2 habitat categories (FWC, 2015) were used to delineate geographic boundaries for 10 habitat 7 7 2.3 Elevation- and Volume-Change Analyses A 2-m XY grid was created in Global Mapper and clipped to the same footprint. Elevation values were then extracted \nfrom each of the three DEMs at the center points of co-aligned 2-m grid boxes. Elevation change between time periods was \ncalculated for each of 4,007,961 paired elevation values (e.g., 2017 elevation – 2016 elevation, and 2019 elevation – 2017 extent of each DEM was then clipped to the areal extent of the common overlapping footprint prior to elevation change analysis \n185 \nusing the ‘Clip’ tool in ArcMap. The following steps were performed in Global Mapper 22.1 due to file size limitations in \nArcMap. A 2-m XY grid was created in Global Mapper and clipped to the same footprint. Elevation values were then extracted \nfrom each of the three DEMs at the center points of co-aligned 2-m grid boxes. Elevation change between time periods was \ncalculated for each of 4,007,961 paired elevation values (e.g., 2017 elevation – 2016 elevation, and 2019 elevation – 2017 elevation). Elevation-change (XYZ) point maps were generated as shapefiles for each time-period of change for the total study \n190 \nsite; positive values indicate an increase in elevation and negative values indicate a decrease in elevation. Data are available \nfrom Fehr et al. (2021). Vertical uncertainty of elevation change analyses were estimated using methods of Yates et al. 2017 \nand the reported vertical accuracy of the lidar and multibeam data sets (typically reported as the 95% root-mean-square error, \nRMSE, Table 1) to calculate a composite RMSE (RMSETotal) for each elevation change analysis (Fig. 3b). The RMSE of lidar and multibeam data sets used for elevation-change analyses in our study ranged from 0.11 to 0.15 m (Table 1). These values \n195 \nare consistent with RMSEs determined in performance evaluations of lidar sensors that ranged from 0.08 to 0.52 m (Fernandez-\nDiaz et al., 2014; Legleiter et al., 2016; Kinzel et al., 2013; Tonina et al., 2019; Yoshida et al., 2022). Composite RMSE values \nfor elevation-change analyses based on comparison of lidar to multibeam DEMs ranged from 0.19 to 0.21m in our study. These \nvalues are consistent with RMSEs determined in performance evaluations of lidar sensors against multibeam echosounders and multibeam data sets used for elevation-change analyses in our study ranged from 0.11 to 0.15 m (Table 1). 2.3 Elevation- and Volume-Change Analyses The RMSE of lidar \nand multibeam data sets used for elevation-change analyses in our study ranged from 0.11 to 0.15 m (Table 1). These values \n195 \nare consistent with RMSEs determined in performance evaluations of lidar sensors that ranged from 0.08 to 0.52 m (Fernandez-\nDiaz et al., 2014; Legleiter et al., 2016; Kinzel et al., 2013; Tonina et al., 2019; Yoshida et al., 2022). Composite RMSE values \nfor elevation-change analyses based on comparison of lidar to multibeam DEMs ranged from 0.19 to 0.21m in our study. These \nvalues are consistent with RMSEs determined in performance evaluations of lidar sensors against multibeam echosounders \nthat ranged from 0.02 to 0.23 m (Awadallah et al., 2023). The FWC UFRT habitat map was clipped to the intersect footprint \n200 \nfor each elevation-change analysis using ArcMap 10.7. Each total-study-site elevation-change data set was then clipped to \nindividual habitat polygons to create individual elevation-change shapefiles for each habitat type. of the three 1-m resolution digital elevation models (DEMs) in ArcMap 10.7 and were used to create a common footprint \n180 \npolygon shapefile for the total LKR study site encompassing the overlapping area among the three datasets. The original (full \nareal extent, or unclipped) 2016–2017 elevation-change data set was 19.71 km2 and included 4,934,364 data points. The \noverlapping areal extent for the 2016, 2017, and 2019 DEMs was 15.98 km2 and excluded areas where water depths were too \nshallow for boat access to collect multibeam data in 2017 and areas of coarse interpolation within the 2017 DEM. The areal of the three 1-m resolution digital elevation models (DEMs) in ArcMap 10.7 and were used to create a common footprint \n180 \npolygon shapefile for the total LKR study site encompassing the overlapping area among the three datasets. The original (full \nareal extent, or unclipped) 2016–2017 elevation-change data set was 19.71 km2 and included 4,934,364 data points. The \noverlapping areal extent for the 2016, 2017, and 2019 DEMs was 15.98 km2 and excluded areas where water depths were too \nshallow for boat access to collect multibeam data in 2017 and areas of coarse interpolation within the 2017 DEM. The areal 180 extent of each DEM was then clipped to the areal extent of the common overlapping footprint prior to elevation change analysis \n185 \nusing the ‘Clip’ tool in ArcMap. The following steps were performed in Global Mapper 22.1 due to file size limitations in \nArcMap. 2.3 Elevation- and Volume-Change Analyses Seafloor elevation- and volume-change analyses were conducted using the methods of Yates et al. techniques of Murphy et al. (2022) (Fig. 3a). Briefly, individual geographic footprint areas (poly Seafloor elevation- and volume-change analyses were conducted using the methods of Yates et al. (2017) and 2-m grid spacing \ntechniques of Murphy et al. (2022) (Fig. 3a). Briefly, individual geographic footprint areas (polygons) were created for each Seafloor elevation- and volume-change analyses were conducted using the methods of Yates et al. (2017) and 2-m grid spacing \ntechniques of Murphy et al. (2022) (Fig. 3a). Briefly, individual geographic footprint areas (polygons) were created for each \nof the three 1-m resolution digital elevation models (DEMs) in ArcMap 10.7 and were used to create a common footprint \n180 \npolygon shapefile for the total LKR study site encompassing the overlapping area among the three datasets. The original (full \nareal extent, or unclipped) 2016–2017 elevation-change data set was 19.71 km2 and included 4,934,364 data points. The \noverlapping areal extent for the 2016, 2017, and 2019 DEMs was 15.98 km2 and excluded areas where water depths were too \nshallow for boat access to collect multibeam data in 2017 and areas of coarse interpolation within the 2017 DEM. The areal \nextent of each DEM was then clipped to the areal extent of the common overlapping footprint prior to elevation change analysis \n185 \nusing the ‘Clip’ tool in ArcMap. The following steps were performed in Global Mapper 22.1 due to file size limitations in \nArcMap. A 2-m XY grid was created in Global Mapper and clipped to the same footprint. Elevation values were then extracted \nfrom each of the three DEMs at the center points of co-aligned 2-m grid boxes. Elevation change between time periods was \ncalculated for each of 4,007,961 paired elevation values (e.g., 2017 elevation – 2016 elevation, and 2019 elevation – 2017 \nelevation). Elevation-change (XYZ) point maps were generated as shapefiles for each time-period of change for the total study \n190 \nsite; positive values indicate an increase in elevation and negative values indicate a decrease in elevation. Data are available \nfrom Fehr et al. (2021). Vertical uncertainty of elevation change analyses were estimated using methods of Yates et al. 2017 \nand the reported vertical accuracy of the lidar and multibeam data sets (typically reported as the 95% root-mean-square error, \nRMSE, Table 1) to calculate a composite RMSE (RMSETotal) for each elevation change analysis (Fig. 3b). 2.3 Elevation- and Volume-Change Analyses These values \n195 \nare consistent with RMSEs determined in performance evaluations of lidar sensors that ranged from 0.08 to 0.52 m (Fernandez-\nDiaz et al., 2014; Legleiter et al., 2016; Kinzel et al., 2013; Tonina et al., 2019; Yoshida et al., 2022). Composite RMSE values \nfor elevation-change analyses based on comparison of lidar to multibeam DEMs ranged from 0.19 to 0.21m in our study. These \nvalues are consistent with RMSEs determined in performance evaluations of lidar sensors against multibeam echosounders that ranged from 0.02 to 0.23 m (Awadallah et al., 2023). The FWC UFRT habitat map was clipped to the intersect footprint \n200 \nfor each elevation-change analysis using ArcMap 10.7. Each total-study-site elevation-change data set was then clipped to \nindividual habitat polygons to create individual elevation-change shapefiles for each habitat type. that ranged from 0.02 to 0.23 m (Awadallah et al., 2023). The FWC UFRT habitat map was clipped to the intersect footprint \n200 \nfor each elevation-change analysis using ArcMap 10.7. Each total-study-site elevation-change data set was then clipped to \nindividual habitat polygons to create individual elevation-change shapefiles for each habitat type. Elevation-change data from each time-period were then used to generate TIN (Triangulated Irregular Network) surface mode Elevation-change data from each time-period were then used to generate TIN (Triangulated Irregular Network) surface models \nin ArcMap for calculation of volume change. TIN models were clipped to the original overall study site intersect footprint to \n205 \nremove interpolation across areas where no data were collected. Lower bound (conservative) volume-change was calculated Elevation-change data from each time-period were then used to generate TIN (Triangulated Irregular Network) surface models in ArcMap for calculation of volume change. TIN models were clipped to the original overall study site intersect footprint to \n205 \nremove interpolation across areas where no data were collected. Lower bound (conservative) volume-change was calculated in ArcMap for calculation of volume change. TIN models were clipped to the original overall study site intersect footprint to \n205 \nremove interpolation across areas where no data were collected. Lower bound (conservative) volume-change was calculated 8 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 𝑅𝑀𝑆𝐸𝑡𝑜𝑡𝑎𝑙= √(𝑅𝑀𝑆𝐸𝑆𝑜𝑢𝑟𝑐𝑒 𝐷𝑎𝑡𝑎𝑠𝑒𝑡 1\n2\n+ 𝑅𝑀𝑆𝐸𝑆𝑜𝑢𝑟𝑐𝑒 𝐷𝑎𝑡𝑎𝑠𝑒𝑡 2\n2\n) 𝑅𝑀𝑆𝐸𝑡𝑜𝑡𝑎𝑙= √(𝑅𝑀𝑆𝐸𝑆𝑜𝑢𝑟𝑐𝑒 𝐷𝑎𝑡𝑎𝑠𝑒𝑡 1\n2\n+ 𝑅𝑀𝑆𝐸𝑆𝑜𝑢𝑟𝑐𝑒 𝐷𝑎𝑡𝑎𝑠𝑒𝑡 2\n2\n) \nation and volume change methods. 2.3 Elevation- and Volume-Change Analyses (a) Flowchart outlining generalized geoprocessing steps in ArcMap and Global \nh 3), and in the Seafloor Elevation Change Analysis Tool (SECAT, step 4) for seafloor elevation and volume change \ns et al. (2017), Murphy et al. (2022), and Zieg and Zawada (2021). (b) Composite RMSE (RMSETotal) for each \nis (2016 to 2017 and 2017 to 2019) calculated using reported RMSE for lidar and multibeam source data and methods \nBlack boxes indicate source data files. Blue boxes indicate steps using geoprocessing tools from ArcMap or Global \ndicate data analysis conducted using SECAT. 𝑅𝑀𝑆𝐸𝑡𝑜𝑡𝑎𝑙= √(𝑅𝑀𝑆𝐸𝑆𝑜𝑢𝑟𝑐𝑒 𝐷𝑎𝑡𝑎𝑠𝑒𝑡 1\n2\n+ 𝑅𝑀𝑆𝐸𝑆𝑜𝑢𝑟𝑐𝑒 𝐷𝑎𝑡𝑎𝑠𝑒𝑡 2\n2\n) \nand volume change methods. (a) Flowchart outlining generalized geoprocessin\nnd in the Seafloor Elevation Change Analysis Tool (SECAT, step 4) for seafloor\nl. (2017), Murphy et al. (2022), and Zieg and Zawada (2021). (b) Composite\n6 to 2017 and 2017 to 2019) calculated using reported RMSE for lidar and multib\nboxes indicate source data files. Blue boxes indicate steps using geoprocessing \ndata analysis conducted using SECAT. 𝑅𝑀𝑆𝐸𝑡𝑜𝑡𝑎𝑙= √(𝑅𝑀𝑆𝐸𝑆𝑜𝑢𝑟𝑐𝑒 𝐷𝑎𝑡𝑎𝑠𝑒𝑡 1\n2\n+ 𝑅𝑀𝑆𝐸𝑆𝑜𝑢𝑟𝑐𝑒 𝐷𝑎𝑡𝑎𝑠𝑒𝑡 2\n2\n) 𝑅𝑀𝑆𝐸𝑡𝑜𝑡𝑎𝑙= √(𝑅𝑀𝑆𝐸𝑆𝑜𝑢\n2 𝑅𝑀𝑆𝐸𝑡𝑜𝑡𝑎𝑙= √(𝑅𝑀𝑆𝐸𝑆𝑜𝑢𝑟𝑐𝑒 𝐷𝑎𝑡𝑎𝑠𝑒𝑡 1\n2\n+ 𝑅𝑀𝑆𝐸𝑆𝑜𝑢𝑟𝑐𝑒 𝐷𝑎𝑡𝑎𝑠𝑒𝑡 2\n2\n) Figure 3. Seafloor elevation and volume change methods. (a) Flowchart outlining generalized geop\n0 Figure 3. Seafloor elevation and volume change methods. (a) Flowchart outlining generalized geoprocessing steps in ArcMap and Global \nMapper (steps 1 through 3), and in the Seafloor Elevation Change Analysis Tool (SECAT, step 4) for seafloor elevation and volume change \n210 \nanalyses based on Yates et al. (2017), Murphy et al. (2022), and Zieg and Zawada (2021). (b) Composite RMSE (RMSETotal) for each \nelevation change analysis (2016 to 2017 and 2017 to 2019) calculated using reported RMSE for lidar and multibeam source data and methods \nof Yates et al. (2017). Black boxes indicate source data files. Blue boxes indicate steps using geoprocessing tools from ArcMap or Global \nMapper. Green boxes indicate data analysis conducted using SECAT. Figure 3. Seafloor elevation and volume change methods. (a) Flowchart outlining generalized geoprocessing steps in ArcMap and Global \nMapper (steps 1 through 3), and in the Seafloor Elevation Change Analysis Tool (SECAT, step 4) for seafloor elevation and volume change \n210 \nanalyses based on Yates et al. (2017), Murphy et al. (2022), and Zieg and Zawada (2021). 2.4 Geomorphic Feature Analyses Sub-areas or geomorphic features of high-magnitude elevation change (greater than approximately ±0.5 m) were delineated \non each total-study-site elevation-change point map by manually drawing polygons in ArcMap 10.7 and creating elevation-\nchange shapefiles for each sub-area. Each sub-area was clipped to individual habitat polygons to create individual shapefiles \n230 \nfor each habitat type within a given sub-area. Elevation- volume-change statistics were computed for each geomorphic feature \nof interest, and each habitat within sub-areas of interest using SECAT and methods described in section 2.3. Sub-areas or geomorphic features of high-magnitude elevation change (greater than approximately ±0.5 m) were delineated \non each total-study-site elevation-change point map by manually drawing polygons in ArcMap 10.7 and creating elevation-\nchange shapefiles for each sub-area. Each sub-area was clipped to individual habitat polygons to create individual shapefiles \n230 \nfor each habitat type within a given sub area Elevation volume change statistics were computed for each geomorphic feature Sub-areas or geomorphic features of high-magnitude elevation change (greater than approximately ±0.5 m) were delineated \non each total-study-site elevation-change point map by manually drawing polygons in ArcMap 10.7 and creating elevation- on each total study site elevation change point map by manually drawing polygons in ArcMap 10.7 and creating elevation\nchange shapefiles for each sub-area. Each sub-area was clipped to individual habitat polygons to create individual shapefiles \n230 \nfor each habitat type within a given sub-area. Elevation- volume-change statistics were computed for each geomorphic feature \nof interest, and each habitat within sub-areas of interest using SECAT and methods described in section 2.3. We examined elevation and elevation-change along four 200 to 300 m transects across exam We examined elevation and elevation-change along four 200 to 300 m transects across examples of high-elevation change \ngeomorphic features. Elevation profiles for 2016, 2017, and 2019 were created for each feature of interest by extracting \n235 \nelevation values from each DEM along transect lines across the areas of greatest elevation change for each feature using \nArcMap. Points were selected using the Select Feature by Line tool in ArcMap, and the selected features were then exported \nas a new shapefile. Positions and types of geomorphic features of interest were verified through in-situ observation by SCUBA \ndivers using methods of Fehr and Yates (2020) at 30 diver reconnaissance sites throughout the total study site. https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 based on areal volume above and below surface plane heights corresponding to plus and minus the RMSETotal of the elevation \nchange analysis (RMSETotal = 21 cm for 2016 to 2017, and 19 cm for 2017 to 2019 change analyses). Upper bound volume \nchanges were calculated based on area volume above and below a plane height of 0 m. The attribute values stored within the \nelevation-change and TIN surface shapefiles were then used to compute elevation and volume change statistics for the total LKR study site and each habitat type using the Seafloor Elevation Change Analysis Tool (SECAT) custom Python script of \n220 \nZieg and Zawada (2021). Pearson correlation and linear regression analyses were performed using Microsoft Excel Version \n2302 (build 16.0.16130.20690) to assess relationships between mean habitat water depth, elevation change, and area-\nnormalized volume change for each habitat type including: 1) 2016 mean water depth (estimated from mean elevation) and \nmean elevation change; 2) 2016 mean water depth and area-normalized volume change; 3) 2017 mean water depth and mean LKR study site and each habitat type using the Seafloor Elevation Change Analysis Tool (SECAT) custom Python script of \n220 \nZieg and Zawada (2021). Pearson correlation and linear regression analyses were performed using Microsoft Excel Version \n2302 (build 16.0.16130.20690) to assess relationships between mean habitat water depth, elevation change, and area-\nnormalized volume change for each habitat type including: 1) 2016 mean water depth (estimated from mean elevation) and \nmean elevation change; 2) 2016 mean water depth and area-normalized volume change; 3) 2017 mean water depth and mean elevation change; 4) 2017 mean water depth and area-normalized volume change; and 5) 2017 to 2019 mean elevation change \n225 \nand 2016 to 2017 mean elevation. 2.3 Elevation- and Volume-Change Analyses (b) Composite RMSE (RMSETotal) for each \nelevation change analysis (2016 to 2017 and 2017 to 2019) calculated using reported RMSE for lidar and multibeam source data and methods \nof Yates et al. (2017). Black boxes indicate source data files. Blue boxes indicate steps using geoprocessing tools from ArcMap or Global \nMapper. Green boxes indicate data analysis conducted using SECAT. 215 9 3.1 Elevation and Volume Change Analyses Elevation-change results for 4,007,961 point-locations at LKR between 2016–2017 (approximately 13.5 months before and 3 \nto 6 months after Hurricane Irma) and between 2017–2019 (from approximately 3 to 16.5 months after Hurricane Irma) are \nshown in Fig. 4a and b, respectively. Mean elevation-change for the total LKR study site from 2016–2017 was 0.34 m ± 0.21; \nand all ten habitat types (Fig. 4c) showed increases in mean elevation (accretion) ranging from 0.20 m to 0.54 m (Table 2). 245 \nLargest mean elevation changes were associated with ‘aggregate reef’ (mean 2016 elevation -13.41 m) and ‘not classified’ 10 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 Table 2. Elevation change data by habitat type associated with each period and geomorphic feature subarea. Habitat type \nTotal points \nArea Mean elevation (m) \nMean elevation change (m) (SD) \n(no.) \n(km2) \n2016 \n2017 \n2019 \n2016 to 2017 2017 to 2019 \nOverall Looe Key Study Site \n Total Study Site \n4007961 \n15.98 \n-8.87 \n-8.53 \n-8.69 \n0.34 (0.21) \n-0.15 (0.11) \n Aggregate Reef \n76647 \n0.30 \n-13.41 -12.91 \n-13.16 \n0.51 (0.20) \n-0.25 (0.20) \n Colonized Pavement \n750 \n0.0028 \n-10.65 -10.33 \n-10.44 \n0.32 (0.12) \n-0.11 (0.08) \n Individual or Aggregate Patch Reef \n54414 \n0.22 \n-8.66 \n-8.33 \n-8.51 \n0.34 (0.15) \n-0.19 (0.10) \n Not Classified \n6932 \n0.026 \n-15.84 -15.30 \n-15.55 \n0.54 (0.25) \n-0.25 (0.17) \n Pavement \n645001 \n2.57 \n-10.00 \n-9.62 \n-9.79 \n0.37 (0.16) \n-0.16 (0.11) \n Reef Rubble \n80987 \n0.32 \n-6.19 \n-5.99 \n-6.17 \n0.20 (0.36) \n-0.18 (0.12) \n Seagrass Continuous \n402458 \n1.60 \n-7.69 \n-7.42 \n-7.54 \n0.27 (0.18) \n-0.12 (0.09) \n Seagrass Discontinuous \n1067504 \n4.26 \n-7.24 \n-6.96 \n-7.10 \n0.28 (0.21) \n-0.14 (0.10) \n Spur and Groove \n184875 \n0.74 \n-9.82 \n-9.45 \n-9.65 \n0.37 (0.25) \n-0.19 (0.19) \n Unconsolidated Sediment \n1488416 \n5.94 \n-9.63 \n-9.26 \n-9.42 \n0.37 (0.21) \n-0.16 (0.09) \nSand Wave \n Total Accretion Area \n15336 \n0.060 \n-6.32 \n-5.53 \n-5.68 \n0.79 (0.45) \n-0.15 (0.12) \n Seagrass Discontinuous \n7345 \n0.029 \n-5.98 \n-5.08 \n-5.23 \n0.90 (0.49) \n-0.15 (0.13) \n Unconsolidated Sediment \n7991 \n0.031 \n-6.63 \n-5.95 \n-6.09 \n0.68 (0.37) \n-0.14 (0.10) \n Total Erosion Area \n11265 \n0.043 \n-5.40 \n-5.75 \n-5.90 \n-0.36 (0.28) \n-0.15 (0.06) \n Seagrass Discontinuous \n580 \n0.002 \n-5.72 \n-5.87 \n-6.02 \n-0.15 (0.15) \n-0.15 (0.08) \n Unconsolidated Sediment \n10685 \n0.041 \n-5.38 \n-5.74 \n-5.90 \n-0.37 (0.29) \n-0.15 (0.05) \nScour Marks \n Scour Mark 1 \n202 \n0.00071 \n-7.03 \n-7.51 \n-7.41 \n-0.49 (0.26) \n0.10 (0.12) \n Seagrass Discontinuous \n197 \n0.00071 \n-7.02 \n-7.51 \n-7.41 \n-0.49 (0.26) \n0.11 (0.12) \n Unconsolidated Sediment \n5 <0.00001 \n-7.34 \n-7.47 \n-7.52 \n-0.12 (0.03) \n-0.05 (0.02) \n Scour Mark 2 \n388 \n0.0014 \n-5.41 \n-5.91 \n-5.71 \n-0.50 (0.27) \n0.20 (0.20) \n Seagrass Continuous \n338 \n0.00124 \n-5.41 \n-5.94 \n-5.70 \n-0.53 (0.27) \n0.24 (0.18) \n Unconsolidated Sediment \n50 \n0.00016 \n-5.42 \n-5.67 \n-5.72 \n-0.26 (0.16) \n-0.05 (0.13) \n Scour Mark 3 \n518 \n0.00188 \n-5.64 \n-6.14 \n-6.02 \n-0.50 (0.29) \n0.12 (0.19) \n Seagrass Continuous \n518 \n0.00188 \n-5.64 \n-6.14 \n-6.02 \n-0.50 (0.29) \n0.12 (0.19) \n Scour Mark 4 \n417 \n0.00152 \n-5.20 \n-5.74 \n-5.63 \n-0.54 (0.28) \n0.12 (0.21) \n Seagrass Continuous \n411 \n0.00151 \n-5.19 \n-5.74 \n-5.62 \n-0.55 (0.27) \n0.12 (0.21) \n Unconsolidated Sediment \n6 \n0.00001 \n-5.69 \n-5.74 \n-5.84 \n-0.06 (0.05) \n-0.10 (0.05) \nReef Rubble Field \n Total Accretion Area \n7216 \n0.028 \n-4.22 \n-3.32 \n-3.57 \n0.89 (0.45) \n-0.24 (0.30) \n Reef Rubble \n3102 \n0.012 \n-3.71 \n-2.84 \n-3.05 \n0.87 (0.44) \n-0.21 (0.36) \n Seagrass Discontinuous \n3489 \n0.014 \n-4.66 \n-3.67 \n-3.97 \n0.99 (0.42) \n-0.30 (0.24) \n Unconsolidated Sediment \n628 \n0.00237 \n-4.25 \n-3.82 \n-3.91 \n0.43 (0.26) \n-0.10 (0.12) \n Total Erosion Area \n6043 \n0.023 \n-3.00 \n-3.64 \n-3.74 \n-0.63 (0.48) \n-0.10 (0.19) \n Reef Rubble \n3409 \n0.013 \n-2.61 \n-3.39 \n-3.44 \n-0.77 (0.50) \n-0.06 (0.20) \n Seagrass Discontinuous \n1941 \n0.00708 \n-3.51 \n-4.05 \n-4.22 \n-0.54 (0.43) \n-0.17 (0.15) \n Unconsolidated Sediment \n694 \n0.00248 \n-3.50 \n-3.70 \n-3.82 \n-0.20 (0.15) \n-0.12 (0.13) \nSand Lobe \n Total Area \n67389 \n0.266 \n-12.41 -11.90 \n-12.10 \n0.51 (0.29) -0.20 (0.09) \n Unconsolidated Sediment \n67389 \n0.266 \n-12.41 -11.90 \n-12.10 \n0.51 (0.29) -0.20 (0.09) \n *58 data points fell on borders between habitats and were counted twice during habitat analysis. https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 Figure 4. Elevation-change results for 4,007,961 point-locations at Looe Key Reef. Elevation change between (a) 2016 \nmonths before and 3 to 6 months after Hurricane Irma) and (b) between 2017 to 2019 (from approximately 3 to 16.5 months af\nrma), and (c) corresponding seafloor habitats (FWC, 2015). The Hurricane Irma best track data in the panel b’s inset is fro\nNHC Irma Storm Track resource page (NHC, 2018, see also Figure 2b). Boundaries for the Looe Key Sanctuary Protection Ar\npecial Protection Unit (SPU) are shown as pink polygons. Geomorphic features of interest are indicated with black polygons\nreas indicate locations where water depth was too shallow for collection of multibeam bathymetric data. Figure 4. Elevation-change results for 4,007,961 point-locations at Looe Key Reef. Elevation change between (a) 2016 to 2017 (13.5 \nmonths before and 3 to 6 months after Hurricane Irma) and (b) between 2017 to 2019 (from approximately 3 to 16.5 months after Hurricane \nIrma), and (c) corresponding seafloor habitats (FWC, 2015). The Hurricane Irma best track data in the panel b’s inset is from the NOAA \n250 \nNHC Irma Storm Track resource page (NHC, 2018, see also Figure 2b). Boundaries for the Looe Key Sanctuary Protection Area (SPA) and \nSpecial Protection Unit (SPU) are shown as pink polygons. Geomorphic features of interest are indicated with black polygons. Gaps in map \nareas indicate locations where water depth was too shallow for collection of multibeam bathymetric data. 255 11 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 habitat types showed increases in net volume (accretion) with upper bound ranges from 0.001 to 2.19 Mm3 (Table 3). Largest \nnet volume changes were associated with habitats covering the largest areal extent of the study area including ‘pavement,’ \n‘discontinuous seagrass,’ and ‘unconsolidated sediment.’ Pearson correlation analysis also indicated a very strong positive \ncorrelation (r(8) = 0.99, p = 0.000) and linear relationship (r2 = 0.92, Fig. 5b)) between 2016 mean habitat water depth and habitat types showed increases in net volume (accretion) with upper bound ranges from 0.001 to 2.19 Mm3 (Table 3). Largest \nnet volume changes were associated with habitats covering the largest areal extent of the study area including ‘pavement,’ \n‘discontinuous seagrass,’ and ‘unconsolidated sediment.’ Pearson correlation analysis also indicated a very strong positive \ncorrelation (r(8) = 0.99, p = 0.000) and linear relationship (r2 = 0.92, Fig. 5b)) between 2016 mean habitat water depth and \narea-normalized volume change; area-normalized volume gains increased significantly with increasing water depth. Largest \n270 \narea-normalized volume changes of 0.51 mM3 and 0.54 mM3 were observed for ‘aggregate reef’ and ‘not classified’ habitats, \nrespectively; and smallest changes of 0.20 to 0.27 mM3 were observed for ‘reef rubble’ and ‘continuous seagrass’ habitats \n(Table 3), consistent with mean elevation changes for those habitats. Mean elevation-change values of the 2016–2017 elevation \nchange data set that was clipped to an area of 15.98 km2 and used for this analysis were within ±0.01 m, and area-normalized \nvolumes were within ±0.016 Mm3 km-2, of values calculated in the original 19.71 km2 published data set (unclipped) for the \n275 \noverall study site and all habitats (Yates et al., 2019). area-normalized volume change; area-normalized volume gains increased significantly with increasing water depth. Largest \n270 \narea-normalized volume changes of 0.51 mM3 and 0.54 mM3 were observed for ‘aggregate reef’ and ‘not classified’ habitats, \nrespectively; and smallest changes of 0.20 to 0.27 mM3 were observed for ‘reef rubble’ and ‘continuous seagrass’ habitats \n(Table 3), consistent with mean elevation changes for those habitats. SD = standard deviation. Elevation change data by habitat type associated with each period and geomorphic feature subarea. elevation -6.19 m) and ‘seagrass continuous’ (mean 2016 elevation -7.69 m) habitats (Table 2). Only 4% of all data points \n260 \nshowed losses in elevation (erosion) ranging from -0.01 m to -0.44 m, while 96% of all data points showed gains in elevation \nranging from 0.31 m to 0.55 m across all habitats. Pearson correlation analysis showed a very strong positive correlation (r(8) \n= 0.96, p = 0.000) and linear relationship (r2 = 0.92, Fig. 5a) between 2016 mean habitat water depth (estimated from mean \nelevation) and mean elevation change; mean elevation gains increased significantly with increasing water depth (i.e., \ndecreasing seafloor elevation). Net volume change was up to 5.36 mM3 over the total 15.98 km2 Looe Key study site; and all \n265 260 12 5c) between estimated to -0.25 m (Fig. 4b, Table 2). Largest mean elevation changes were associated with ‘aggregate reef’ and ‘not classified’ habitat \n280 \ntypes, and smallest changes were associated with ‘colonized pavement’ and ‘continuous seagrass’ habitats (Table 2). Only 5% \nof all data points showed gains in elevation with mean accretion ranging from 0.04 m to 0.19 m, while 95% of all data points \nshowed losses in elevation with mean erosion ranging from -0.13 m to -0.27 m across all habitat types. Pearson correlation \nanalysis indicated a moderate correlation (r(8) = -0.67, p = 0.035) and linear relationship (r2 = 0.45, Fig. 5c) between estimated 2017 mean habitat water depth and mean elevation change; mean elevation loss generally increased with increasing water \n285 \ndepth. Net volume change was up to -2.46 mM3 over the total 15.98 km2 Looe Key study site and area-normalized volume \nchange was -0.15 Mm3km-2. Losses in net volume up to -0.931 Mm3 (erosion) were observed across all habitat types (Table \n4). 2017 mean habitat water depth and mean elevation change; mean elevation loss generally increased with increasing water \n285 \ndepth. Net volume change was up to -2.46 mM3 over the total 15.98 km2 Looe Key study site and area-normalized volume \nchange was -0.15 Mm3km-2. Losses in net volume up to -0.931 Mm3 (erosion) were observed across all habitat types (Table \n4). Largest net volume changes were associated with habitats covering the largest areal extent of the study area including \n290 \n‘pavement,’ ‘discontinuous seagrass,’ and ‘unconsolidated sediment.’ Pearson correlation analysis indicated a moderate \ncorrelation (r(8) = -0.67, p = 0.035) and linear relationship (r2 = 0.45, Fig. 5d) between 2017 mean habitat water depth and \narea-normalized volume change; area-normalized volume losses generally increased with increasing water depth. Largest area-\nnormalized volume changes were observed for ‘aggregate reef’ and ‘not classified’ habitats, -0.254 and -0.247 Mm3 km-2, Largest net volume changes were associated with habitats covering the largest areal extent of the study area including \n290 \n‘pavement,’ ‘discontinuous seagrass,’ and ‘unconsolidated sediment.’ Pearson correlation analysis indicated a moderate \ncorrelation (r(8) = -0.67, p = 0.035) and linear relationship (r2 = 0.45, Fig. 5d) between 2017 mean habitat water depth and \narea-normalized volume change; area-normalized volume losses generally increased with increasing water depth. Mean elevation-change values of the 2016–2017 elevation \nchange data set that was clipped to an area of 15.98 km2 and used for this analysis were within ±0.01 m, and area-normalized area-normalized volume change; area-normalized volume gains increased significantly with increasing water depth. Largest \n270 \narea-normalized volume changes of 0.51 mM3 and 0.54 mM3 were observed for ‘aggregate reef’ and ‘not classified’ habitats, \nrespectively; and smallest changes of 0.20 to 0.27 mM3 were observed for ‘reef rubble’ and ‘continuous seagrass’ habitats \n(Table 3), consistent with mean elevation changes for those habitats. Mean elevation-change values of the 2016–2017 elevation \nchange data set that was clipped to an area of 15.98 km2 and used for this analysis were within ±0.01 m, and area-normalized \nvolumes were within ±0.016 Mm3 km-2, of values calculated in the original 19.71 km2 published data set (unclipped) for the \n275 \noverall study site and all habitats (Yates et al 2019) Mean elevation-change during a 13-month time-period between December 2017 to June 2019 (up to approximately 16.5 \nmonths after Hurricane Irma) was -0.15 ± 0.11 m, and all habitat types showed losses in mean elevation ranging from -0.11 m )\n,\nyp\ng g\nto -0.25 m (Fig. 4b, Table 2). Largest mean elevation changes were associated with ‘aggregate reef’ and ‘not classified’ habitat \n280 \ntypes, and smallest changes were associated with ‘colonized pavement’ and ‘continuous seagrass’ habitats (Table 2). Only 5% \nof all data points showed gains in elevation with mean accretion ranging from 0.04 m to 0.19 m, while 95% of all data points \nshowed losses in elevation with mean erosion ranging from -0.13 m to -0.27 m across all habitat types. Pearson correlation \nanalysis indicated a moderate correlation (r(8) = -0.67, p = 0.035) and linear relationship (r2 = 0.45, Fig. 5c) between estimated to -0.25 m (Fig. 4b, Table 2). Largest mean elevation changes were associated with ‘aggregate reef’ and ‘not classified’ habitat \n280 \ntypes, and smallest changes were associated with ‘colonized pavement’ and ‘continuous seagrass’ habitats (Table 2). Only 5% \nof all data points showed gains in elevation with mean accretion ranging from 0.04 m to 0.19 m, while 95% of all data points \nshowed losses in elevation with mean erosion ranging from -0.13 m to -0.27 m across all habitat types. Pearson correlation \nanalysis indicated a moderate correlation (r(8) = -0.67, p = 0.035) and linear relationship (r2 = 0.45, Fig. Largest area-\nnormalized volume changes were observed for ‘aggregate reef’ and ‘not classified’ habitats, -0.254 and -0.247 Mm3 km-2, Largest net volume changes were associated with habitats covering the largest areal extent of the study area including \n290 \n‘pavement,’ ‘discontinuous seagrass,’ and ‘unconsolidated sediment.’ Pearson correlation analysis indicated a moderate \ncorrelation (r(8) = -0.67, p = 0.035) and linear relationship (r2 = 0.45, Fig. 5d) between 2017 mean habitat water depth and \narea-normalized volume change; area-normalized volume losses generally increased with increasing water depth. Largest area-\nnormalized volume changes were observed for ‘aggregate reef’ and ‘not classified’ habitats, -0.254 and -0.247 Mm3 km-2, respectively; smallest changes were observed for ‘colonized pavement’ and ‘continuous seagrass’ habitats, -0.112 to -0.118 \n295 \nMm3 km-2 respectively (Table 4), consistent with mean elevation changes for those habitats. Pearson correlation analysis \nindicated a strong negative correlation (r(8) = -0.74, p = 0.014) and linear relationship (r2 = 0.55, Fig. 5e) between 2017 to \n2019 mean habitat elevation change and 2016 to 2017 mean habitat elevation change; mean elevation losses during 2017 to \n2019 were significantly greater in habitats with larger mean elevation gains during 2016 to 2017. Mean elevation change (loss) respectively; smallest changes were observed for ‘colonized pavement’ and ‘continuous seagrass’ habitats, -0.112 to -0.118 \n295 \nMm3 km-2 respectively (Table 4), consistent with mean elevation changes for those habitats. Pearson correlation analysis \nindicated a strong negative correlation (r(8) = -0.74, p = 0.014) and linear relationship (r2 = 0.55, Fig. 5e) between 2017 to \n2019 mean habitat elevation change and 2016 to 2017 mean habitat elevation change; mean elevation losses during 2017 to \n2019 were significantly greater in habitats with larger mean elevation gains during 2016 to 2017. Mean elevation change (loss) 13 during 2017 to 2019 was 35 to 55% of the mean elevation change (gain) during 2016 to 2017 for all habitats except for reef \n00 \nrubble which was 92% and had the shallowest mean depth (6.0 m) of all habitats. https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 Table 3. Compiled volume change data by habitat type for all study areas during the 2016 to 2017 study period (storm period). Habitat Type \nHabitat Area \n(km2) \nGross erosion \n(Mm3) \nGross accretion \n(Mm3) \nNet volume change \n(Mm3 study-area-1) \nArea-normalized volume \nchange (Mm3 km-2) \nLower \nUpper \nLower Upper \n Lower \nUpper \nLower \nUpper \nOverall Looe Key Study Site \n Total Study Site \n15.98 \n0.053 \n0.134 \n2.456 \n5.490 \n2.403 \n5.356 \n0.150 \n0.335 \n Aggregate Reef \n0.30 \n0 \n0 \n0.090 \n0.154 \n0.090 \n0.154 \n0.296 \n0.505 \n Colonized Pavement \n0.0028 \n0 \n0 \n0 \n0.001 \n0 \n0.001 \n0.124 \n0.323 \n Individual or Aggregate Patch Reef \n0.22 \n0 \n0 \n0.029 \n0.073 \n0.029 \n0.072 \n0.136 \n0.336 \n Not Classified \n0.026 \n0 \n0 \n0.009 \n0.014 \n0.009 \n0.014 \n0.337 \n0.540 \n Pavement \n2.57 \n0 \n0.003 \n0.445 \n0.962 \n0.445 \n0.959 \n0.173 \n0.373 \n Reef Rubble \n0.32 \n0.013 \n0.021 \n0.033 \n0.085 \n0.020 \n0.064 \n0.062 \n0.199 \n Seagrass Continuous \n1.60 \n0.008 \n0.021 \n0.152 \n0.449 \n0.144 \n0.428 \n0.090 \n0.267 \n Seagrass Discontinuous \n4.26 \n0.015 \n0.045 \n0.477 \n1.250 \n0.462 \n1.205 \n0.109 \n0.283 \n Spur and Groove \n0.74 \n0.003 \n0.007 \n0.136 \n0.278 \n0.133 \n0.271 \n0.181 \n0.367 \n Unconsolidated Sediment \n5.94 \n0.013 \n0.037 \n1.083 \n2.226 \n1.070 \n2.189 \n0.180 \n0.369 \nSand Wave \n Total Accretion Area \n0.060 \n0 \n0 \n0.036 \n0.048 \n0.036 \n0.048 \n0.598 \n0.800 \n Seagrass Discontinuous \n0.029 \n0 \n0 \n0.020 \n0.026 \n0.020 \n0.026 \n0.709 \n0.914 \n Unconsolidated Sediment \n0.031 \n0 \n0 \n0.015 \n0.021 \n0.015 \n0.021 \n0.494 \n0.694 \n Total Erosion Area \n0.043 \n0.009 \n0.016 \n0 \n0 \n-0.009 \n-0.016 \n-0.198 \n-0.370 \n Seagrass Discontinuous \n0.002 \n<0.001 \n<0.001 \n0 \n0 \n<-0.001 \n<-0.001 \n-0.045 \n-0.162 \n Unconsolidated Sediment \n0.041 \n0.008 \n0.016 \n0 \n0 \n-0.008 \n-0.016 \n-0.205 \n-0.380 \nScour Marks \n Scour Mark 1 \n0.00071 \n0.0002 \n0.0004 \n0 \n0 \n-0.0002 \n-0.0004 \n-0.3083 \n-0.5114 \n Seagrass Discontinuous \n0.00071 \n0.0002 \n0.0004 \n0 \n0 \n-0.0002 \n-0.0004 \n-0.3118 \n-0.5154 \n Unconsolidated Sediment \n<0.00001 \n0 \n0 \n0 \n0 \n0 <-0.0001 \n-0.0001 \n-0.1479 \n Scour Mark 2 \n0.0014 \n0.0005 \n0.0007 \n0 \n0 \n-0.0005 \n-0.0007 \n-0.3255 \n-0.5271 \n Seagrass Continuous \n0.00124 \n0.0004 \n0.0007 \n0 \n0 \n-0.0004 \n-0.0007 \n-0.3558 \n-0.5595 \n Unconsolidated Sediment \n0.00016 \n0 <-0.0001 \n0 \n0 \n0 <-0.0001 \n-0.0943 \n-0.2790 \n Scour Mark 3 \n0.00188 \n0.0006 \n0.0010 \n0 \n0 \n-0.0006 \n-0.0010 \n-0.3247 \n-0.5232 \n Seagrass Continuous \n0.00188 \n0.0006 \n0.0010 \n0 \n0 \n-0.0006 \n-0.0010 \n-0.3247 \n-0.5232 \n Scour Mark 4 \n0.00152 \n0.0006 \n0.0009 \n0 \n0 \n-0.0006 \n-0.0009 \n-0.3631 \n-0.5661 \n Seagrass Continuous \n0.00151 \n0.0006 \n0.0009 \n0 \n0 \n-0.0006 \n-0.0009 \n-0.3658 \n-0.5697 \n Unconsolidated Sediment \n0.00001 \n0 <-0.0001 \n0 \n0 \n0 <-0.0001 \n0.0000 \n-0.0748 \nReef Rubble Field \n Total Accretion Area \n0.028 \n0 \n0 \n0.020 \n0.025 \n0.020 \n0.025 \n0.707 \n0.914 \n Reef Rubble \n0.012 \n0 \n0 \n0.008 \n0.011 \n0.008 \n0.011 \n0.690 \n0.897 \n Seagrass Discontinuous \n0.014 \n0 \n0 \n0.011 \n0.014 \n0.011 \n0.014 \n0.802 \n1.011 \n Unconsolidated Sediment \n0.002 \n0 \n0 \n0.001 \n0.001 \n0.001 \n0.001 \n0.252 \n0.446 \n Total Erosion Area \n0.023 \n0.011 \n0.015 \n0 \n0 \n-0.011 \n-0.015 \n-0.464 \n-0.661 \n Reef Rubble \n0.013 \n0.008 \n0.010 \n0 \n0 \n-0.008 \n-0.010 \n-0.584 \n-0.788 \n Seagrass Discontinuous \n0.007 \n0.003 \n0.004 \n0 \n0 \n-0.003 \n-0.004 \n-0.382 \n-0.577 \n Unconsolidated Sediment \n0.002 \n0.000 \n0.001 \n0 \n0 \n0.000 \n-0.001 \n-0.064 \n-0.221 \nSand Lobe \n Total Area \n0.27 \n0 \n0.002 \n0.089 \n0.139 \n0.089 \n0.137 \n0.332 \n0.513 \n Unconsolidated Sediment \n0.27 \n0 \n0.002 \n0.089 \n0.139 \n0.089 \n0.137 \n0.332 \n0.513 \n‘Upper’ and ‘lower’ headings refer to the upper and lower bounds of volume change based on total RMSE root mean square error). https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 during 2017 to 2019 was 35 to 55% of the mean elevation change (gain) during 2016 to 2017 for all habitats except for reef \n300 \nrubble which was 92% and had the shallowest mean depth (6.0 m) of all habitats. during 2017 to 2019 was 35 to 55% of the mean elevation change (gain) during 2016 to 2017 for all habitats except for reef \n300 \nrubble which was 92% and had the shallowest mean depth (6.0 m) of all habitats. during 2017 to 2019 was 35 to 55% of the mean elevation change (gain) during 2016 to 2017 for all habitats except for reef \n300 \nrubble which was 92% and had the shallowest mean depth (6.0 m) of all habitats. (a) \n \n(b) \n \n(c) \n \n(d) \n \n (e) \n \nFigure 5. Linear relationships between elevation change, volume change, and water depth. Linear relationships and coeffici\ndetermination between (a) mean elevation change, (b) mean area-normalized volume change, and estimated 2016 mean water de\nseafloor habitats of the Looe Key study site between 2016 to 2017. Linear relationships and coefficients of determination between (c\n5 \nelevation change, (d) mean area-normalized volume change, and estimated 2017 mean water depth for seafloor habitats of the Loo\nstudy site between 2017 to 2019 (a, b). Linear relationship between 2017 to 2019 mean elevation change and 2016 to 2017 mean ele\nchange (e). (a) (b) (a) (d) (c) (d) (c) (e) (e) Figure 5. Linear relationships between elevation change, volume change, and water depth. Linear relationships and coefficients of \ndetermination between (a) mean elevation change, (b) mean area-normalized volume change, and estimated 2016 mean water depth for \nseafloor habitats of the Looe Key study site between 2016 to 2017. Linear relationships and coefficients of determination between (c) mean \nelevation change, (d) mean area-normalized volume change, and estimated 2017 mean water depth for seafloor habitats of the Looe Key \nstudy site between 2017 to 2019 (a, b). Linear relationship between 2017 to 2019 mean elevation change and 2016 to 2017 mean elevation \nchange (e). 14 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 Table 4. Compiled volume change data by habitat type for all study areas during the 2017 to 2019 study period (post-storm re-\nequilibration period). Table 4. Compiled volume change data by habitat type for all study areas during the 2017 to 2019 study period (post-storm re-\nequilibration period). 4. Compiled volume change data by habitat type for all study areas during the 2017 to 2019 study \nbration period). hange data by habitat type for all study areas during the 2017 to 2019 study period (post-storm re- 320 Lower bounds use tota\nRMSE as a plane height in calculating volume. Table 3. Compiled volume change data by habitat type for all study areas during the 2016 to 2017 study period (storm period). 310 Table 3. Compiled volume change data by habitat type for all study areas during the 2016 to 2017 stud e change data by habitat type for all study areas during the 2016 to 2017 study period (storm period). 315 15 Habitat Type \nHabitat Area \n(km2) \nGross erosion \n(Mm3) \nGross accretion \n(Mm3) \nNet volume change \n(Mm3 study-area-1) \nArea-normalized volume \nchange (Mm3 km-2) \nLower \nUpper \nLower Upper \n Lower \nUpper \nLower \nUpper \nOverall Looe Key Study Site \n Total Study Site \n15.98 \n0.316 \n2.502 \n0.005 \n0.041 \n-0.311 \n-2.461 \n-0.019 \n-0.154 \n Aggregate Reef \n0.30 \n0.028 \n0.078 \n0 <0.001 \n-0.028 \n-0.077 \n-0.093 \n-0.254 \n Colonized Pavement \n0.0028 \n<0.001 \n<0.001 \n0 \n0 \n<-0.001 \n<-0.001 \n-0.004 \n-0.112 \n Individual or Aggregate Patch Reef \n0.22 \n0.006 \n0.040 \n0 \n0 \n-0.006 \n-0.040 \n-0.028 \n-0.186 \n Not Classified \n0.026 \n0.002 \n0.007 \n0 <0.001 \n-0.002 \n-0.006 \n-0.083 \n-0.247 \n Pavement \n2.57 \n0.059 \n0.424 \n0 \n0.004 \n-0.059 \n-0.420 \n-0.023 \n-0.163 \n Reef Rubble \n0.32 \n0.010 \n0.061 \n0.001 \n0.002 \n-0.009 \n-0.059 \n-0.029 \n-0.182 \n Seagrass Continuous \n1.60 \n0.012 \n0.197 \n0.001 \n0.008 \n-0.011 \n-0.189 \n-0.007 \n-0.118 \n Seagrass Discontinuous \n4.26 \n0.064 \n0.612 \n0.001 \n0.015 \n-0.063 \n-0.597 \n-0.015 \n-0.140 \n Spur and Groove \n0.74 \n0.032 \n0.145 \n0.001 \n0.003 \n-0.031 \n-0.141 \n-0.042 \n-0.192 \n Unconsolidated Sediment \n5.94 \n0.102 \n0.938 \n0.000 \n0.007 \n-0.102 \n-0.931 \n-0.017 \n-0.157 \nSand Wave \n Total Accretion Area \n0.060 \n0.0015 \n0.0093 \n0 0.0005 \n-0.0015 \n-0.0088 \n-0.0245 \n-0.1479 \n Seagrass Discontinuous \n0.029 \n0.0010 \n0.0048 \n0 0.0003 \n-0.0010 \n-0.0044 \n-0.0336 \n-0.1544 \n Unconsolidated Sediment \n0.031 \n0.0005 \n0.0045 \n0 0.0001 \n-0.0005 \n-0.0044 \n-0.0159 \n-0.1419 \n Total Erosion Area \n0.043 \n0.0003 \n0.0066 \n0 \n0 \n-0.0003 \n-0.0066 \n-0.0074 \n-0.1529 \n Seagrass Discontinuous \n0.002 \n<0.0001 \n0.0003 \n0 \n0 \n<-0.0001 \n-0.0003 \n-0.0158 \n-0.1521 \n Unconsolidated Sediment \n0.041 \n0.0003 \n0.0063 \n0 \n0 \n-0.0003 \n-0.0063 \n-0.0070 \n-0.1529 \nScour Marks \n Scour Mark 1 \n0.00071 \n0.0000 \n0.0000 \n<0.0001 0.0001 \n<0.0001 \n0.0001 \n0.0171 \n0.1201 \n Seagrass Discontinuous \n0.00071 \n0.0000 \n0.0000 \n<0.0001 0.0001 \n<0.0001 \n0.0001 \n0.0173 \n0.1219 \n Unconsolidated Sediment \n<0.00001 \n0.0000 \n<0.0001 \n0.0000 0.0000 \n0.0000 <-0.0001 \n0.0000 \n-0.0447 \n Scour Mark 2 \n0.0014 \n0 \n0 \n0.0001 0.0003 \n0.0001 \n0.0003 \n0.0880 \n0.2226 \n Seagrass Continuous \n0.00124 \n0 \n0 \n0.0001 0.0003 \n0.0001 \n0.0003 \n0.0996 \n0.2550 \n Unconsolidated Sediment \n0.00016 \n<0.0001 \n<0.0001 \n0 \n0 \n<-0.0001 <-0.0001 \n-0.0009 \n-0.0254 \n Scour Mark 3 \n0.00188 \n0 \n0 \n0.0001 0.0003 \n0.0001 \n0.0003 \n0.0524 \n0.1380 \n Seagrass Continuous \n0.00188 \n0 \n0 \n0.0001 0.0003 \n0.0001 \n0.0003 \n0.0524 \n0.1380 \n Scour Mark 4 \n0.00152 \n0 \n0 \n0.0001 0.0002 \n0.0001 \n0.0002 \n0.0615 \n0.1334 \n Seagrass Continuous \n0.00151 \n0 \n0 \n0.0001 0.0002 \n0.0001 \n0.0002 \n0.0620 \n0.1351 \n Unconsolidated Sediment \n0.00001 \n0.0000 \n<0.0001 \n0.0000 0.0000 \n0.0000 <-0.0001 \n0.0000 \n-0.1029 \nReef Rubble Field \n Total Accretion Area \n0.028 \n0.0040 \n0.0080 \n0.0010 0.0010 \n-0.0040 \n-0.0070 \n-0.1310 \n-0.2480 \n Reef Rubble \n0.012 \n0.0020 \n0.0040 \n0 0.0010 \n-0.0010 \n-0.0030 \n-0.1260 \n-0.2180 \n Seagrass Discontinuous \n0.014 \n0.0020 \n0.0040 \n0 \n0 \n-0.0020 \n-0.0040 \n-0.1560 \n-0.3020 \n Unconsolidated Sediment \n0.002 \n<0.0001 \n<0.0001 \n0 \n0 \n<-0.0001 <-0.0001 \n-0.0070 \n-0.0910 \n Total Erosion Area \n0.023 \n0.0005 \n0.0031 \n0.0003 0.0008 \n-0.0002 \n-0.0023 \n-0.0084 \n-0.1026 \n Reef Rubble \n0.013 \n0.0001 \n0.0015 \n0.0003 0.0007 \n0.0002 \n-0.0008 \n0.0113 \n-0.0612 \n Seagrass Discontinuous \n0.007 \n0.0003 \n0.0013 \n0 0.0001 \n-0.0003 \n-0.0012 \n-0.0429 \n-0.1741 \n Unconsolidated Sediment \n0.002 \n<0.0001 \n0.0003 \n0 \n0 \n<-0.0001 \n-0.0003 \n-0.0152 \n-0.1188 \nSand Lobe \n Total Area \n0.27 \n0.010 \n0.055 \n0 <0.001 \n-0.010 \n-0.054 \n-0.038 \n-0.204 \n Unconsolidated Sediment \n0.27 \n0.010 \n0.055 \n0 <0.001 \n-0.010 \n-0.054 \n-0.038 \n-0.204 \n‘Upper’ and ‘lower’ headings refer to the upper and lower bounds of volume change based on total RMSE (root mean square error). https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 Several scour features developed in ‘discontinuous seagrass’ and ‘unconsolidated sediment’ habitats of the Looe Key back \nreef, indicated by areas of erosion that appear as pits. Rubble fields within and near the Looe Key SPA area were displaced, \nas indicated by adjacent areas of accretion and erosion. Substantial deposition of sediments occurred along a sand lobe at the \nbase of the Looe Key Reef ‘spur and groove’ habitat. 330 330 3.2.1 Sand Wave Total net volume change for the accretion area of the feature was 0.048 Mm3 and area-normalized \nvolume change was 0.800 Mm3 km-2 (Table 3). Mean elevation-change and area-normalized volume change was greatest within the discontinuous seagrass habitat (0.90 m and 0.914 Mm3 km-2, respectively), approximately 2.7 times greater than \n345 \nmean elevation change and area-normalized volume change for the overall Looe Key study site. It accounted for 55% of total \nnet volume gain, indicating burial of seagrass habitat during migration of the sand wave. Net volume change of the erosion \narea was approximately -0.016 Mm3 and area-normalized volume change -0.37 Mm3 km-2 with 98% of net volume change \nassociated with erosion of unconsolidated sediment habitat (Table 3). within the discontinuous seagrass habitat (0.90 m and 0.914 Mm3 km-2, respectively), approximately 2.7 times greater than \n345 \nmean elevation change and area-normalized volume change for the overall Looe Key study site. It accounted for 55% of total \nnet volume gain, indicating burial of seagrass habitat during migration of the sand wave. Net volume change of the erosion \narea was approximately -0.016 Mm3 and area-normalized volume change -0.37 Mm3 km-2 with 98% of net volume change \nassociated with erosion of unconsolidated sediment habitat (Table 3). 350 \nBetween 2017 and 2019, the sand wave (accretion area) showed mean elevation and net volume change of approximately -\n0.15 m and approximately -0.009 Mm3, respectively (Table 2, 4 and Fig. 6b). Similar mean elevation change values were \nobserved for discontinuous seagrass and unconsolidated sediment habitats associated with the feature, and net volume change \nfor each habitat was approximately 50% of the total net volume change (Table 4). Area-normalized volume change was similar 350 \nBetween 2017 and 2019, the sand wave (accretion area) showed mean elevation and net volume change of approximately -\n0.15 m and approximately -0.009 Mm3, respectively (Table 2, 4 and Fig. 6b). Similar mean elevation change values were \nobserved for discontinuous seagrass and unconsolidated sediment habitats associated with the feature, and net volume change \nfor each habitat was approximately 50% of the total net volume change (Table 4). Area-normalized volume change was similar 350 \nBetween 2017 and 2019, the sand wave (accretion area) showed mean elevation and net volume change of approximately -\n0.15 m and approximately -0.009 Mm3, respectively (Table 2, 4 and Fig. 6b). 3.2 Geomorphic Feature Analyses Large-scale geomorphic features that were 10s to 100s of m2 in areal extent and showed extensive erosion and/or accretion \nwith elevation-changes greater than 0.5 m were observed between 2016 and 2017 (Fig. 6 and 7). Examples of these features \n325 \nincluded migration of a sand wave in the back reef area of Looe Key reef indicated by adjacent areas of erosion and accretion. 16 3.2.1 Sand Wave An accretion of 0.060 km2 included \napproximately 50% discontinuous seagrass and 50% unconsolidated sediment habitat. of approximately 5.6 m. Transect elevation profiles showed the location of this feature in 2016, westward migration of \n335 \napproximately 78 m (crest to crest) in 2017, and minor erosion in 2019 (Fig. 7a). An accretion of 0.060 km2 included \napproximately 50% discontinuous seagrass and 50% unconsolidated sediment habitat. of approximately 5.6 m. Transect elevation profiles showed the location of this feature in 2016, westward migration of \n335 \napproximately 78 m (crest to crest) in 2017, and minor erosion in 2019 (Fig. 7a). An accretion of 0.060 km2 included \napproximately 50% discontinuous seagrass and 50% unconsolidated sediment habitat. Between 2016 and 2017, mean elevation change of the accretion area (2017 location of the sand wave) was 0.79 m (Table 2) \nwith a maximum elevation gain at the crest of 1.84m. An adjacent area of erosion was approximately 630 x 122 m in length \n340 \nand width (0.043 km2) and included approximately 5% discontinuous seagrass and 95% unconsolidated sediment. Mean \nelevation-change of the erosion area was -0.36 m (Table 2) with a maximum elevation loss of -1.23 m near the 2016 location \nof the sand wave crest. Total net volume change for the accretion area of the feature was 0.048 Mm3 and area-normalized \nvolume change was 0.800 Mm3 km-2 (Table 3). Mean elevation-change and area-normalized volume change was greatest g\nwith a maximum elevation gain at the crest of 1.84m. An adjacent area of erosion was approximately 630 x 122 m in length \n340 \nand width (0.043 km2) and included approximately 5% discontinuous seagrass and 95% unconsolidated sediment. Mean \nelevation-change of the erosion area was -0.36 m (Table 2) with a maximum elevation loss of -1.23 m near the 2016 location \nof the sand wave crest. Total net volume change for the accretion area of the feature was 0.048 Mm3 and area-normalized \nvolume change was 0.800 Mm3 km-2 (Table 3). Mean elevation-change and area-normalized volume change was greatest with a maximum elevation gain at the crest of 1.84m. An adjacent area of erosion was approximately 630 x 122 m in length \n340 \nand width (0.043 km2) and included approximately 5% discontinuous seagrass and 95% unconsolidated sediment. Mean \nelevation-change of the erosion area was -0.36 m (Table 2) with a maximum elevation loss of -1.23 m near the 2016 location \nof the sand wave crest. 3.2.1 Sand Wave Migration of a sand wave was observed in the back reef area of Looe Key Reef between 2016 and 2017, with minor erosion \nof this feature occurring between 2017 and 2019 (Fig. 6a, b, and c). The sand wave was approximately 733 m long and 104 m \nwide at its widest point in 2017, 2 m in height from the crest to base on the deepest (western) edge, with average water depth \nof approximately 5.6 m. Transect elevation profiles showed the location of this feature in 2016, westward migration of \n335 \napproximately 78 m (crest to crest) in 2017, and minor erosion in 2019 (Fig. 7a). An accretion of 0.060 km2 included \napproximately 50% discontinuous seagrass and 50% unconsolidated sediment habitat. Migration of a sand wave was observed in the back reef area of Looe Key Reef between 2016 and 2017, with minor erosion \nof this feature occurring between 2017 and 2019 (Fig. 6a, b, and c). The sand wave was approximately 733 m long and 104 m \nwide at its widest point in 2017, 2 m in height from the crest to base on the deepest (western) edge, with average water depth \nof approximately 5.6 m. Transect elevation profiles showed the location of this feature in 2016, westward migration of \n335 \ni\nl\n78\n(\n) i\n2017\nd\ni\ni\ni\n2019 (Fi\n7 ) A\ni\nf 0 060 k\n2 i\nl d d Migration of a sand wave was observed in the back reef area of Looe Key Reef between 2016 and 2017, with minor erosion \nof this feature occurring between 2017 and 2019 (Fig. 6a, b, and c). The sand wave was approximately 733 m long and 104 m \nwide at its widest point in 2017, 2 m in height from the crest to base on the deepest (western) edge, with average water depth of this feature occurring between 2017 and 2019 (Fig. 6a, b, and c). The sand wave was approximately 733 m long and 104 m \nwide at its widest point in 2017, 2 m in height from the crest to base on the deepest (western) edge, with average water depth \nof approximately 5.6 m. Transect elevation profiles showed the location of this feature in 2016, westward migration of \n335 \napproximately 78 m (crest to crest) in 2017, and minor erosion in 2019 (Fig. 7a). 3.2.1 Sand Wave Similar mean elevation change values were \nobserved for discontinuous seagrass and unconsolidated sediment habitats associated with the feature, and net volume change \nfor each habitat was approximately 50% of the total net volume change (Table 4). Area-normalized volume change was similar for the total area of the sand wave and the sub-areas within it, including discontinuous seagrass and unconsolidated sediment \n355 \nhabitats, ranging from approximately -0.148 to -0.154 Mm3/km2. The adjacent erosion area (original 2016 location of the sand \nwave) also showed a mean elevation change of -0.15 m with similar values for the associated discontinuous seagrass and \nunconsolidated sediment habitats. Net volume change of the erosion area was approximately -0.007 Mm3 with approximately for the total area of the sand wave and the sub-areas within it, including discontinuous seagrass and unconsolidated sediment \n355 \nhabitats, ranging from approximately -0.148 to -0.154 Mm3/km2. The adjacent erosion area (original 2016 location of the sand \nwave) also showed a mean elevation change of -0.15 m with similar values for the associated discontinuous seagrass and \nunconsolidated sediment habitats. Net volume change of the erosion area was approximately -0.007 Mm3 with approximately 17 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 95% of this loss associated with unconsolidated sediment (Table 4). Area-normalized volume change was also consistent \nacross the total erosion feature area, discontinuous seagrass, and unconsolidated sediment habitats at -0.15 Mm3 km-2. 360 95% of this loss associated with unconsolidated sediment (Table 4). Area-normalized volume change was also consistent \nacross the total erosion feature area, discontinuous seagrass, and unconsolidated sediment habitats at -0.15 Mm3 km-2. 360 360 Figure 6. Elevation change data and transect positions for each geomorphic feature subarea. Geomorphic features included a s\nwave (a, b, c), scour marks (d, e, f), western rubble field (g, h, i), and sand lobe subareas (j, k, l). These feature locations and correspond\nhabitat are also shown in Fig. 4. Elevation-change from 2016 to 2017 (a, d, g, j), 2017 to 2019 (b, e, h, k), and corresponding reconnaissa\n5 \nimagery (c, f, i, l) Transect positions are indicated by black lines and lowercase letters in the elevation change panels (see also Fig. 7). Sc\nmarks in panels d and e are labelled SM1 through 4. Photo credit: Mitch Lemon, Cherokee Nations System Solutions for U.S. Geolog\nSurvey. Figure 6. Elevation change data and transect positions for each geomorphic feature subarea. Geomorphic features included a sand \nwave (a, b, c), scour marks (d, e, f), western rubble field (g, h, i), and sand lobe subareas (j, k, l). These feature locations and corresponding \nhabitat are also shown in Fig. 4. Elevation-change from 2016 to 2017 (a, d, g, j), 2017 to 2019 (b, e, h, k), and corresponding reconnaissance \n365 \nimagery (c, f, i, l) Transect positions are indicated by black lines and lowercase letters in the elevation change panels (see also Fig. 7). Scour \nmarks in panels d and e are labelled SM1 through 4. Photo credit: Mitch Lemon, Cherokee Nations System Solutions for U.S. Geological \nSurvey. 18 Figure 7. Elevation transects across geomorphic features in 2016, 2017, and 2019. Geomorphic features included a sand wave (a, e\nscour marks (b, f), western reef rubble field (c, g), and a sand lobe (d, h). Lowercase letters indicate direction of transects as shown in Figur\n6. Vertical red lines indicate areas of erosion and vertical blue lines indicate areas of accretion between (a-d) 2016 and 2017 (before an\nafter Hurricane Irma) and between (e-f) 2017 and 2019. SM = scour mark. Figure 7. Elevation transects across geomorphic features in 2016, 2017, and 2019. Geomorphic features included a sand wave (a, e), \n370 \nscour marks (b, f), western reef rubble field (c, g), and a sand lobe (d, h). Lowercase letters indicate direction of transects as shown in Figure \n6. Vertical red lines indicate areas of erosion and vertical blue lines indicate areas of accretion between (a-d) 2016 and 2017 (before and \nafter Hurricane Irma) and between (e-f) 2017 and 2019. SM = scour mark. 3.2.2 Scour Marks \n375 Development of scour marks was observed in seagrass and unconsolidated sediment habitats in the back reef area of Looe Key \nReef between 2016 and 2017 (Fig. 6d, e, and f). These features ranged from approximately 30 to 60 m in length and width \nwith average depths of approximately 5.7 to 7.5 m in 2017. Visual validation of select scour features indicated they developed 19 mean elevation change of 0.10 m and a net volume change of less than 0.001 Mm3 (Tables 2 and 4). Area-normalized volume \n390 \nchange was approximately 0.12 Mm3 km-2. Scour mark 2 was 1,400 m2 with 88% of the area consisting of continuous seagrass \nand 12% unconsolidated sediment. Between 2016 and 2017, mean elevation change was -0.50 m (Table 2) with maximum \nobserved change of -1.28 m. Net volume change was less than -0.001 Mm3 and area-normalized volume change was \napproximately -0.53 Mm3 km-2 (Table 3). Ninety-four percent of net volume change was associated with continuous seagrass mean elevation change of 0.10 m and a net volume change of less than 0.001 Mm3 (Tables 2 and 4). Area-normalized volume \n390 \nchange was approximately 0.12 Mm3 km-2. Scour mark 2 was 1,400 m2 with 88% of the area consisting of continuous seagrass \nand 12% unconsolidated sediment. Between 2016 and 2017, mean elevation change was -0.50 m (Table 2) with maximum \nobserved change of -1.28 m. Net volume change was less than -0.001 Mm3 and area-normalized volume change was \napproximately -0.53 Mm3 km-2 (Table 3). Ninety-four percent of net volume change was associated with continuous seagrass habitat, which also had the highest area-normalized volume change of -0.56 Mm3 km-2. Between 2017 and 2019, this feature \n395 \nshowed accretion with mean elevation change of 0.20 m and net volume change of 0.0003 Mm3 (Tables 2 and 4). Continuous \nseagrass showed an increase in mean elevation (0.24 m) and net volume (0.0003 Mm3) while unconsolidated sediment showed \na decrease in mean elevation (-0.05 m) and net volume (less than -0.0001 Mm3). Area-normalized volume change across the \nentire scour mark was approximately 0.22 Mm3 km-2. Scour mark 3 was 1,882 m2 with 100% of the area consisting of habitat, which also had the highest area-normalized volume change of -0.56 Mm3 km-2. Between 2017 and 2019, this feature \n395 \nshowed accretion with mean elevation change of 0.20 m and net volume change of 0.0003 Mm3 (Tables 2 and 4). Continuous \nseagrass showed an increase in mean elevation (0.24 m) and net volume (0.0003 Mm3) while unconsolidated sediment showed \na decrease in mean elevation (-0.05 m) and net volume (less than -0.0001 Mm3). Area-normalized volume change across the \nentire scour mark was approximately 0.22 Mm3 km-2. Scour mark 3 was 1,882 m2 with 100% of the area consisting of continuous seagrass. https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 between 2016 and 2017 at the edges of seagrass beds where small (approximately 0.5 m) ledges marked the transition between \nthe slightly higher elevation of seagrass beds and lower elevation of adjacent unconsolidated sediment (Fig. 6f). Transect \n380 \nanalyses showed considerable erosion of the western boundaries of seagrass beds, development of pit-like features up to \napproximately 20 m in diameter and 1 m deep, transport of sediment westward, and burial of seagrass between scour features \n(Fig. 7b). Scour marks showed some infilling between 2017 and 2019. Validation imagery showed exposed rhizomatous \ngrowth at the western edges of seagrass beds (Fig. 8). 380 385 \nElevation- and volume-change analyses were performed on four examples of these features (Fig. 6d and e). Scour mark 1 was \n714 m2 with 99% of the area consisting of discontinuous seagrass. Between 2016 and 2017, mean elevation change was -0.49 \nm (Table 2) with a maximum observed change of -1.09 m. Net volume change was less than -0.001 Mm3, and area-normalized \nvolume change was approximately -0.51 Mm3 km-2 (Table 3). Between 2017 and 2019, this feature showed accretion with 385 \nElevation- and volume-change analyses were performed on four examples of these features (Fig. 6d and e). Scour mark 1 was \n714 m2 with 99% of the area consisting of discontinuous seagrass. Between 2016 and 2017, mean elevation change was -0.49 \nm (Table 2) with a maximum observed change of -1.09 m. Net volume change was less than -0.001 Mm3, and area-normalized \nvolume change was approximately -0.51 Mm3 km-2 (Table 3). Between 2017 and 2019, this feature showed accretion with Elevation- and volume-change analyses were performed on four examples of these features (Fig. 6d and e). Scour mark 1 was \n714 m2 with 99% of the area consisting of discontinuous seagrass. Between 2016 and 2017, mean elevation change was -0.49 \nm (Table 2) with a maximum observed change of -1.09 m. Net volume change was less than -0.001 Mm3, and area-normalized \nvolume change was approximately -0.51 Mm3 km-2 (Table 3). Between 2017 and 2019, this feature showed accretion with \nmean elevation change of 0.10 m and a net volume change of less than 0.001 Mm3 (Tables 2 and 4). Area-normalized volume \n390 \nchange was approximately 0.12 Mm3 km-2. Scour mark 2 was 1,400 m2 with 88% of the area consisting of continuous seagrass \nand 12% unconsolidated sediment. Between 2016 and 2017, mean elevation change was -0.50 m (Table 2) with maximum \nobserved change of -1.28 m. Net volume change was less than -0.001 Mm3 and area-normalized volume change was \napproximately -0.53 Mm3 km-2 (Table 3). Ninety-four percent of net volume change was associated with continuous seagrass \nhabitat, which also had the highest area-normalized volume change of -0.56 Mm3 km-2. Between 2017 and 2019, this feature \n395 \nshowed accretion with mean elevation change of 0.20 m and net volume change of 0.0003 Mm3 (Tables 2 and 4). Continuous \nseagrass showed an increase in mean elevation (0.24 m) and net volume (0.0003 Mm3) while unconsolidated sediment showed \na decrease in mean elevation (-0.05 m) and net volume (less than -0.0001 Mm3). Area-normalized volume change across the \nentire scour mark was approximately 0.22 Mm3 km-2. Scour mark 3 was 1,882 m2 with 100% of the area consisting of \ncontinuous seagrass. Between 2016 and 2017, mean elevation change was -0.50 m with a maximum observed change of -1.25 \n400 \nm (Table 2). Net volume change was -0.001 Mm3 and area-normalized volume change was approximately -0.52 Mm3 km-2 \n(Table 3). Between 2017 to 2019, this feature showed accretion with mean elevation change of 0.12 m and net volume change \nof 0.0003 Mm3 (Tables 2 and 4). Area-normalized volume change was approximately 0.14 Mm3 km-2. Scour mark 4 was 1,520 \nm2 with 99% of area consisting of continuous seagrass and 1% unconsolidated sediment. Between 2016 and 2017, mean \nelevation change was -0.54 m with a maximum observed change of -1.29 m (Table 2). Net volume change was -0.0009 Mm3 \n405 \nand area-normalized volume change was approximately -0.57 Mm3 km-2 (Table 3). Ninety-nine percent of net volume change \nwas associated with continuous seagrass habitat which also had the highest area-normalized volume change of -0.57 Mm3 km-\n2. Between 2017 to 2019, this feature showed accretion with mean elevation change of 0.12 m and net volume change of -\n0.0002 Mm3 (Tables 2 and 4). Area-normalized volume change was approximately 0.13 Mm3 km-2. More than 99% of net \nvolume change was associated with continuous seagrass habitat which also had the highest area-normalized volume change of \n410 \napproximately 0.14 Mm3 km-2. High erosion was noted between 2016 and 201\non the sand flat (western) side of the habitat transition and minimal accretion was noted on the seagrass bed (eastern) side of the habit Figure 8. Cardinal orientation imagery (a, b, d, and e represent north, west, east, and south, respectively) and elevation change (c) \nat a scour mark location used to validate benthic features observed in elevation change data. East and west arrows show the boundaries \n415 \nbetween seagrass beds and sand flats in the elevation change data (c) and imagery (b and d). High erosion was noted between 2016 and 2017 \non the sand flat (western) side of the habitat transition and minimal accretion was noted on the seagrass bed (eastern) side of the habitat \ntransition. Photo credit: Mitch Lemon, Cherokee Nations System Solutions for U.S. Geological Survey. 415 Between 2016 and 2017, mean elevation change was -0.50 m with a maximum observed change of -1.25 \n400 \nm (Table 2). Net volume change was -0.001 Mm3 and area-normalized volume change was approximately -0.52 Mm3 km-2 \n(Table 3). Between 2017 to 2019, this feature showed accretion with mean elevation change of 0.12 m and net volume change \nof 0.0003 Mm3 (Tables 2 and 4). Area-normalized volume change was approximately 0.14 Mm3 km-2. Scour mark 4 was 1,520 \nm2 with 99% of area consisting of continuous seagrass and 1% unconsolidated sediment. Between 2016 and 2017, mean elevation change was -0.54 m with a maximum observed change of -1.29 m (Table 2). Net volume change was -0.0009 Mm3 \n405 \nand area-normalized volume change was approximately -0.57 Mm3 km-2 (Table 3). Ninety-nine percent of net volume change \nwas associated with continuous seagrass habitat which also had the highest area-normalized volume change of -0.57 Mm3 km-\n2. Between 2017 to 2019, this feature showed accretion with mean elevation change of 0.12 m and net volume change of -\n0.0002 Mm3 (Tables 2 and 4). Area-normalized volume change was approximately 0.13 Mm3 km-2. More than 99% of net volume change was associated with continuous seagrass habitat which also had the highest area-normalized volume change of \n410 \napproximately 0.14 Mm3 km-2. 20 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 Figure 8. Cardinal orientation imagery (a, b, d, and e represent north, west, east, and south, respectively) and elevation change (c) \nat a scour mark location used to validate benthic features observed in elevation change data. East and west arrows show the boundaries \n415 \nbetween seagrass beds and sand flats in the elevation change data (c) and imagery (b and d). High erosion was noted between 2016 and 2017 \non the sand flat (western) side of the habitat transition and minimal accretion was noted on the seagrass bed (eastern) side of the habitat \ntransition. Photo credit: Mitch Lemon, Cherokee Nations System Solutions for U.S. Geological Survey. Figure 8. Cardinal orientation imagery (a, b, d, and e represent north, west, east, and south, respectively) and elevation change (\nat a scour mark location used to validate benthic features observed in elevation change data. East and west arrows show the boundarie\n415 \nbetween seagrass beds and sand flats in the elevation change data (c) and imagery (b and d). The adjacent erosion \n440 \narea (original 2016 location of the rubble field) showed a mean elevation change of -0.10 m (Table 2) with a maximum \nelevation loss of -0.52 m. Total net volume change was approximately -0.002 Mm3 and area-normalized volume change was \n-0.103 Mm3 km-2 with 53% of net volume change associated with discontinuous seagrass (Table 4). Highest mean elevation \nand area-normalized volume changes were also associated with discontinuous seagrass. Mean elevation and volume losses \ngenerally decreased with increasing mean habitat depth in the erosion area (Tables 2 and 4). 445 loss in mean elevation and area-normalized volume change and accounted for 59% of net volume change. The adjacent erosion \n440 \narea (original 2016 location of the rubble field) showed a mean elevation change of -0.10 m (Table 2) with a maximum \nelevation loss of -0.52 m. Total net volume change was approximately -0.002 Mm3 and area-normalized volume change was \n-0.103 Mm3 km-2 with 53% of net volume change associated with discontinuous seagrass (Table 4). Highest mean elevation \nand area-normalized volume changes were also associated with discontinuous seagrass. Mean elevation and volume losses \ngenerally decreased with increasing mean habitat depth in the erosion area (Tables 2 and 4). 445 3.2.3 Rubble Fields \n420 Migration of reef rubble fields was observed in areas north and northeast of Looe Key Reef between 2016 and 2017. The \nlargest of these features was approximately 418 m long and x 122 m wide at its widest point in 2017, 3 m in height from the \ncrest to base on the deepest (western) edge, with average water depth of approximately 3.3 m (Fig 6g, h, and i). Transect \nelevation profiles showed the location of this feature in 2016, westward migration of approximately 80 m (crest to crest) in Migration of reef rubble fields was observed in areas north and northeast of Looe Key Reef between 2016 and 2017. The \nlargest of these features was approximately 418 m long and x 122 m wide at its widest point in 2017, 3 m in height from the \ncrest to base on the deepest (western) edge, with average water depth of approximately 3.3 m (Fig 6g, h, and i). Transect \nelevation profiles showed the location of this feature in 2016, westward migration of approximately 80 m (crest to crest) in 2017, and minor eastward migration of 8 m (crest to crest) in 2019 (Fig. 7c). The accretion area of this feature covered an area \n425 \nof about 0.03 km2 including approximately 43% reef rubble, 49% discontinuous seagrass, and 9% unconsolidated sediment. Between 2016 and 2017, mean elevation change of the accretion area (2017 location of the rubble field) was 0.89 m (Table 2) \nwith a maximum elevation gain of 2.21 m. Total net volume change was 0.025 Mm3 and area-normalized volume change was 21 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 0.914 Mm3 km-2 with discontinuous seagrass accounting for 54% of net volume change indicating burial of seagrass during \nmigration of the rubble field (Table 3). Highest mean elevation and area-normalized volume changes were also associated with \n430 \ndiscontinuous seagrass habitat. An area of erosion (0.023 km2) was observed in 2017 at the original 2016 location of the rubble \nfield that was approximately 428 m long and x 78 m wide including 58% reef rubble, 31% discontinuous seagrass, and 11% \nunconsolidated sediment. Mean elevation-change of the erosion area between 2016 and 2017 was -0.63 m (Table 2) with a \nmaximum elevation loss of -2.11 m. Total net volume change was approximately -0.015 Mm3 and area-normalized volume \nchange was -0.661 Mm3 km-2 with 69% of net volume change associated with reef rubble (Table 3). Highest mean elevation \n435 \nand area-normalized volume changes were also associated with reef rubble. 0.914 Mm3 km-2 with discontinuous seagrass accounting for 54% of net volume change indicating burial of seagrass during \nmigration of the rubble field (Table 3). Highest mean elevation and area-normalized volume changes were also associated with \n430 \ndiscontinuous seagrass habitat. An area of erosion (0.023 km2) was observed in 2017 at the original 2016 location of the rubble \nfield that was approximately 428 m long and x 78 m wide including 58% reef rubble, 31% discontinuous seagrass, and 11% \nunconsolidated sediment. Mean elevation-change of the erosion area between 2016 and 2017 was -0.63 m (Table 2) with a \nmaximum elevation loss of -2.11 m. Total net volume change was approximately -0.015 Mm3 and area-normalized volume change was -0.661 Mm3 km-2 with 69% of net volume change associated with reef rubble (Table 3). Highest mean elevation \n435 \nand area-normalized volume changes were also associated with reef rubble. Between 2017 and 2019, the rubble field (accretion area) showed mean elevation change of -0.24 m, net volume change of -\n0.007 Mm3, and area-normalized volume change of -0.248 Mm3 km-2 (Tables 2 and 4). Discontinuous seagrass showed greatest 0.007 Mm3, and area-normalized volume change of -0.248 Mm3 km-2 (Tables 2 and 4). Discontinuous seagrass showed greatest \nloss in mean elevation and area-normalized volume change and accounted for 59% of net volume change. 3.2.4 Sand Lobe Substantial accretion was observed along a sand lobe located near the base of the fore-reef slope of Looe Key Reef between \n2016 and 2017 (Fig. 6j, k, and l). This feature was approximately 1,383 m long and 344 m wide (approximately 0.27 km2) at \nthe widest point with an average water depth of approximately 11.9 m in 2017 and included only unconsolidated sediment \nhabitat. Between 2016 and 2017, mean elevation change was 0.51 m (Table 2) with maximum gains in elevation up to 1.5 m \n450 \nalong the southern (seaward) downslope section of this feature and maximum elevation losses of -0.58 m along the northern \nlandward section, nearest to the base of the of the fore-reef slope (Fig. 7d). Total net volume change was 0.14 Mm3 and area-\nnormalized volume change was 0.51 Mm3 km-2 (Table 3). Between 2017and 2019, mean elevation change was -0.20 m with \nmaximum elevation losses up to -1.12 m (Table 2, Fig. 7d). Only 852 of 67,389 elevation points analysed for this feature \nshowed gains in elevation after 2017, averaging 0.05 m. Transect elevation profiles showed relatively consistent losses in \n455 \nelevation (erosion) across the sand lobe north to south (landward to seaward) during this time-period. Total net volume change \nwas -0.05 Mm3 and area-normalized volume change was -0.20 Mm3 km-2 (Table 4). Substantial accretion was observed along a sand lobe located near the base of the fore-reef slope of Looe Key Reef between \n2016 and 2017 (Fig. 6j, k, and l). This feature was approximately 1,383 m long and 344 m wide (approximately 0.27 km2) at \nthe widest point with an average water depth of approximately 11.9 m in 2017 and included only unconsolidated sediment (\ng\nj, ,\n)\npp\ny ,\ng\n( pp\ny\n)\nthe widest point with an average water depth of approximately 11.9 m in 2017 and included only unconsolidated sediment \nhabitat. Between 2016 and 2017, mean elevation change was 0.51 m (Table 2) with maximum gains in elevation up to 1.5 m \n450 \nalong the southern (seaward) downslope section of this feature and maximum elevation losses of -0.58 m along the northern \nlandward section, nearest to the base of the of the fore-reef slope (Fig. 7d). Total net volume change was 0.14 Mm3 and area-\nnormalized volume change was 0.51 Mm3 km-2 (Table 3). https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 3.2.4 Sand Lobe Between 2017and 2019, mean elevation change was -0.20 m with \nmaximum elevation losses up to -1.12 m (Table 2, Fig. 7d). Only 852 of 67,389 elevation points analysed for this feature \nshowed gains in elevation after 2017, averaging 0.05 m. Transect elevation profiles showed relatively consistent losses in \n455 \nelevation (erosion) across the sand lobe north to south (landward to seaward) during this time-period. Total net volume change \nwas -0.05 Mm3 and area-normalized volume change was -0.20 Mm3 km-2 (Table 4). habitat. Between 2016 and 2017, mean elevation change was 0.51 m (Table 2) with maximum gains in elevation up to 1.5 m \n450 \nalong the southern (seaward) downslope section of this feature and maximum elevation losses of -0.58 m along the northern \nlandward section, nearest to the base of the of the fore-reef slope (Fig. 7d). Total net volume change was 0.14 Mm3 and area-\nnormalized volume change was 0.51 Mm3 km-2 (Table 3). Between 2017and 2019, mean elevation change was -0.20 m with \nmaximum elevation losses up to -1.12 m (Table 2, Fig. 7d). Only 852 of 67,389 elevation points analysed for this feature showed gains in elevation after 2017, averaging 0.05 m. Transect elevation profiles showed relatively consistent losses in\n455 \nelevation (erosion) across the sand lobe north to south (landward to seaward) during this time-period. Total net volume change\nwas -0.05 Mm3 and area-normalized volume change was -0.20 Mm3 km-2 (Table 4). 22 4 Discussion Our 2016 to 2017 elevation change results showed general movement of sediment \nand migration of major geomorphic features from ENE to WSW in shallow areas (ranging from approximately 2 to 5.5 m \nwater depth in 2016) of the reef proper and back reef area, consistent with the direction of sustained, high magnitude winds \nduring the passing of Hurricane Irma (Fig. 4; Fig. 6a, d, and g). For example, large sand waves and rubble fields (approximately \n475 \n0.02 to 0.06 km2 in area) migrated westward approximately 80 m (Fig. 6a and g) causing burial of seagrass habitat. Scour \nmarks developed due to erosion of the western edges of seagrass beds and westward transport of sediment, causing burial of \nadjacent seagrass beds between scour marks (Fig. 6d). Numerical modelling of the impact of hurricane-induced wave-current \ninteractions on the transport of material along the FRT during Hurricane Irma showed that wave radiation stress primarily \naffected particle transport trajectories during the passage of the hurricane (Dobbelaere et al., 2022). Additionally, wave energy \n480 \ndissipation occurred through depth-induced wave breaking and bottom dissipation at the shelf break and over the coral reefs. Furthermore, after the passage of the hurricane, suspended particles were transported northeastward by the Florida Current \n(Fig. 1d) and were advected (via Stokes drift) from the outer shelf to inshore for approximately 2 days (Dobbelaere et al., storm and, therefore, included any persistent change that occurred during quiescent sea state conditions before and after the \n465 \npassing of Irma. However, observations during several rapid reef assessments after the storm indicated broad-scale sediment \ndeposition as a direct result of Hurricane Irma (Viehman et al., 2018; Walker, 2018; Wilson et al., 2020; Kobelt et al. 2019), \nwhich corroborates our findings of increased mean elevation and sediment accretion resulting from this storm event. Furthermore, wind conditions were relatively quiescent from the 2016 lidar acquisition date up to the passing of Hurricane storm and, therefore, included any persistent change that occurred during quiescent sea state conditions before and after the \n465 \npassing of Irma. However, observations during several rapid reef assessments after the storm indicated broad-scale sediment \ndeposition as a direct result of Hurricane Irma (Viehman et al., 2018; Walker, 2018; Wilson et al., 2020; Kobelt et al. 2019), \nwhich corroborates our findings of increased mean elevation and sediment accretion resulting from this storm event. 4 Discussion Furthermore, wind conditions were relatively quiescent from the 2016 lidar acquisition date up to the passing of Hurricane Irma and after the storm, and historical aerial imagery of LKR from 2014 and 18 March 2017 (3 years and 6 months prior to \n470 \nHurricane Irma, respectively, Fig. 9) show that patterns of major sedimentary features were mostly static (Finkl and Vollmer, \n2017) in the few years prior to the storm. Our 2016 to 2017 elevation change results showed general movement of sediment \nand migration of major geomorphic features from ENE to WSW in shallow areas (ranging from approximately 2 to 5.5 m \nwater depth in 2016) of the reef proper and back reef area, consistent with the direction of sustained, high magnitude winds Irma and after the storm, and historical aerial imagery of LKR from 2014 and 18 March 2017 (3 years and 6 months prior to \n470 \nHurricane Irma, respectively, Fig. 9) show that patterns of major sedimentary features were mostly static (Finkl and Vollmer, \n2017) in the few years prior to the storm. Our 2016 to 2017 elevation change results showed general movement of sediment \nand migration of major geomorphic features from ENE to WSW in shallow areas (ranging from approximately 2 to 5.5 m \nwater depth in 2016) of the reef proper and back reef area, consistent with the direction of sustained, high magnitude winds Irma and after the storm, and historical aerial imagery of LKR from 2014 and 18 March 2017 (3 years and 6 months prior to \n470 \nHurricane Irma, respectively, Fig. 9) show that patterns of major sedimentary features were mostly static (Finkl and Vollmer, \n2017) in the few years prior to the storm. Our 2016 to 2017 elevation change results showed general movement of sediment \nand migration of major geomorphic features from ENE to WSW in shallow areas (ranging from approximately 2 to 5.5 m \nwater depth in 2016) of the reef proper and back reef area, consistent with the direction of sustained, high magnitude winds during the passing of Hurricane Irma (Fig. 4; Fig. 6a, d, and g). For example, large sand waves and rubble fields (approximately \n475 \n0.02 to 0.06 km2 in area) migrated westward approximately 80 m (Fig. 6a and g) causing burial of seagrass habitat. 4 Discussion There are few comprehensive assessments of the effects of major hurricanes on seafloor elevation and geomorphology on coral \nreefs; and no quantitative studies of reef-scale seafloor elevation change resulting from tropical storm impacts have previously \n460 \nbeen conducted in the Florida Keys. Our results showed Hurricane Irma was primarily a depositional event that increased mean \nseafloor elevation and volume over a 15.98 km2 section of Looe Key Reef by 0.34 m (annualized elevation-change rate of up \nto 247 mm yr-1) and up to 5.4 Mm3, respectively, with area-normalized volume change of approximately 0.34 Mm3km-2. Our \nobservations were based on elevation measurements collected 13.5 months before the storm and three to six months after the There are few comprehensive assessments of the effects of major hurricanes on seafloor elevation and geomorphology on coral \nreefs; and no quantitative studies of reef-scale seafloor elevation change resulting from tropical storm impacts have previously \n460 \nbeen conducted in the Florida Keys. Our results showed Hurricane Irma was primarily a depositional event that increased mean \nseafloor elevation and volume over a 15.98 km2 section of Looe Key Reef by 0.34 m (annualized elevation-change rate of up \nto 247 mm yr-1) and up to 5.4 Mm3, respectively, with area-normalized volume change of approximately 0.34 Mm3km-2. Our \nobservations were based on elevation measurements collected 13.5 months before the storm and three to six months after the \nstorm and, therefore, included any persistent change that occurred during quiescent sea state conditions before and after the \n465 \npassing of Irma. However, observations during several rapid reef assessments after the storm indicated broad-scale sediment \ndeposition as a direct result of Hurricane Irma (Viehman et al., 2018; Walker, 2018; Wilson et al., 2020; Kobelt et al. 2019), \nwhich corroborates our findings of increased mean elevation and sediment accretion resulting from this storm event. Furthermore, wind conditions were relatively quiescent from the 2016 lidar acquisition date up to the passing of Hurricane \nIrma and after the storm, and historical aerial imagery of LKR from 2014 and 18 March 2017 (3 years and 6 months prior to \n470 \nHurricane Irma, respectively, Fig. 9) show that patterns of major sedimentary features were mostly static (Finkl and Vollmer, \n2017) in the few years prior to the storm. 4 Discussion Scour \nmarks developed due to erosion of the western edges of seagrass beds and westward transport of sediment, causing burial of \nadjacent seagrass beds between scour marks (Fig. 6d). Numerical modelling of the impact of hurricane-induced wave-current \ninteractions on the transport of material along the FRT during Hurricane Irma showed that wave radiation stress primarily during the passing of Hurricane Irma (Fig. 4; Fig. 6a, d, and g). For example, large sand waves and rubble fields (approximately \n475 \n0.02 to 0.06 km2 in area) migrated westward approximately 80 m (Fig. 6a and g) causing burial of seagrass habitat. Scour \nmarks developed due to erosion of the western edges of seagrass beds and westward transport of sediment, causing burial of \nadjacent seagrass beds between scour marks (Fig. 6d). Numerical modelling of the impact of hurricane-induced wave-current \ninteractions on the transport of material along the FRT during Hurricane Irma showed that wave radiation stress primarily affected particle transport trajectories during the passage of the hurricane (Dobbelaere et al., 2022). Additionally, wave energy \n480 \ndissipation occurred through depth-induced wave breaking and bottom dissipation at the shelf break and over the coral reefs. Furthermore, after the passage of the hurricane, suspended particles were transported northeastward by the Florida Current \n(Fig. 1d) and were advected (via Stokes drift) from the outer shelf to inshore for approximately 2 days (Dobbelaere et al., \n2022). affected particle transport trajectories during the passage of the hurricane (Dobbelaere et al., 2022). Additionally, wave energy \n480 \ndissipation occurred through depth-induced wave breaking and bottom dissipation at the shelf break and over the coral reefs. Furthermore, after the passage of the hurricane, suspended particles were transported northeastward by the Florida Current \n(Fig. 1d) and were advected (via Stokes drift) from the outer shelf to inshore for approximately 2 days (Dobbelaere et al., \n2022). 485 \nSimilar geomorphic seafloor changes have been documented for other category 4 hurricanes in the Florida Keys based on \nphotographic air and ground surveys, maps, sediment cores, and bottom markers. 4 Discussion In 1967, Hurricane Donna approached from \nthe southeast and passed over the central islands of the Florida Keys in September 1960 with sustained winds of 226 km h-1 \n(category 4) and with breaking waves and storm currents causing broken coral rubble up to a meter in diameter, shoreward 485 \nSimilar geomorphic seafloor changes have been documented for other category 4 hurricanes in the Florida Keys based on \nphotographic air and ground surveys, maps, sediment cores, and bottom markers. In 1967, Hurricane Donna approached from \nthe southeast and passed over the central islands of the Florida Keys in September 1960 with sustained winds of 226 km h-1 \n(category 4) and with breaking waves and storm currents causing broken coral rubble up to a meter in diameter, shoreward \ntransport of gravel to boulder sized rubble and sand approximately 60 to 150 m shoreward, and burial of seagrass with 15 cm \n490 485 \nSimilar geomorphic seafloor changes have been documented for other category 4 hurricanes in the Florida Keys based on \nphotographic air and ground surveys, maps, sediment cores, and bottom markers. In 1967, Hurricane Donna approached from \nthe southeast and passed over the central islands of the Florida Keys in September 1960 with sustained winds of 226 km h-1 \n(category 4) and with breaking waves and storm currents causing broken coral rubble up to a meter in diameter, shoreward \ntransport of gravel to boulder sized rubble and sand approximately 60 to 150 m shoreward, and burial of seagrass with 15 cm \n490 transport of gravel to boulder sized rubble and sand approximately 60 to 150 m shoreward, and burial of seagrass with 15 cm \n490 23 Figure 9. Historical satellite and aerial imagery of Looe Key Reef. Imagery from (a) 17 December 2014, before Hurricane Irma; (b) 18\nMarch 2017, before Hurricane Irma; (c) 30 December 2017, 3 months after Hurricane Irma; (d) 2019, 16.5 months after Hurricane Irma; \n2023. Panel d source: 2019 NOAA National Geodetic Survey via NOAA Digital Coast, downloaded 11 September 2023, \nhttps://www.fisheries.noaa.gov/inport/item/63292. and (e) from 1975 (Lidz et al., 2016). Panels a, b, and c source: Maxar 2023 via © Google Earth Pro 7.3.6.9345, downloaded 11 Septembe Figure 9. Historical satellite and aerial imagery of Looe Key Reef. 4 Discussion Imagery from (a) 17 December 2014, before Hurricane Irma; (b) 18 \nMarch 2017, before Hurricane Irma; (c) 30 December 2017, 3 months after Hurricane Irma; (d) 2019, 16.5 months after Hurricane Irma; \n2023. Panel d source: 2019 NOAA National Geodetic Survey via NOAA Digital Coast, downloaded 11 September 2023, \nhttps://www.fisheries.noaa.gov/inport/item/63292. 495 \nand (e) from 1975 (Lidz et al., 2016). Panels a, b, and c source: Maxar 2023 via © Google Earth Pro 7.3.6.9345, downloaded 11 September of sediment (Ball et al., 1967). Hurricane Betsy approached from the west and passed over the Florida Keys approximately 25 \nkm north of Hurricane Donna’s landfall in September 1965 with sustained winds of up to 226 km h-1. While both storms had \n500 \nsimilar destructive effects to corals on the outer reefs, Hurricane Betsy produced less rubble, showed an overall effect of \nerosion and recycling of sediment in the environment, and caused sediment plumes from the mainland to the edge of the Gulf km north of Hurricane Donna’s landfall in September 1965 with sustained winds of up to 226 km h-1. While both storms had \n500 \nsimilar destructive effects to corals on the outer reefs, Hurricane Betsy produced less rubble, showed an overall effect of \nerosion and recycling of sediment in the environment, and caused sediment plumes from the mainland to the edge of the Gulf 24 Fourqurean and Rutten (2004) showed that seagrass recovery was slowest at sites that were \neroded; losses by mechanical thinning and burial with only a few centimeters of sediment recovered quickly; and seagrass \nburied with 10s of centimeters of sediment hadn’t recovered by three years after the storm. Results from these studies show \n515 \nthe variability in storm impacts due complex interactions among factors such as location, fetch, wind speed, duration, storm \nhistory, and water depth (Fourqurean and Rutten, 2004), and demonstrate the value of comprehensive, quantitative post storm \nassessments of geological and ecological impacts. with maximum sustained winds of only 145 km h-1. However, data from 30 seagrass monitoring transects showed a 3% decline \n510 \nin density of Thalassia testudinum and 19% decline in density of Syringodium filiforme seagrasses, with complete loss of \nseagrass beds at 3 monitoring stations, burial of one station with 50cm of sediment, substantial erosion at two stations \n(Fourqurean and Rutten, 2004). Fourqurean and Rutten (2004) showed that seagrass recovery was slowest at sites that were \neroded; losses by mechanical thinning and burial with only a few centimeters of sediment recovered quickly; and seagrass with maximum sustained winds of only 145 km h-1. However, data from 30 seagrass monitoring transects showed a 3% decline \n510 \nin density of Thalassia testudinum and 19% decline in density of Syringodium filiforme seagrasses, with complete loss of \nseagrass beds at 3 monitoring stations, burial of one station with 50cm of sediment, substantial erosion at two stations \n(Fourqurean and Rutten, 2004). Fourqurean and Rutten (2004) showed that seagrass recovery was slowest at sites that were \neroded; losses by mechanical thinning and burial with only a few centimeters of sediment recovered quickly; and seagrass buried with 10s of centimeters of sediment hadn’t recovered by three years after the storm. Results from these studies show \n515 \nthe variability in storm impacts due complex interactions among factors such as location, fetch, wind speed, duration, storm \nhistory, and water depth (Fourqurean and Rutten, 2004), and demonstrate the value of comprehensive, quantitative post storm \nassessments of geological and ecological impacts. buried with 10s of centimeters of sediment hadn’t recovered by three years after the storm. Results from these studies show \n515 \nthe variability in storm impacts due complex interactions among factors such as location, fetch, wind speed, duration, storm \nhistory, and water depth (Fourqurean and Rutten, 2004), and demonstrate the value of comprehensive, quantitative post storm \nassessments of geological and ecological impacts. 520 \nA previous analysis of seafloor elevation change at LKR during the decade prior to Hurricane Irma (from 2004–2016, during \nwhich only one minor tropical storm impacted this location in 2008) indicated an increase in mean elevation of 0.39 m \n(annualized elevation-change rate of 32.5 mm yr-1), net volume gain of up to 6.4 Mm3 and area-normalized volume change of \n0.39 Mm3 km-2, with accretion observed across all habitat types and some WSW movement of sand waves (Yates et al., 2019). 520 \nA previous analysis of seafloor elevation change at LKR during the decade prior to Hurricane Irma (from 2004–2016, during \nwhich only one minor tropical storm impacted this location in 2008) indicated an increase in mean elevation of 0.39 m \n(annualized elevation-change rate of 32.5 mm yr-1), net volume gain of up to 6.4 Mm3 and area-normalized volume change of \n0.39 Mm3 km-2, with accretion observed across all habitat types and some WSW movement of sand waves (Yates et al., 2019). 520 \nA previous analysis of seafloor elevation change at LKR during the decade prior to Hurricane Irma (from 2004–2016, during \nwhich only one minor tropical storm impacted this location in 2008) indicated an increase in mean elevation of 0.39 m \n(annualized elevation-change rate of 32.5 mm yr-1), net volume gain of up to 6.4 Mm3 and area-normalized volume change of \n0.39 Mm3 km-2, with accretion observed across all habitat types and some WSW movement of sand waves (Yates et al., 2019). Our results showed that sediment deposited during the approximately 16.5 to 19.5-month time-period including impacts from \n525 \nHurricane Irma caused changes in seafloor elevation and volume across all habitat types similar in magnitude to net changes \nobserved over the past decade and at accumulation rates one order of magnitude greater. Previous studies on several coral reefs \naround St. Croix, U.S. https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 Stream for several days after the Hurricane passed (Perkins and Enos, 1968). Perkins and Enos (1968) noted the difference in \nwind directions for the two storms caused different effects, and that it is difficult to extrapolate quantitative sedimentation rates \nfrom the sedimentary record of one hurricane and frequency of recorded hurricanes. Hurricane Andrew made landfall along \n505 \nthe southeast coast of Florida just south of Miami also with sustained winds of 226 km h-1 with maximum wave heights of less \nthan 2 m. Branching corals were broken, massive coral heads were toppled, seafans and sponges were ripped loose, and shallow \nreefs sustained the most damage (Orr and Ogden, 1992); however, there was little damage to seagrass beds immediately \nseaward of coastal mangroves (Tilmant et al., 1994). Hurricane Georges was a category 2 storm that passed over Key West Stream for several days after the Hurricane passed (Perkins and Enos, 1968). Perkins and Enos (1968) noted the difference in \nwind directions for the two storms caused different effects, and that it is difficult to extrapolate quantitative sedimentation rates \nfrom the sedimentary record of one hurricane and frequency of recorded hurricanes. Hurricane Andrew made landfall along \n505 \nthe southeast coast of Florida just south of Miami also with sustained winds of 226 km h-1 with maximum wave heights of less \nthan 2 m. Branching corals were broken, massive coral heads were toppled, seafans and sponges were ripped loose, and shallow \nreefs sustained the most damage (Orr and Ogden, 1992); however, there was little damage to seagrass beds immediately \nseaward of coastal mangroves (Tilmant et al., 1994). Hurricane Georges was a category 2 storm that passed over Key West \nwith maximum sustained winds of only 145 km h-1. However, data from 30 seagrass monitoring transects showed a 3% decline \n510 \nin density of Thalassia testudinum and 19% decline in density of Syringodium filiforme seagrasses, with complete loss of \nseagrass beds at 3 monitoring stations, burial of one station with 50cm of sediment, substantial erosion at two stations \n(Fourqurean and Rutten, 2004). https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0.\nl\n.\n0 Virgin Islands showed that physical transport of sediment is primarily due to wave-induced oscillatory \nand unidirectional currents, and that storms can increase sediment transport by an order of magnitude higher than during non- Our results showed that sediment deposited during the approximately 16.5 to 19.5-month time-period including impacts from \n525 \nHurricane Irma caused changes in seafloor elevation and volume across all habitat types similar in magnitude to net changes \nobserved over the past decade and at accumulation rates one order of magnitude greater. Previous studies on several coral reefs \naround St. Croix, U.S. Virgin Islands showed that physical transport of sediment is primarily due to wave-induced oscillatory \nand unidirectional currents, and that storms can increase sediment transport by an order of magnitude higher than during non- storm conditions (Hubbard et al., 1981; Hubbard, 1986). Measurements from 15 locations around St. Croix showed sediment \n530 \ntransport rates ranging from 0.009 to 0.3 Mm3 km-2yr-1 during non-storm conditions, and 0.09 to 1.5 Mm3 km-2yr-1 during storm \nconditions (Hubbard et al., 1981; Yates et al., 2017). Sediment trap studies along the southwest coast of Puerto Rico showed \nmedian sediment accumulation rates increased by an order of magnitude (from approximately 6 to 68 mg m-2 d-1) after the \npassage of Hurricane Maria in September of 2017 (a category 4 storm) and a large October 2017 storm that caused resuspension storm conditions (Hubbard et al., 1981; Hubbard, 1986). Measurements from 15 locations around St. Croix showed sediment \n530 \ntransport rates ranging from 0.009 to 0.3 Mm3 km-2yr-1 during non-storm conditions, and 0.09 to 1.5 Mm3 km-2yr-1 during storm \nconditions (Hubbard et al., 1981; Yates et al., 2017). Sediment trap studies along the southwest coast of Puerto Rico showed \nmedian sediment accumulation rates increased by an order of magnitude (from approximately 6 to 68 mg m-2 d-1) after the \npassage of Hurricane Maria in September of 2017 (a category 4 storm) and a large October 2017 storm that caused resuspension 25 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 which is considered heavy sedimentation and has been associated with fewer coral species, less live coral, lower coral growth \nrates, reduced coral recruitment and calcification rates, and slower rates of reef accretion (Rogers, 1990). Mean elevation- and area-normalized volume-change from 2016–2017 for habitats examined in our study increased \nsignificantly with water depth suggesting that, in addition to broad-scale sediment deposition across the study site, sediment \n540 \nwas also transported from shallower to deeper habitats (Fig. 5a and b). Notably, greatest increases in elevation (accretion) were \nassociated with habitats in water depths exceeding 11 m including aggregate reef, a sand lobe consisting of unconsolidated \nsediment, and ‘not classified’ habitat located seaward and near the base of the reef’s spur-and-groove formation, suggesting \nsome movement of sediment offshore and downslope (Fig. 4a, Table 2). Additionally, erosion was observed in the shallower, upslope grooves of the spur-and-groove formation, and accretion was observed in the deeper, downslope areas of the grooves \n545 \nfrom 2016 to 2017 further suggesting downslope, offshore movement of sediments (Fig. 10). The sand lobe at the base of the \nspur and groove formation also showed upslope erosion and considerable downslope (seaward) accretion, further suggesting \noffshore transport of sediments (Fig. 7d). Our observations are consistent with previous bathymetric change analyses \nconducted along the northern FRT from 2001 to 2008 (approximately 3 years before Hurricane Ivan and 3 years after Hurricane Katrina) that showed movement of up to 1.8 Mm3 of sediment between these time periods and transport of sediment from the \n550 \ninner shelf to offshore and beyond the shelf edge through gaps in the barrier reef and diabathic (cross-shore) channels during \nhigh-energy events or when the back reef overfills with sand (Finkl, 2004; Finkl and Vollmer, 2017). These observations are \nalso consistent with results of Yates et al. (2017) that show a multi-decadal trend along the FRT of reef sediment transport \ndown the fore-reef-slope and export offshore. Field observations of currents, waves, and reef sediment grain-size analyses Katrina) that showed movement of up to 1.8 Mm3 of sediment between these time periods and transport of sediment from the \n550 \ninner shelf to offshore and beyond the shelf edge through gaps in the barrier reef and diabathic (cross-shore) channels during \nhigh-energy events or when the back reef overfills with sand (Finkl, 2004; Finkl and Vollmer, 2017). These observations are \nalso consistent with results of Yates et al. (2017) that show a multi-decadal trend along the FRT of reef sediment transport \ndown the fore-reef-slope and export offshore. Field observations of currents, waves, and reef sediment grain-size analyses Katrina) that showed movement of up to 1.8 Mm3 of sediment between these time periods and transport of sediment from the \n550 \ninner shelf to offshore and beyond the shelf edge through gaps in the barrier reef and diabathic (cross-shore) channels during \nhigh-energy events or when the back reef overfills with sand (Finkl, 2004; Finkl and Vollmer, 2017). These observations are \nalso consistent with results of Yates et al. (2017) that show a multi-decadal trend along the FRT of reef sediment transport \ndown the fore-reef-slope and export offshore. Field observations of currents, waves, and reef sediment grain-size analyses coupled with integrated ocean-atmosphere-wave-sediment transport modelling during a one-year study at Crocker reef in the \n555 \nUpper Florida Keys showed that sediment mobility was primarily driven by wave stress exceeding critical shear stress; current \nstress alone only exceeded the critical shear stress for sediment mobility 5% of the time usually due to Florida Current eddies \n(Torres-Garcia et al., 2018). Torres-Garcia (2018) showed that nonbreaking wave stress (characteristic of quiescent sea states) \nmobilizes sand approximately 23 to 59% of the time; and fine-grained material is winnowed from the shallow areas of the reef and deposited to the flanks and offshore, particularly to the southwest. Furthermore, the critical stress threshold of gravel-sized \n560 \nmaterial was exceeded only 1 to 13% of the time, particularly during near-field tropical storm conditions (similar to Hurricane \nWilma, a category 3 hurricane) that cause breaking waves, mobilize and transport gravel material, and can cause physical reef \ndegradation (Torres-Garcia et al., 2018). https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 570 \n \nFigure 10. Elevation-change along Looe Key Reef spur and groove formation. (a) Upslope to downslope transects along Looe Key\nReef spur and groove formation (green lines); image source: 2019 NOAA National Geodetic Survey via NOAA Digital Coast, downlo\n11 September 2023, https://www.fisheries.noaa.gov/inport/item/63292 with structure from motion overlay image of Hatcher et al. (202\nAreas of erosion (red circles) and accretion (blue circles) along transect 1 between (b) 2016 and 2017, and between (c) 2017 and 2019. 575 \nAreas of erosion (red circles) and accretion (blue circles) along transect 2 between (d) 2016 and 2017, and between (e) 2017 and 2019. Elevation profiles from 2016, 2017, and 2019 for (f) transect 1 and (g) transect 2. Vertical red lines indicate net erosion and vertical blu\nlines indicate net accretion between 2016 and 2017. 570 Figure 10. Elevation-change along Looe Key Reef spur and groove formation. (a) Upslope to downslope transects along Looe Key \nReef spur and groove formation (green lines); image source: 2019 NOAA National Geodetic Survey via NOAA Digital Coast, downloaded \n11 September 2023, https://www.fisheries.noaa.gov/inport/item/63292 with structure from motion overlay image of Hatcher et al. (2022). Areas of erosion (red circles) and accretion (blue circles) along transect 1 between (b) 2016 and 2017, and between (c) 2017 and 2019. 575 \nAreas of erosion (red circles) and accretion (blue circles) along transect 2 between (d) 2016 and 2017, and between (e) 2017 and 2019. Elevation profiles from 2016, 2017, and 2019 for (f) transect 1 and (g) transect 2. Vertical red lines indicate net erosion and vertical blue \nlines indicate net accretion between 2016 and 2017. Figure 10. Elevation-change along Looe Key Reef spur and groove formation. (a) Upslope to downslope transects along Looe Key \nReef spur and groove formation (green lines); image source: 2019 NOAA National Geodetic Survey via NOAA Digital Coast, downloaded \n11 September 2023, https://www.fisheries.noaa.gov/inport/item/63292 with structure from motion overlay image of Hatcher et al. (2022). Areas of erosion (red circles) and accretion (blue circles) along transect 1 between (b) 2016 and 2017, and between (c) 2017 and 2019. 575 \nAreas of erosion (red circles) and accretion (blue circles) along transect 2 between (d) 2016 and 2017, and between (e) 2017 and 2019. Elevation profiles from 2016, 2017, and 2019 for (f) transect 1 and (g) transect 2. Southwest counter currents due to the formation of Florida Current eddies (Lee and \nWilliams, 1999) and WSW movement of sand wave features over a decadal time-period (Yates et al., 2019b) have also been and deposited to the flanks and offshore, particularly to the southwest. Furthermore, the critical stress threshold of gravel-sized \n560 \nmaterial was exceeded only 1 to 13% of the time, particularly during near-field tropical storm conditions (similar to Hurricane \nWilma, a category 3 hurricane) that cause breaking waves, mobilize and transport gravel material, and can cause physical reef \ndegradation (Torres-Garcia et al., 2018). Southwest counter currents due to the formation of Florida Current eddies (Lee and \nWilliams, 1999) and WSW movement of sand wave features over a decadal time-period (Yates et al., 2019b) have also been observed near LKR. Results from these previous studies suggest that some sediment transport observed in our study could be \n565 \ndue to persistent transport of sand during quiescent sea state conditions; however, the large volume of material transported \n(including gravel-sized and larger reef rubble) during the short time-period of our study from 2016 to 2017 was likely due \nprimarily to storm conditions caused by Hurricane Irma. observed near LKR. Results from these previous studies suggest that some sediment transport observed in our study could be \n565 \ndue to persistent transport of sand during quiescent sea state conditions; however, the large volume of material transported \n(including gravel-sized and larger reef rubble) during the short time-period of our study from 2016 to 2017 was likely due \nprimarily to storm conditions caused by Hurricane Irma. observed near LKR. Results from these previous studies suggest that some sediment transport observed in our study could be \n565 \ndue to persistent transport of sand during quiescent sea state conditions; however, the large volume of material transported \n(including gravel-sized and larger reef rubble) during the short time-period of our study from 2016 to 2017 was likely due \nprimarily to storm conditions caused by Hurricane Irma. 26 Vertical red lines indicate net erosion and vertical blue \nlines indicate net accretion between 2016 and 2017. 580 27 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 Approximately 16.5 months after Hurricane Irma (during a 13-month period between 2017 and 2019), net erosion was observed \nacross all habitats with mean elevation-change of -0.15 (annualized elevation change-rate of -139 mm yr-1), net volume change \nup to -2.46 Mm3, and area-normalized volume change of -0.15 Mm3km-2. Newly deposited carbonate sediments typically have \nporosities of 40 to 70% (Choquette and Pray, 1970) at shallow sediment depths of a few hundreds of meters (Schmoker and Halley, 1982). Porosity of carbonate sands on the FRT and in Hawk Channel ranges from 60 to 72% in the upper 22 cm of \n585 \ndeposited sediment (Walter et al. 2007). Schmoker and Halley (1982) showed that there is little or no sediment porosity loss \nat near-surface sediment depths. Application of their exponential function for porosity versus depth of sediment (porosity (%) \n= 41.73e-z/2498, where z = depth below sediment surface) indicates that the decrease in porosity of deposited carbonate sediments \nat 2 m below the sediment-surface is only 0.03%. Carbonate sands have settling velocities ranging from 0.025 to 0.364 m s-1 Halley, 1982). Porosity of carbonate sands on the FRT and in Hawk Channel ranges from 60 to 72% in the upper 22 cm of \n585 \ndeposited sediment (Walter et al. 2007). Schmoker and Halley (1982) showed that there is little or no sediment porosity loss \nat near-surface sediment depths. Application of their exponential function for porosity versus depth of sediment (porosity (%) \n= 41.73e-z/2498, where z = depth below sediment surface) indicates that the decrease in porosity of deposited carbonate sediments \nat 2 m below the sediment-surface is only 0.03%. Carbonate sands have settling velocities ranging from 0.025 to 0.364 m s-1 585 (Riazi et al., 2020). Satellite imagery shows the sediment plume caused by resuspension of sediment during Hurricane Irma \n590 \ncleared within approximately 5 days of the storm’s passing (NASA, 2023). Therefore, it is likely that resuspended sediment \nsettled quickly (within days) when storm conditions subsided; and it is unlikely that the decrease in elevation observed between \n2017 and 2019 was caused by compaction of sediment after the storm. This suggests that approximately 50% of sediment \ndeposited between 2016 and 2017 was eroded by 2019 due to physical transport away from the study site. The sand wave and (Riazi et al., 2020). Satellite imagery shows the sediment plume caused by resuspension of sediment during Hurricane Irma \n590 \ncleared within approximately 5 days of the storm’s passing (NASA, 2023). Therefore, it is likely that resuspended sediment \nsettled quickly (within days) when storm conditions subsided; and it is unlikely that the decrease in elevation observed between \n2017 and 2019 was caused by compaction of sediment after the storm. This suggests that approximately 50% of sediment \ndeposited between 2016 and 2017 was eroded by 2019 due to physical transport away from the study site. The sand wave and reef rubble field showed continued erosion between 2017 to 2019 with some evidence for migration of the crest of the rubble \n595 \nfield back toward its original 2016 position indicated in the elevation profile (Fig. 6 and 7). Shallow areas between the scour \nmarks showed erosion, while the scour mark pits showed infilling (Fig. 6 and 7). Spurs of the spur-and-groove formation \nprimarily showed erosion, while shallow (landward) sections of grooves showed some accretion, likely due to transport of \nsediments from spurs to grooves and downslope from the shallow reef (Fig. 10). Deeper (seaward) areas of grooves and the reef rubble field showed continued erosion between 2017 to 2019 with some evidence for migration of the crest of the rubble \n595 \nfield back toward its original 2016 position indicated in the elevation profile (Fig. 6 and 7). Shallow areas between the scour \nmarks showed erosion, while the scour mark pits showed infilling (Fig. 6 and 7). Spurs of the spur-and-groove formation \nprimarily showed erosion, while shallow (landward) sections of grooves showed some accretion, likely due to transport of \nsediments from spurs to grooves and downslope from the shallow reef (Fig. 10). Deeper (seaward) areas of grooves and the reef rubble field showed continued erosion between 2017 to 2019 with some evidence for migration of the crest of the rubble \n595 \nfield back toward its original 2016 position indicated in the elevation profile (Fig. 6 and 7). Shallow areas between the scour \nmarks showed erosion, while the scour mark pits showed infilling (Fig. 6 and 7). Spurs of the spur-and-groove formation \nprimarily showed erosion, while shallow (landward) sections of grooves showed some accretion, likely due to transport of \nsediments from spurs to grooves and downslope from the shallow reef (Fig. 10). Deeper (seaward) areas of grooves and the sand lobe located at the base of the spur and groove formation showed erosion (Fig. 4 and 6k) suggesting continued downslope, \n600 \noffshore transport of sediments. Historical aerial and satellite imagery from before and after the passing of Hurricane Irma \ncorroborates our elevation-change observations (Fig. 9). Imagery from 2014 and March 2017 shows that major geomorphic \nfeatures of Looe Key proper such as distribution of seagrass beds and the size and position of the sand lobe and rubble fields \nwere relatively static between these time periods leading up to Hurricane Irma (Fig. 9a and b). Imagery from December 2017, sand lobe located at the base of the spur and groove formation showed erosion (Fig. 4 and 6k) suggesting continued downslope, \n600 \noffshore transport of sediments. Historical aerial and satellite imagery from before and after the passing of Hurricane Irma \ncorroborates our elevation-change observations (Fig. 9). Imagery from 2014 and March 2017 shows that major geomorphic \nfeatures of Looe Key proper such as distribution of seagrass beds and the size and position of the sand lobe and rubble fields \nwere relatively static between these time periods leading up to Hurricane Irma (Fig. 9a and b). Imagery from December 2017, 3 months after Hurricane Irma passed, shows broad scale sediment deposition and burial of seagrass beds in the shallow areas \n605 \nof the reef proper, erosion and exposure of deeper, downslope spur-and-groove formation and downslope deposition on the \nsand lobe (Fig. 9c). Imagery from 2019 shows re-exposure of some shallow seagrass beds and deep spur-and-groove formation \nas sediments were eroded (Fig. 9d). Historical areal imagery from 1975 (Fig. 9e, Lidz et al., 2016) shows a distribution of \nseagrass, presence of rubble fields, and patterns of sediment along the sand lobe similar to 2014 and 2017 imagery (before 3 months after Hurricane Irma passed, shows broad scale sediment deposition and burial of seagrass beds in the shallow areas \n605 \nof the reef proper, erosion and exposure of deeper, downslope spur-and-groove formation and downslope deposition on the \nsand lobe (Fig. 9c). Imagery from 2019 shows re-exposure of some shallow seagrass beds and deep spur-and-groove formation \nas sediments were eroded (Fig. 9d). Historical areal imagery from 1975 (Fig. 9e, Lidz et al., 2016) shows a distribution of \nseagrass, presence of rubble fields, and patterns of sediment along the sand lobe similar to 2014 and 2017 imagery (before 3 months after Hurricane Irma passed, shows broad scale sediment deposition and burial of seagrass beds in the shallow areas \n605 \nof the reef proper, erosion and exposure of deeper, downslope spur-and-groove formation and downslope deposition on the \nsand lobe (Fig. 9c). Imagery from 2019 shows re-exposure of some shallow seagrass beds and deep spur-and-groove formation \nas sediments were eroded (Fig. 9d). Historical areal imagery from 1975 (Fig. 9e, Lidz et al., 2016) shows a distribution of \nseagrass, presence of rubble fields, and patterns of sediment along the sand lobe similar to 2014 and 2017 imagery (before Hurricane Irma) indicating these features have persisted over the past several decades despite repeated impact from tropical \n610 \nand seasonal storms. Lidz et al. (2016) suggested the formation of rubble fields in the shallow back reef area is mainly due to \nhistorical passage of hurricanes and winter storms, and our elevation change results suggest that these structures continue to \nmigrate in response to storm conditions. Lidz et al. (2016) also suggested that transport of sediment during hurricanes was Hurricane Irma) indicating these features have persisted over the past several decades despite repeated impact from tropical \n610 \nand seasonal storms. Lidz et al. (2016) suggested the formation of rubble fields in the shallow back reef area is mainly due to \nhistorical passage of hurricanes and winter storms, and our elevation change results suggest that these structures continue to \nmigrate in response to storm conditions. Lidz et al. (2016) also suggested that transport of sediment during hurricanes was Hurricane Irma) indicating these features have persisted over the past several decades despite repeated impact from tropical \n610 \nand seasonal storms. Lidz et al. (2016) suggested the formation of rubble fields in the shallow back reef area is mainly due to \nhistorical passage of hurricanes and winter storms, and our elevation change results suggest that these structures continue to \nmigrate in response to storm conditions. Lidz et al. (2016) also suggested that transport of sediment during hurricanes was 28 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 primarily to the north; however, our observations showed primary sediment movement during Hurricane Irma was WSW and \ndownslope from shallow to deep habitats with apparent seaward movement of the sand lobe after the storm passed. 615 615 elevation-change rate of -1.4 mm yr-1 between 1934 and 2004 (Yates et al., 2017). Furthermore, six of nine habitats at LKR \n620 \nshowed elevation loss over those periods, with greatest losses associated with shallow habitats, and mean elevation and \nvolume gains in deep-water habitats including at the base of the spur-and-groove habitat, indicating transport of reef \nsediments down the fore-reef-slope and export offshore (Yates et al., 2017). Our observed rate of mean elevation loss \nbetween 2017 and 2019 (-139 mm yr-1) was two orders of magnitude higher than the multi-decadal rates of Yates et al. (2017). Additionally, elevation loss (erosion) showed a moderate correlation with water depth, and mean elevation losses \n625 \nduring 2017 to 2019 were significantly greater in habitats with larger mean elevation gains during 2016 to 2017, suggesting \nthat sediment distribution was re-equilibrating or stabilizing to quiescent sea-state conditions up to 16.5 months after the \nstorm. The annualized mean rate of elevation-change for LKR from the 2.5-year period between July 2016 to January 2019 examined \nin our study, including sediment accretion from Hurricane Irma and the post-storm erosion and re-equilibration, was \n630 \napproximately 72 mm yr-1, which is almost double the rate of accretion observed in the previous decade of 32.5 mm yr-1 (Yates \net al. 2019b). Numerous field reconnaissance observations immediately after the passing of Hurricane Irma indicated \nbroadscale sediment deposition across the FRT due to the storm (e.g., Viehman et al., 2018; Walker, 2018; Wilson et al., 2020; \nKobelt et al. 2019). Our 2016 to 2019 elevation-change rate is consistent with annualized mean elevation-change rates from The annualized mean rate of elevation-change for LKR from the 2.5-year period between July 2016 to January 2019 examined \nin our study, including sediment accretion from Hurricane Irma and the post-storm erosion and re-equilibration, was \n630 \napproximately 72 mm yr-1, which is almost double the rate of accretion observed in the previous decade of 32.5 mm yr-1 (Yates \net al. 2019b). 5 Conclusion High-resolution lidar and multibeam bathymetric data were used to quantify seafloor elevation and volume change within the \nLooe Key Reef system of the Florida Keys Reef Tract over a 2.5-year period from 2016–2019 and to examine impacts from \ncategory-4 Hurricane Irma and post-storm re-equilibration of seafloor sediments. Analysis of seafloor elevation and volume \nchange over a 16.5-month period from July 2016 to December 2017 showed Hurricane Irma caused broadscale deposition of \n655 \nsediments across all benthic habitats of this reef system and burial of seagrass and coral dominated habitat. Rates of net \nelevation change were one order of magnitude greater during this short-term period that included storm impacts from Hurricane \nIrma than for the previous decade (Yates et al., 2019). Major seafloor geomorphic features such as sand waves and rubble \nfields migrated 10s of meters to the WSW in response to predominant wind conditions during the passing of Hurricane Irma, \nand sediment accretion was significantly greater in deep habitats than shallow habitats, suggesting downslope and offshore \n660 \ntransport of seafloor sediment. High-resolution lidar and multibeam bathymetric data were used to quantify seafloor elevation and volume change within the \nLooe Key Reef system of the Florida Keys Reef Tract over a 2.5-year period from 2016–2019 and to examine impacts from \ncategory-4 Hurricane Irma and post-storm re-equilibration of seafloor sediments. Analysis of seafloor elevation and volume change over a 16.5-month period from July 2016 to December 2017 showed Hurricane Irma caused broadscale deposition of \n655 \nsediments across all benthic habitats of this reef system and burial of seagrass and coral dominated habitat. Rates of net \nelevation change were one order of magnitude greater during this short-term period that included storm impacts from Hurricane \nIrma than for the previous decade (Yates et al., 2019). Major seafloor geomorphic features such as sand waves and rubble \nfields migrated 10s of meters to the WSW in response to predominant wind conditions during the passing of Hurricane Irma, \nand sediment accretion was significantly greater in deep habitats than shallow habitats, suggesting downslope and offshore \n660 \ntransport of seafloor sediment. 655 Loss of mean elevation and volume in all habitats in the period following the storm (from December 2017 to January 2019) \nindicated that 35% to 50% of sediment deposited during the storm had eroded by approximately 16.5 months after the storm \nand that erosion rates were two orders of magnitude greater than historical, multi-decadal rates of erosion. https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 Table 5. Annualized mean elevation-change rates (mm yr-1) for event-driven to multi-decadal time periods at the Florida Keys \nReef Tract. Annualized mean elevation change rate (mm yr-1) \nLocation \nEvent-drivena \n2016 to 2017 \nPost-stormb \n2017 to 2019 \nShort-termc \n2016 to 2019 \nDecadal \n2004 to 2016 \nMulti-decadal \n1930s to 2000s \nLooe Key Reef \n247 \n-139 \n72 \n32.5d \n-4.5e \nLower Florida Reef Tract \n(south of Big Pine) \nna \nna \n84f \nna \nna \nFlorida Reef Tract (Miami to \nKey West) \nna \nna \n76f \nna \nna \nUpper Florida Reef Tract \n(Elliott Key to Tavernier Key) \nna \nna \nna \nna \n-1.4e \na = calculated assuming a total time-period of 16.5 months (13.5 month pre- to 3 months post-storm); b = total time period 13.5 months (3 to 16.5 months \npost-storm); c = total time-period approximately 30 months (13.5 months pre- to 16.5 months post-storm); d = using data from Yates et al., 2019; e = using \ndata from Yates et al., 2017; f = using data from Fehr et al., 2021; na = no data available. 0 Table 5. Annualized mean elevation-change rates (mm yr-1) for event-driven to multi-decadal time periods at the Florida Keys \nReef Tract Table 5. Annualized mean elevation-change rates (mm yr-1) for event-driven to multi-decadal time per\nReef Tract. elevation-change rates (mm yr-1) for event-driven to multi-decadal time periods at the Florida Keys a = calculated assuming a total time-period of 16.5 months (13.5 month pre- to 3 months post-storm); b = total time period 13.5 months (3 to 16.5 months \npost-storm); c = total time-period approximately 30 months (13.5 months pre- to 16.5 months post-storm); d = using data from Yates et al., 2019; e = using \ndata from Yates et al., 2017; f = using data from Fehr et al., 2021; na = no data available. 650 Numerous field reconnaissance observations immediately after the passing of Hurricane Irma indicated \nbroadscale sediment deposition across the FRT due to the storm (e.g., Viehman et al., 2018; Walker, 2018; Wilson et al., 2020; \nKobelt et al. 2019). Our 2016 to 2019 elevation-change rate is consistent with annualized mean elevation-change rates from 2016 to 2019 for the Lower FRT from approximately Big Pine Key to Key West of 84 mm yr-1, and for the FRT from Miami \n635 \nto Key West of 76 mm yr-1 (Fehr et al., 2021), further suggesting that sediment distribution may have still been undergoing \npost-storm re-equilibration at our study site and along the broader FRT (Table 5). Collection and analysis of additional elevation-change data sets over shorter time-periods (e.g., seasonal to annual) could Collection and analysis of additional elevation-change data sets over shorter time-periods (e.g., seasonal to annual) could \nimprove characterization of post-storm elevation-change rates and duration of post-storm sediment re-equilibration periods \n640 \nrelative to persistent seasonal, interannual, decadal, and multi-decadal time periods. Our results also suggest that caution should \nbe used in selection of DEMs for use in elevation change and projection modelling to minimize bias that could result from \nselecting elevation surfaces that reflect periods of rapid elevation change due to storm impacts and periods of post-storm re-\nequilibration. improve characterization of post-storm elevation-change rates and duration of post-storm sediment re-equilibration periods \n640 \nrelative to persistent seasonal, interannual, decadal, and multi-decadal time periods. Our results also suggest that caution should \nbe used in selection of DEMs for use in elevation change and projection modelling to minimize bias that could result from \nselecting elevation surfaces that reflect periods of rapid elevation change due to storm impacts and periods of post-storm re-\nequilibration. 645 29 5 Conclusion Sediment erosion \n665 \nafter the storm (2017–2019) was moderately correlated with depth and was significantly greater in habitats that showed greater \naccumulation during the period including Hurricane Irma from 2016–2017, suggesting a period of rapid sediment re-\nequilibration after the storm. Historical satellite and aerial imagery show that major geomorphic features at this location \nincluding rubble fields, sand waves, and a sand lobe at the base of the spur-and-groove formation have persisted over the past several decades despite impacts from storms. However, our elevation-change results indicate these features are highly \n670 \nephemeral, migrating rapidly during storms, re-equilibrating to non-storm sea state conditions between storms, and periodically \nburying seafloor habitat such as seagrass. Such features and the area surrounding them likely represent localized areas of long- \nand short-term seafloor instability that could be less suitable for restoration of slow growing benthic species. Our observed several decades despite impacts from storms. However, our elevation-change results indicate these features are highly \n670 \nephemeral, migrating rapidly during storms, re-equilibrating to non-storm sea state conditions between storms, and periodically \nburying seafloor habitat such as seagrass. Such features and the area surrounding them likely represent localized areas of long- \nand short-term seafloor instability that could be less suitable for restoration of slow growing benthic species. Our observed 30 Data availability Elevation-change and multibeam bathymetric data are publicly available in U.S. Geological Survey Data Releases at \nhttps://doi.org/10.5066/P9CHC95D, \nhttps://doi.org/10.5066/P937LNZF, \nhttps://doi.org/10.5066/P9NXNX61, \nhttps://doi.org/10.5066/P9JTOOMB and https://doi.org/10.5066/P9P2V7L0. Lidar topobathymetric data are publicly available \n690 \nfrom \nthe \nNOAA \nOffice \nfor \nCoastal \nManagement \nat \nhttps://www.fisheries.noaa.gov/inport/item/63018 \nand \nhttps://www.fisheries.noaa.gov/inport/item/48373. Seafloor habitat data are publicly available from the Florida Fish and \nWildlife \nConservation \nCommission, \nFish \nand \nWildlife \nResearch \nInstitute \nat \nhttp://ocean.floridamarine.org/IntegratedReefMap/UnifiedReefTract.htm. p\ng\ng\np\n \n695 \nAuthor contribution \nKY and DZ conceptualized the research, data acquisition approach, and methodology for analysis of seafloor elevation data. KY, ZF, and DZ performed formal analysis of data. DZ developed the SECAT software for statistical analysis of elevation- \nand volume-change data. KY and ZF developed data interpretations and prepared the original manuscript draft with \ncontributions from SJ and DZ. All authors contributed to preparation of the final, published manuscript. 700 \n \nCompeting interests 695 \nAuthor contribution \nKY and DZ conceptualized the research, data acquisition approach, and methodology for analysis of seafloor elevation data. KY, ZF, and DZ performed formal analysis of data. DZ developed the SECAT software for statistical analysis of elevation- \nand volume-change data. KY and ZF developed data interpretations and prepared the original manuscript draft with \ncontributions from SJ and DZ. All authors contributed to preparation of the final, published manuscript. 700 Code availability Python script for the Seafloor Elevation Change Analysis Tool (SECAT), intended to be applied in ArcMap or ArcGIS Pro, is \npublicly \navailable \nas \na \nU. S. Geological \nSurvey \nsoftware \nrelease, \ndoi: \n10.5066/P9D5UUZ0, \nhttps://www.usgs.gov/software/seafloor-elevation-change-analysis-tool. Python script for the Seafloor Elevation Change Analysis Tool (SECAT), intended to be applied in ArcMap or ArcGIS Pro, is \npublicly \navailable \nas \na \nU. S. Geological \nSurvey \nsoftware \nrelease, \ndoi: \n10.5066/P9D5UUZ0, \nhttps://www.usgs.gov/software/seafloor-elevation-change-analysis-tool. https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 rates of elevation change in the 16-month period after Hurricane Irma were one to two orders of magnitude greater than during \nthe past decade or multi-decadal period (Yates et al., 2017; 2019b) indicating seafloor sediments across all habitats may have \n675 \nstill been re-equilibrating to non-storm sea state conditions up January 2019. Higher resolution elevation-change data collected \nover seasonal and annual time periods could improve characterization and understanding of short-term (event-driven, seasonal, \ninterannual) and long-term (decadal to multi-decadal) rates and processes of seafloor change and help guide benthic habitat \npost-storm recovery and restoration efforts in topographically complex coral reef systems. https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 Acknowledgement Funding for this study was provided by the U.S. Geological Survey, Coastal and Marine Hazards and Resources Program and \n710 \nby 2018 Hurricane and Wildfire Supplemental Funding provided to the U.S. Geological Survey from the Additional \nSupplemental Appropriations for Disaster Relief Requirements Act of 2018 (P.L. 115-123). We would like to thank J.J. Fredericks, B.J. Reynolds, and A.S. Farmer for collection of the multibeam bathymetry data and J.J. Fredericks for \ndevelopment of the associated multibeam digital elevation model. We also thank J. Zieg for assistance with development of Funding for this study was provided by the U.S. Geological Survey, Coastal and Marine Hazards and Resources Program and \n710 \nby 2018 Hurricane and Wildfire Supplemental Funding provided to the U.S. Geological Survey from the Additional \nSupplemental Appropriations for Disaster Relief Requirements Act of 2018 (P.L. 115-123). We would like to thank J.J. Fredericks, B.J. Reynolds, and A.S. Farmer for collection of the multibeam bathymetry data and J.J. Fredericks for \ndevelopment of the associated multibeam digital elevation model. We also thank J. Zieg for assistance with development of the SECAT software and K. Murphy for assistance with development of methods for sub-sampling large-scale digital elevation \n715 \nmodels. We greatly appreciate reviews of the original draft manuscript and constructive comments from L. Toth and G. Hatcher. Competing interests The authors declare that they have no conflict of interest. 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Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 Yates, K.K., Zawada, D. G., and Arsenault, S.R.: Seafloor elevation change from 2016 to 2017 at Looe Key, Florida Keys—\nImpacts from Hurricane Irma (ver. 2.0, November 2020), U.S. Geological Survey data release, USGS [data set], \nhttps://doi.org/10.5066/P937LNZF, 2019a. 1045 \n \nYates, K. K., Zawada, D. G., Murphy, K. A., and Arsenault, S. R.: Seafloor elevation change from 2004 to 2016 at Looe Key, \nFlorida Keys, U.S. Geological Survey data release, USGS [data set], https://doi.org/10.5066/P9JTOOMB, 2019b. Yates, K.K., Zawada, D. 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bèj’ ﺩ ﺍﳋﺎﻣﺲ
ﺍﻟﻌﺪﺩ ﺣﻮﻟﻴﺔ
•ﺣ ﻀﻰ ﺍﻟﻠﻐﺔ،)(١
ﻑ ﺍﻟﺒﻴﺎﻥ ﺇﱃ
ﻡﺎﻜﺣﻷﺍ
«. ﺎﻥ ﻋﻨﻪ ﻋﻠﻴﻪ
ﺍﻟﻘﺮﺁﻥ ﲟﻘﺘﻀ
ﻢﻬﻨ . ﻲﻫﻭ
:
، ﻓﻘﺪ ﺃﺿﺎﻑ
ﻮﻝ ﻋﻠﻰ ﺑﻴﺎﻥ
ﺎ ﻓﻴﻪ ﺑﻴﺎ ﻭﺭﺩ
ﺯﺍﻮﺟ ﲑﺴﻔﺗ
ﺔ
ﻘﻘﶈﺍﻭ
ﲔ ﻨﻣ
n
ﻞﺤﻨﻟﺍ
٤٤ :
ﻚ) .(٢
ﻪ :
»ﻪﻧﺇ ﻮﻤﳏ
ﻪﻨﻋ
ﺎﻤﻴﻓ
ﺐ ﺇﱃ ﻋﺪﻡ
ﺟ
ﲨﻬﻮﺭ ﺍﳊﻨﺎﺑﻠﺔ
Z
[
\
n
ﳎﺎﻭﺯﺓ ﺫﻟﻚ
ﻲ)٣(ﻪﻨﻋ ﻪﻟﻮﻘﺑ
ﺻﺢ ﻭﺭﻭﺩﻩ
ﺎﺑﻠﺔ ﻣﻦ ﺫﻫﺐ
ﲨ ﺑﺄﺩﻟﺔ ﺭﺩﻫﺎ
o
X
Y
ﻓﻠﻴﺲ
ﺪﺣﻷ
ﳋﻄﺎﺏ ﺍﳊﻨﺒﻠﻲ
ﺻ ﻗﻔﻪ ﻋﻠﻰ ﻣﺎ
ﺕﺎ . ﺇ
ﻥ ﻣﻦ ﺍﳊﻨﺎ
ﻮﺍ ﻋﻠﻰ ﺫﻟﻚ
ﻗﺎﻝ ﺗﻌﺎﱃ:
o
ﷲ
،
ﺍﺬﻟﻭ
ﺃﺟﺎﺏ ﺃﺑﻮ ﺍﳋ
ﻧﺰﺍﻉ ﰲ ﺗﻮﻗ
ﺎﻫﺎﲡﻻ
ﻻﻭﺃ
:
ﻮﻟﺪﺘﺳ
١- ﻗ
ﺳﻮﻝ ﺍﷲ
ﺪﻗﻭ ﺃ
ﺍﺬﻫ ﻻ ،ﺔ)١( ﺩ ﺍﳋﺎﻣﺲ
٨٥١ /٢:
ﻭﻧﻘﻞ
ﺍﻟﻌﺪﺩ
ﻭﺍﻟﻜﻮﻛﺐ ﺍﳌﻨﲑ
٧ ﺓﺩﻮﺴﳌﺍﻭ
:
١٧٦
ﲨﺎﻋﺔ ﻣﻦ ﺍﳊﻨﺎﺑﻠﺔ. ﺣﻮﻟﻴﺔ
ﺍﺑﻦ ﺍﻟﻠﺤﺎﻡ
٣٧ :
ﺿﻲ ﺃﰊ ﻳﻌﻠﻰ ﻭﲨ
. •ﺣ
، ﻳﻨﻈﺮ
:
ﺮﺼﺘﺨﳌﺍ
:
ﰊ ﺍﳊﺴﲔ ﺑﻦ ﺍﻟﻘﺎ
ﻲﻠﺒﻨﳊ :٢/ ٢٨٢
. ٢
ﺣﺪ ﻗﻮﻟﲔ ﻟﻠﺤﻨﺎﺑﻠﺔ
ﺮﺃﻱ ﻋﻦ ﺍﻟﻘﺎﺿﻲ ﺃ
ﺪ :ﺃﺑﻮ ﺍﳋﻄﺎﺏ ﺍﳊ
ﺭ
ﻖﺑﺎﺴﻟﺍ
٢٨/٢:
)( ١
ﻫﻮ ﺃﺣ
ﺍﻟﺮﻫﺬﺍ
)٢(
ﺪﻴﻬﻤﺘﻟﺍ
)٣(
ﺭﺪﺼﳌﺍ ﺩ ﺍﳋﺎﻣﺲ
٨٥١ /٢:
ﻭﻧﻘﻞ
ﺍﻟﻌﺪﺩ
ﻭﺍﻟﻜﻮﻛﺐ ﺍﳌﻨﲑ
٧ ﺓﺩﻮﺴﳌﺍﻭ
:
١٧٦
ﲨﺎﻋﺔ ﻣﻦ ﺍﳊﻨﺎﺑﻠﺔ. ﺣﻮﻟﻴﺔ
ﺍﺑﻦ ﺍﻟﻠﺤﺎﻡ
٣٧ :
ﺿﻲ ﺃﰊ ﻳﻌﻠﻰ ﻭﲨ
. •ﺣ
، ﻳﻨﻈﺮ
:
ﺮﺼﺘﺨﳌﺍ
:
ﰊ ﺍﳊﺴﲔ ﺑﻦ ﺍﻟﻘﺎ
ﻲﻠﺒﻨﳊ :٢/ ٢٨٢
. ٢
ﺣﺪ ﻗﻮﻟﲔ ﻟﻠﺤﻨﺎﺑﻠﺔ
ﺮﺃﻱ ﻋﻦ ﺍﻟﻘﺎﺿﻲ ﺃ
ﺪ :ﺃﺑﻮ ﺍﳋﻄﺎﺏ ﺍﳊ
ﺭ
ﻖﺑﺎﺴﻟﺍ
٢٨/٢:
)( ١
ﻫﻮ ﺃﺣ
ﺍﻟﺮﻫﺬﺍ
)٢(
ﺪﻴﻬﻤﺘﻟﺍ
)٣(
ﺭﺪﺼﳌﺍ ٨٥١ /٢:
ﻭﻧﻘ ﻭﺍﻟﻜﻮﻛﺐ ﺍﳌﻨﲑ
٧ ﺓﺩﻮﺴﳌﺍﻭ
:
١٧٦
ﲨﺎﻋﺔ ﻣﻦ ﺍﳊﻨﺎﺑﻠﺔ. ﺍﺑﻦ ﺍﻟﻠﺤﺎﻡ
٣٧ :
ﺿﻲ ﺃﰊ ﻳﻌﻠﻰ ﻭﲨ
. ، ﻳﻨﻈﺮ
:
ﺮﺼﺘﺨﳌﺍ
:
ﰊ ﺍﳊﺴﲔ ﺑﻦ ﺍﻟﻘﺎ
ﻲﻠﺒﻨﳊ :٢/ ٢٨٢
. ٢
ﺣﺪ ﻗﻮﻟﲔ ﻟﻠﺤﻨﺎﺑﻠﺔ
ﺮﺃﻱ ﻋﻦ ﺍﻟﻘﺎﺿﻲ ﺃ
ﺪ :ﺃﺑﻮ ﺍﳋﻄﺎﺏ ﺍﳊ
ﺭ
ﻖﺑﺎﺴﻟﺍ
٢٨/٢:
)( ١
ﻫﻮ ﺃﺣ
ﺍﻟﺮﻫﺬﺍ
)٢(
ﺪﻴﻬﻤﺘﻟﺍ
)٣(
ﺭﺪﺼﳌﺍ
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bèj’ ﺩ ﺍﳋﺎﻣﺲ
ﻋﻦ ﺭﺳﻮﻝ
ﺮﺩ ﻓﻴﻪ ﺑﻴﺎﻥ،
ﺔﲨ
،ﻅﺎﻔﻟﻷﺍ
ﺰﻧﹶ
ﺎ ﺃ ﻣﻭﺩﺪ
ﹶ
ﻝ
ﺯ ﺍﻻﺣﺘﺠﺎﺝ
ﰲ ﺍﳊﺪﻭﺩ
ﺽ ﻭﺍﻹﻧﺴﺎﻥ،
ﰲ
ﺗﻔﺼﻴﻞ ﺍﳌﺮﺍﺩ
ﺍﻟﻌﺪﺩ
ﻓﺈﻥ ﻣﺎ ﻭﺭﺩ
ﻭﺃﻣﺎ ﻣﺎ ﱂ ﻳﺮ
ﻤﺎ ﻳﺘﻌﻠﻖ
ﲨﺮﺘﺑ
ﹼ
ﻻ
ﺪﻮﺍ ﺣﹶﻤ
ﻠﻌﻳ
ﺪ ﻋﺪﻡ ﺟﻮﺍﺯ
ﳓﺘﺞ ﺑﻘﻮﳍﻢ
ﺴﻮﺍﺩ ﻭﺍﻟﺒﻴﺎﺽ
ﺴﻠﻢ ﻟﻮﺟﻮﻩ:
:٢/ ١٦٢
ﺮﻈﻨﻳﻭ
ﺎﻤ،ﺪﻌﺑ
ﺍﺬﻟﻭ
ﻃﺮﻳﻖ ﺁﺧﺮ. ﻟﱰﺍﻉ ﻫﻮ ﻓﻴﻤ
ﻥ ﻭﺗﻮﻗﻴﻒ .
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bèj’ ﻻﹶ
ﺃ ﺭﺪﺟﹶ
ﺃﹰ ﻭ
ﺎﻗﺎ
٩٧
، ﻓﻬﻲ ﺗﻔﻴﺪ
ﻪ :
»ﺎﻧﺇ ﻻ
ﻨﺎﻫﺎ، ﻣﺜﻞ ﺍﻟﺴ
ﻉﻮﺿ
ﲑﻏ ﺴﻣ
ﰲ ﻋﻠﻮﻡ ﺍﻟﻘﺮﺁﻥ
ﺣﻮﻟﻴﺔ
ﻣﺎﺳﺔ ﺇﻟﻴﻪ ﻓﻴﻤ
ﻃ ﻋﻦ ﺍﻟﻨﻈﺮ ﰲ
ﻩ.)١( ﻞﳏﻭ
ﱰﻟﺍ
ﱂ ﻳﺮﺩ ﻓﻴﻪ ﺑﻴﺎﻥ
ﺎﹶ
ﻔﹺ
ﻧﹰ ﻭ
ﺍﺮﹾ
ﻔﹸ
ﻛ ﺪ
ﻢ ﺔﺑﻮﺘﻟﺍ
: ٧
.) (٢
ﻲﻠﺒﻨﳊ)٣( ﻪﻟﻮﻘﺑ
ﻅﺎﻔﻟﻷﺍ ﺎﻨﻌﻣﻭ
. «
ﻣﺜﻞ ﻫﺬﺍ ﺍﳌﻮﺿ
:١٨٦
، ﺍﻟﱪﻫﺎﻥ
. •ﺣ
ﻣ ﻩ، ﻭﺍﳊﺎﺟﺔ
ﻋ ﻓﻔﻴﻪ ﻛﻔﺎﻳﺔ
ﻞ ﺍﻟﻌﻠﻢ ﺑﻌﺪﻩ
ﱂ ﺍﻟﻠﻐﺔ ﻓﻴﻤﺎ
ﺍ
َ
ﻷ
ﺪﺷﹶ
ﺃ ﺏﺍﺮﻋ
ﻢﻴﻜﺣ ﻢﻴﻠﻋ ﻪ
ﺴﲑ ﻛﻼﻡ ﺍﷲ. ﺍﳋﻄﺎﺏ ﺍﳊﻨ
ﺞ ﺑﻘﻮﳍﻢ ﰲ
ﺍ
ﻤﻬﻢ ﻓﻼ ﺗﻘﺒﻞ
ﺬﻩ ﺍﻵﻳﺔ ﰲ
ﺪﻣﺔ ﻛﺘﺎﺏ ﺍﳌﺒﺎﱐ
ﻥﺁﺮﻘﻟ :١٢ -٢٠ . . ٢
ﺇﻟﻴﻪ ﰲ ﻋﺼﺮﻩ
ﻦﻣ ﻥﺎﻴﺑ
ﻓ ﻪﻟ
ﻨﺌﺬ
ﺓﺮﻜﻓ
ﻞﻫﺃ
ﻘﺮﺁﻥ ﲟﻘﺘﻀﻰ
ﻗﺎﻝ ﺗﻌﺎﱃ:
ﻪﱠ
ﺍﻟﻠ ﻭﻪﻮﻟﺳﻰ ﺭ
ﻌﺮﺏ ﰲ ﺗﻔﺴ
ﺏ ﻋﻨﻪ ﺃﺑﻮ
ﺎﻡ، ﻭﺇﳕﺎ ﳛﺘﺞ
ﺎﺭﻫﻢ ﻭﺣﻜﻤ
ﺝﺎﺠﺘﺣﻻﺍ
ﺀ
:١/ ٢٩٠
، ﻣﻘﺪ
:ﺗﻄﻮﺭ ﺗﻔﺴﲑ
ﻘﻟﺍ
ﺪ :٢/ ٢٨٢
. ﺭ
ﻖﺑﺎﺴﻟﺍ
٢٨/٢:
ﺇ ﺔﺟﺎﳊﺍ
ﺍﷲ
ﻓﻔﻴﻪ ﺣﻴﻨ
ﻭﻓﻬﻢ ﺍﻟﻘ
٢- ﻗ
ﻰﹶ
ﻠﻋ ﻪﱠ
ﻠﻟﺍ
ﺑﻜﻼﻡ ﺍﻟﻌ
ﺏﺎﺟﺃﻭ
ﺎﻜﺣﻷﺍﻭ
ﻓﺄﻣﺎ ﺃﺧﺒﺎ
ﰒ
ﻥﺇ
)( ١
ﺀﺎﻴﺣﻹﺍ
ﻪﻧﺎﻴﺒﺑ
)٢(
ﺪﻴﻬﻤﺘﻟﺍ
( ٣)
ﺭﺪﺼﳌﺍ ﰲ
ﺗﻔﺼﻴﻞ ﺍ :٢٦١ /٢
ﺮﻈﻨﻳﻭ
ﰲ ﻋﻠﻮﻡ ﺍﻟﻘﺮﺁﻥ
:١٨٦
، ﺍﻟﱪﻫﺎﻥ
. ﺪﻣﺔ ﻛﺘﺎﺏ ﺍﳌﺒﺎﱐ
ﻥﺁﺮﻘﻟ :١٢ -٢٠ . . ٢
ﺀ
:١/ ٢٩٠
، ﻣﻘﺪ
:ﺗﻄﻮﺭ ﺗﻔﺴﲑ
ﻘﻟﺍ
ﺪ :٢/ ٢٨٢
. ﺭ
ﻖﺑﺎﺴﻟﺍ
٢٨/٢:
)( ١
ﺀﺎﻴﺣﻹﺍ
ﻪﻧﺎﻴﺒﺑ
)٢(
ﺪﻴﻬﻤﺘﻟﺍ
( ٣)
ﺭﺪﺼﳌﺍ :٢/٢
ﻥ ١٤٣
ﺩ ﺍﳋﺎﻣﺲ
ﺎﺩﻳﺔ، ﻣﻘﺎﺑﻠﺔ
ﺑﺎﳌﺪﻳﻨﺔ ﺫﻛﺮ
ﻢ ﺃﺷﺪ، ﻷ
ﻗﻮﻻ، ﻓﺄﺧﻠﻖ
ﺔ ﺝﺎﺠﺘﺣﻻﺍ
ﻢﻭﻓﻖ ﻟﺴﺎ
ﺲ ﻫﻮ ﺍﳌﻔﺴﺮ
ﻥ ﻋﻠﻰ ﻭﻓﻘﻪ
ﻨﻬﻢ ﰲ ﻫﺬﻩ
ﻳﺪﺧﻞ ﻓﻴﻪ
ﻳﺘﺴﺎﻭﻭﺍ ﰲ
ﻦ ﺍﻻﺣﺘﺠﺎﺝ
ﺔ ﺍﻻﺣﺘﺠﺎﺝ
ﺍﻟﻌﺪﺩ
ﻣﻦ ﺃﻫﻞ ﺍﻟﺒﺎ
ﻮﺍﻝ ﺍﳌ
ﺑ ﲔﻘﻓﺎﻨ
ﺃﻥ ﻛﻔﺮﻫﻢ
ﻗﻮ ﻌﺎ، ﻭﺃﺟﻔﻰ
ﺍﻉ ﰲ
ﺔﺤﺻ
ﺪ ﻭ ﻧﺰﻝ ﻋﻠﻰ
ﻣﻪ،
ﻟﻴﺲ ﻷﻧﻪ
ﺃﻟﻔﺎﻅ
ﻥﺁﺮﻘﻟﺍ
ﻣﺴﻜﻮﺕ ﻋﻨ
ﺿﺖ ﳍﻢ ﻻ
ﻻ ﳝﻜﻦ ﺃﻥ
ﻪﻴﻠﻋﻭ ﻦﻜﻤﻴﻓ
ﳑﺎ ﻳﺮﺩ ﺻﺤﺔ
ﲑﺴﻔ .
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bèj’ ﻜﻔﺮ ﻭﺍﻟﻨﻔﺎﻕ
ﺟﻞ ﻭﻋﺰ ﺃﺣﻮ
، ﻓﺄﺧﱪ ﻋﻦ
ﻢ ﺃﻏﻠﻆ ﻃﺒﻌ
ﻋﻦ ﳏﻞ ﺍﻟﱰﺍ
،ﻼﺻﺃ
ﺪﻗﻭ
ﺔ ﻋﻨﻪ ﺇﺳﻼﻣ
ﺃ ﻐﻮﻱ ﻓﻴﻔﺴﺮ
ﻫﻞ ﺍﳊﺎﺿﺮﺓ
ﻋﻠﻰ ﻣﻦ ﺗﻌﺮﺿ
ﻡ ﻏﲑﻫﻢ، ﻭﻻ
،ﲔﻨﻣﺆﻣ
)٢( ﻭ
ﳑ ،ﺺ ﻭﻣﻮﺟﺒﻪ
ﻟﻌﺮﺏ ﰲ ﺍﻟﺘﻔﺴ
ﺣﻮﻟﻴﺔ
ﺣﺎﻝ ﺃﻫﻞ ﺍﻟﻜ
ﺟ ﺪ ﺃﻥ ﺫﻛﺮ
ﺃﻫﻞ ﺍﻟﺒﺎﺩﻳﺔ
ﺴﻨﻦ ﻣﻊ ﻛﻮ
ﺍﺬﻫ ﻋ ﺝﺭﺎﺧ
ﻧﺰﻭﻝ ﺍﻟﻘﺮﺁﻥ
ﻦ ﻧﻘﻠﺖ ﺍﻟﻠﻐﺔ
ﻪ ﺍﻟﻌﺮﰊ ﺍﻟﻠﻐﻮ
ﺍﻟﺒﺎﺩﻳﺔ، ﻭﺃﻫ
ﻋ ﺍﻵﻳﺔ ﻫﺬﻩ
ﺍﻷﻋﺮﺍﺏ ﺃﻡ
ﻦ ﺃﻫﻞ
ﺔﻳﺩﺎﺒﻟﺍ
ﻣﻘﺘﻀﻰ ﺍﻟﻨﺺ
ﺠﺎﺝ ﺑﻜﻼﻡ ﺍﻟ
•ﺣ
À
ﺣ ﺘﺤﺪﺙ ﻋﻦ
ﳊﺎﺿﺮﺓ، ﻓﺒﻌﺪ
ﻦﻣ
ﺏﺍﺮﻋﻷﺍ
ﻞ
ﺔﻓﺮﻌﻣﻭ
ﺴﻟﺍ
،ﻉﺮﺸﻟ)١( ﻫﻭ
ﺩﺍﺕ ﺍﻟﻘ
ﻥﺁﺮ. ﻗﺎﺋﻤﺔ ﻗﺒﻞ
ﻳﺸﺘﺮﻁ ﻓﻴﻤﻦ
ﺍﳌﻔﺴﺮ ﻛﻼﻣﻪ
ﻫﻢ ﺃﻋﺮﺍ
ﺏ
ﻚ ﺃﻥ ﺣﻜﻢ
ﺀ ﻛﺎﻧﻮﺍ
ﻦﻣ
ﺇﻧﻜﺎﺭ ﺃﻥ ﻣﻦ
ﻣ ﻭﺟﻬﻢ ﻋﻦ
ﺯﺍﻮﺟ ﺠﺘﺣﻻﺍ
٢-٢٣٢
. . ٢
- ﻥﺇ
ﺍﻵﻳﺔ ﺗﺘﺤ
ﳍﻢ ﻣﻦ
ﻞﻫﺃ
ﺍ
ﻥ ﻧﺎﺋﻴﺎ ﻋﻨﻬﺎ
ﻦ ﲰﺎﻉ ﺍﻟﺘﱰﻳﻞ
ﻤﻮﺍ ﻓﺮﺍﺋﺾ ﺍﻟ
ﰲ ﻓﻬﻢ ﻣﻔﺮﺩ
ﺎﻬ-
ﺇﻥ ﺍﻟﻠﻐﺔ
ﺍﻟﻘﺎﻃﻊ، ﻓﻼ
ﻭﺇﳕﺎ ﻳﺄﺧﺬ
ﻪﺘﺤ. - ﻥﺇ ﺀﻻﺆﻫ
ﺎ -
ﻻﻭ ﻚﺷ
ﻣﻨﻬﻢ، ﺳﻮﺍ
ﻛﻤﺎ ﻻ ﳝﻜﻦ
،ﲔﻨﻣﺆﳌ ﻭﺮﳋ
ﻳﺔ ﻋﻠﻰ ﻋﺪﻡ
ﱯﻃﺮﻘﻟﺍ
١٣٢ /٨:
ﺭ
ﻖﺑﺎﺴﻟﺍ
٢٣/٨:
è
ﺎﳍﻭﺃ-
ﻣﻊ ﺃﻣﺜﺎﳍ
ﻣﻦ ﻛﺎﻥ
ﺃﺑﻌﺪ ﻋﻦ
ﺃﻻ ﻳﻌﻠﻤﻮ
ﻢﺎﺴﻠﺑ
ﻬﻴﻧﺎﺛ
ﻞﻴﻟﺪﻟﺎﺑ
،ﺮﺷﺎﺒﳌﺍ
ﻋﻨﺪ ﺻﺤ
ﺎﻬﺜﻟﺎﺛ
ﺔﻳﻵﺍ
. ﺎﻬﻌﺑﺍﺭ
ﻥﻮﻨﻣﺆﳌﺍ
ﻚﻟﺫ
ﻛ. ﻠﺑ
ﺍﳌﺴﺎﻥ
ﺬﻩ ﺍﻵﻳﺔ
)( ١
ﲑﺴﻔﺗ
( ٢)
ﺭﺪﺼﳌﺍ ،ﻉ)١( ﻭ،ﲔ)٢( ﻭ
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bèj’ ﳍﻢ ﺑﻘﻮﻟﻪ :ﻻ
ﺍﷲ ﻭﺃﻭﺭﺩ
ﻟﺸﻌﺮ ﺃﺻﻼ
ﻓﻴﻪ ﻫﻮ ﺑﻴﺎﻥ
v
w
x
، ﻭﻟﻮ ﻛﺎﻥ
ﺪ ﺑﺴﻨﺪﻩ ﻋﻦ
ﺪ :ﻳﻌﲏ ﻛﺎﻥ
ﳍ ﻣﻊ ﻭﺻﻔﻪ
ﺗﻔﺴﲑ
ﻡﻼﻛ
ﺸﻌﺮ ﳚﻌﻞ ﺍ
ﺔﻳﺎﻏ ﻓ ﺮﻣﻷﺍ
ﻝ:
o
u
v
n
ﺀﺍﺮﻌﺸﻟﺍ
:
١٩٥
ﻭﻯ ﺃﺑﻮ ﻋﺒﻴﺪ
ﻗﺎﻝ ﺃﺑﻮ ﻋﺒﻴﺪ
ﱂ ﺴﻳﻤ،ﻢﻬ
ﺗ ﺑﺎﻟﻠﻐﺔ ﰲ
ﻭﻣﺸﻜﻠﻪ ﺑﺎﻟﺸ
ﻟﻠﻘﺮﺁﻥ، ﺇﺫ
ﻏ
ﺳﺒﺤﺎﻧﻪ ﻳﻘﻮﻝ
s
t
u
n
،ﻪ)٢( ﱴﺣ
ﻭﺭ
ﺪ ﻓﻴﻪ ﺍﻟﺸﻌﺮ
.
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bèj’ ﻱ ﻋﻦ ﲨﺎﻋﺔ
ﺯ ﺍﻻﺣﺘﺠﺎﺝ
ﻭ ﻳﺐ ﺍﻟﻘﺮﺁﻥ
ﻟﺸﻌﺮ ﺃﺻﻼ
ﻌﺮ، ﻷﻥ ﺍﷲ
ﻝﻮﻘﻳﻭ :
o
s
ﺏ ﻭﻋﻤﻠﻮﺍ ﺑﻪ
ﻥﺁﺮﻘﻟﺍ
ﺪﺸﻨﻴﻓ
ﺮ ﺑﻦ ﺍﻷﻧﺒﺎﺭﻱ
ﱃ ﻋﺪﻡ ﺟﻮﺍﺯ
ﺝ ﻋﻠﻰ ﻏﺮﻳﺐ
ﻚ ﻻ ﳚﻌﻞ ﺍﻟ
ﺍﻟﻘﺮﺁﻥ ﺑﺎﻟﺸﻌ
ﻟﺰﺧﺮﻑ
، ٣:
ﻭ
ﻋﻠﻴﻪ ﺍﻷﺻﺤﺎﺏ
ﺍ ﻥ ﻳﺴﺄﻝ ﻋﻦ
ﺴﲑ«.) (٣
ﻧﻘﻞ ﺃﺑﻮ ﺑﻜﺮ
ﻢ .ﺫﻫﺒﻮﺍ ﺇﱃ
ﻢ ﻭﻫﻲ :
ﻥﺇ
ﺝﺎﺠﺘﺣﻻﺍ
) (١
: ﺑﻪ
ﺇﻥ ﺫﻟﻚ
ﺍﻟﻐﺮﻳﺐ ﻣﻦ
y
z
n
ﺍ
ﻋ ﻎ ﳌﺎ ﺣﺾ
ﺱ :
»ﻪﻧﺃ ﻥﺎﻛ
ﺪ ﺑﻪ ﻋﻠﻰ ﺍﻟﺘﻔﺴ
ﺎﻴﻧﺎﺛ:
ﻋﻠﻢ ﳍﻢ
ﻢﻬﺠﺠﺣ
١-
ﻥﺁﺮﻘﻠﻟ
.)
ﺍﻮﺟﻭ
ﺍﳊﺮﻑ
y
ﻏﲑ ﺳﺎﺋﻎ
ﻦﺑﺍ ﺱﺎﺒﻋ
ﺪﻬﺸﺘﺴﻳ v
w
x
، ﻭﻟﻮ ﻛﺎﻥ
ﺪ ﺑﺴﻨﺪﻩ ﻋﻦ
ﺪ :ﻳﻌﲏ ﻛﺎﻥ
ﻝ:
o
u
v
n
ﺀﺍﺮﻌﺸﻟﺍ
:
١٩٥
ﻭﻯ ﺃﺑﻮ ﻋﺒﻴﺪ
ﻗﺎﻝ ﺃﺑﻮ ﻋﺒﻴﺪ
ﺳﺒﺤﺎﻧﻪ ﻳﻘﻮﻝ
s
t
u
n
،ﻪ)٢( ﱴﺣ
ﻭﺭ
ﺪ ﻓﻴﻪ ﺍﻟﺸﻌﺮ
. ﻌﺮ، ﻷﻥ ﺍﷲ
ﻝﻮﻘﻳﻭ :
o
s
ﺏ ﻭﻋﻤﻠﻮﺍ ﺑﻪ
ﻥﺁﺮﻘﻟﺍ
ﺪﺸﻨﻴﻓ
ﺍﻟﻘﺮﺁﻥ ﺑﺎﻟﺸﻌ
ﻟﺰﺧﺮﻑ
، ٣:
ﻭ
ﻋﻠﻴﻪ ﺍﻷﺻﺤﺎﺏ
ﺍ ﻥ ﻳﺴﺄﻝ ﻋﻦ
ﺴﲑ«.) (٣
ﺍﻟﻐﺮﻳﺐ ﻣﻦ
y
z
n
ﺍ
ﻋ ﻎ ﳌﺎ ﺣﺾ
ﺱ :
»ﻪﻧﺃ ﻥﺎﻛ
ﺪ ﺑﻪ ﻋﻠﻰ ﺍﻟﺘﻔﺴ
ﺍﳊﺮﻑ
y
ﻏﲑ ﺳﺎﺋﻎ
ﻦﺑﺍ ﺱﺎﺒﻋ
ﺪﻬﺸﺘﺴﻳ v
w
x
، ﻭﻟﻮ ﻛﺎﻥ
ﺪ ﺑﺴﻨﺪﻩ ﻋﻦ
ﻝ:
o
u
v
n
ﺀﺍﺮﻌﺸﻟﺍ
:
١٩٥
ﻭﻯ ﺃﺑﻮ ﻋﺒﻴﺪ
ﺳﺒﺤﺎﻧﻪ ﻳﻘﻮﻝ
s
t
u
n
،ﻪ)٢( ﱴﺣ
ﻭﺭ
ﻌﺮ، ﻷﻥ ﺍﷲ
ﻝﻮﻘﻳﻭ :
o
s
ﺏ ﻭﻋﻤﻠﻮﺍ ﺑﻪ
ﺍﻟﻘﺮﺁﻥ ﺑﺎﻟﺸﻌ
ﻟﺰﺧﺮﻑ
، ٣:
ﻭ
ﻋﻠﻴﻪ ﺍﻷﺻﺤﺎﺏ
ﺍﻟﻐﺮﻳﺐ ﻣﻦ
y
z
n
ﺍ
ﻋ ﻎ ﳌﺎ ﺣﺾ
ﺍﳊﺮﻑ
y
ﻏﲑ ﺳﺎﺋﻎ )٢( ﺩ ﺍﳋﺎﻣﺲ
ﺍﻟﻌﺪﺩ
ﺣﻮﻟﻴﺔ
. ١
ﻥﺎ :١/ ١١٩
. ٣ . •ﺣ
ﻥﺎﻘﺗﻹﺍﻭ
:١/١٩
٠٠١ /١:
ﺎﻘﺗﻹﺍﻭ
ﻀﺎﺋﻞ ﺍﻟﻘﺮﺁﻥ :٤٣
ﺡ
ﺍﻟﻮﻗﻒ
:١/ ١٠٠
ﺡ ﺍﻟﻮﻗﻒ ﻭﺍﻻﺑﺘﺪﺍﺀ
ﺭﺍﻥ ﺍﻟﺴﺎﺑﻘﺎﻥ، ﻭﻓﻀ
)١(
ﺡﺎﻀﻳﺇ
)٢(
ﺡﺎﻀﻳﺇ
)٣(
ﺭﺪﺼﳌﺍ ©
ª
n
¨
©ﱃ: o
¨ ﺩ ﺍﳋﺎﻣﺲ
ﻼﻡ، ﻓﻬﻮ ﻻ
ﻣﻦ ﺃﺧﺮﺟﻬﻢ
ﻭﺃﻧﻪ ﻭﺻﻒ
µ
¶
¸
¹
ﳑﻦ ﻳﻘﻮﻟﻮﻥ
ﺍﻟﻌﺪﺩ
ﻪ ﻛﻘﺒﻴﺢ ﺍﻟﻜﻼ
ﻪﺒ ﻦﻣ ﺀﺎﻨﺜﺘﺳﺎﺑ
ﺀﺍﺮﻌﺸ :٢٢٧
،
³
´
µ
ﻋﻠﻰ ﻣﺜﺎﳍﻢ
ﻜﻼﻡ، ﻭﻗﺒﻴﺤﻪ
ﺗﺮﻯ ﺃﻧﻪ
ﺒﻘﻋﺃ
Â
Ã
n
ﺸﻟﺍ
°
±
²
³
ﺸﺮﻛﲔ ﻭﻣﻦ
ﺣﻮﻟﻴﺔ
ﻦﺴﺤﻛ
ﻜﻟﺍ
«.) (٢
ﺗ ﻋﻤﻮﻣﻪ، ﺃﻻ
À
Á
Â
o
¬
®
¯
°
ﻬﻢ ﺷﻌﺮﺍﺀ ﺍﳌﺸ
:١/ ١٢٠
. •ﺣ
ﻜﻼﻡ، ﺣﺴﻨﻪ
ﺮﻩ ﳌﻀﻤﻨﺎﺗﻪ«
ﻋ ﻟﻴﺲ ﻋﻠﻰ
¾
¿
À
ﻮﻣﲔ ﺑﻘﻮﻟﻪ:
o
- ٢٢٦
،
ﻬﻓ
١/ ١٠٠
ﻥﺎﻘﺗﻹﺍﻭ
١٥ .
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bèj’ ﻧﻮﻉ ﻣﻦ ﺍﻟﻜ
ﺍﺗﻪ، ﻭﺇﳕﺎ ﻳﻜﺮ
ﺍﻟﺬﻡ ﻟﻠﺸﻌﺮﺍﺀ
o
¼
½
¾
ﻟﺸﻌﺮﺍﺀ ﺍﳌﺬﻣﻮ
n
ﺀﺍﺮﻌﺸﻟﺍ
:
٢٢٥
ﺡ ﺍﻟﻮﻗﻒ ﻭﺍﻻﺑﺘﺪﺍﺀ
ﺍﻟﻘﺮﻃﱯ
١٥/ ٣١ :
» ﺮﻌﺸﻟﺍ
ﻳﻜﺮﻩ ﻟﺬ
ﻥﺇﻭ ﺍ
ﻪﻟﻮﻘﺑ
: o
ﺃﻭﻟﺌﻚ ﺍﻟ
º
n
)١(
ﺡﺎﻀﻳﺇ
)٢( ﲑﺴﻔﺗ :١/ ١٢٠
. ﻥﺎ
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bèj’
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bèj’ ﻟﻐﲑﻫﺎ ﻣﻦ
ﻪﻟﻮﻗ
» :
ﻷ
ﻥ
ﺇﺫ ﻋﺮﺽ ﻟﻪ
ﻭﺁﺩﺍﺑﻪ، ﺃﻭ
ﺍﻟﺸﻌﺮ ﻣﺜﻞ
«
). (٢
ﺇ ﻪﻟﻮﻗﻭ
(
ﺎﳘﻮﳓﻭ
. ﺩﺉ ﺍﻹﺳﻼﻡ
ﺇﱃ ﺫﻡ ﻩﻇﺎﻫﺮ
ﳝﺘﻠﺊ ﺷﻌﺮﺍ«
ﻥﺎﻄﻴﺸﻟ
«.)٣
ﻭ ﻣﻨﺎﻓﻴﺎ ﳌﺒﺎﺩ
(
ﺎﳑ ﲑﺸﻳ
ﻇ
ﺧﲑ ﻣﻦ ﺃﻥ
ﻭ :ﺃﻣﺴﻜﻮﺍ ﺍ
ﻼﻡ ﻭﺃﻫﻠﻪ، ﺃﻭ
ﺎ ﺍﻹﺳﻼﻡ.)١(
ﺭﺳﻮﻝ ﺍﷲ
ﻳ ﻢ ﻗﻴﺤﺎ
ﹺ
ﺮﻳﻪ
ﺍ ﻥﺎﻄﻴﺸﻟﺍ
ﺃﻭ. ﻌﺎﺩﻳﺎ ﻟﻺﺳﻼ
ﺏ ﺍﻟﱵ ﻳﺮﻓﻀﻬﺎ
ﺭ ﺍﳌﺮﻭﻱ ﻋﻦ
ﻮﻑ ﺃﺣﺪﻛﻢ
ﺪﺸ :
»ﺍﻭﺬﺧ
ﺍﺮﻌﺷ
ﻌﻣ
ﺏﺎﺒﺳﻷ
ﺎﻣﺃﻭ
ﺘﻠﺊ ﺟﻮ
ﺮﻋﺎﺷ
ﺸﻨﻳ ﻟﻐﲑﻫﺎ ﻣﻦ
ﻪﻟﻮﻗ
» :
ﻷ
ﻥ
ﺇﺫ ﻋﺮﺽ ﻟﻪ
ﻭﺁﺩﺍﺑﻪ، ﺃﻭ
ﺍﻟﺸﻌﺮ ﻣﺜﻞ
«
). (٢
ﺇ ﻪﻟﻮﻗﻭ
(
ﺎﳘﻮﳓﻭ
. ﺩﺉ ﺍﻹﺳﻼﻡ
ﺇﱃ ﺫﻡ ﻩﻇﺎﻫﺮ
ﳝﺘﻠﺊ ﺷﻌﺮﺍ«
ﻥﺎﻄﻴﺸﻟ
«.)٣
ﻭ ﻣﻨﺎﻓﻴﺎ ﳌﺒﺎﺩ
(
ﺎﳑ ﲑﺸﻳ
ﻇ
ﺧﲑ ﻣﻦ ﺃﻥ
ﻭ :ﺃﻣﺴﻜﻮﺍ ﺍ
ﻼﻡ ﻭﺃﻫﻠﻪ، ﺃﻭ
ﺎ ﺍﻹﺳﻼﻡ.)١(
ﺭﺳﻮﻝ ﺍﷲ
ﻳ ﻢ ﻗﻴﺤﺎ
ﹺ
ﺮﻳﻪ
ﺍ ﻥﺎﻄﻴﺸﻟﺍ
ﺃﻭ. ﻌﺎﺩﻳﺎ ﻟﻺﺳﻼ
ﺏ ﺍﻟﱵ ﻳﺮﻓﻀﻬﺎ
ﺭ ﺍﳌﺮﻭﻱ ﻋﻦ
ﻮﻑ ﺃﺣﺪﻛﻢ
ﺪﺸ :
»ﺍﻭﺬﺧ
ﺍﺮﻌ
ﻌﻣ
ﺏﺎﺒﺳﻷ
ﺎﻣﺃﻭ
ﻠﺊ ﺟﻮ
ﺮﻋﺎ
ﺸﻨﻳ ﺩ ﺍﳋﺎﻣﺲ
ﻪ ﺭﺳﻮﻝ ﺍﷲ
ﺤﻪ ﺇﺫ ﺑﻮﺏ
ﺮﻌ(،
)٥( ﻮﻬﻓ
ﻛﺘﺎﺏ ﺍﻟﺸﻌﺮ
:
ﺍﻟﻌﺪﺩ
ﻟﺬﻱ ﻳﻬﺠﺎ ﺑﻪ
ﳍﻢ.) (٤
ﺭﻱ ﰲ ﺻﺤﻴﺤ
ﺍﻹﻧﺴﺎﻥ ﺍﻟﺸﻌ
ﺢﻴﺤﺻﻭ ﻢﻠﺴﻣ
:
( . ﻫﻮ ﺍﻟﺸﻌﺮ ﺍﻟ
ﳍ ﳍﺠﺎﺀ ﻏﺮﺿﺎ
ﺍﻹﻣﺎﻡ ﺍﻟﺒﺨﺎﺭ
ﺍ ﻟﻐﺎﻟﺐ ﻋﻠﻰ
ﺏﺍﺩ
ﻭ ٤٧/٤:
/١٤٠
. . ١
ﷲ ﻭﺍﻟﻌﻠﻢ ﻭﺍﻟﻘﺮﺁﻥ
ﺣﻮﻟﻴﺔ
ﺮ
،ﻓﻴﻤﺎ ﺳﺒﻖ
ﺟﻌﻠﻮﺍ ﻫﺬﺍ ﺍﳍ
ﻣﺎ ﺃﺷﺎﺭ ﺇﻟﻴﻪ
ﻩ ﺃﻥ ﻳﻜﻮﻥ ﺍ
ﻒ :١/ ١٠٢
. ﺨﺎﺭﻱ: ﻛﺘﺎﺏ ﺍﻵ
ﺔﻔﲢ ﺍ
ﻷ
ﻱﺫﻮﺣ
٨: /
ﺍﳊﺪﻳﺚ ﺍﻟﺴﺎﺑﻖ
. ﱯﻃﺮﻘﻟﺍ:١٣ /٥٣
ﻳﺼﺪﻩ ﻋﻦ ﺫﻛﺮ ﺍﷲ
•ﺣ
ﻪﻴﻄﻌﻳ.
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bèj’ ﻪﻣ
ﺮﻌﺸﻠﻟ
ﺀﺍﺮﻌﺸ
ﺟ ﻦﻳﺬﻟﺍ
ﻣ ﻪ ﰲ ﺗﺄﻭﻳﻠﻪ
)ﺑﺎﺏ ﻣﺎ ﻳﻜﺮ
٤١ ﻭﺇﻳﻀﺎﺡ ﺍﳌﻮﻗﻒ
ﻠﻢ :ﺻﺤﻴﺢ ﺍﻟﺒﺨ
ﺩﻭ :٤/ ٣٠٤
ﻭﲢ
٧١ ﻭﺗﻜﻤﻠﺘﻪ ﺑﻨﺤﻮ
١-١٠٣
ﺍ ﲑﺴﻔﺗﻭ
٧
ﻪﺘﻠﻤﻜﺗﻭ
ﺣﱴ )
ﻳ ﰲ ﻣﺪﺡ ﻣﻦ
ﻦ ﺃﻥ ﻳﺮﺍﺩ ﺑﺬ
ﻤﻠﺴﻮ،ﻥ ﺸﻟﺍﻭ
ﻥﻮﻜﻳ ﻪﺟﻭﻷﺍ
ﻩ
ﺍﳊﺪﻳﺚ) :
ﱯﻃﺮﻘﻟﺍ
٦٤/ ٣١ :
ﺟﻪ
ﻱﺭﺎﺨﺒﻟﺍ
ﻠﺴﻣﻭ
١٧ ﻦﻨﺳﻭ
ﰊﺃ ﻭﺍﺩ
ﻪ
ﻢﻠﺴﻣ
٩٦٧ /٥:
ﺡ ﻟﺍ
ﻮ
ﻗﻒ
٢٠/١:
ﺢ
ﻱﺭﺎﺨﺒﻟﺍ
:٤/٧٤
ﰲ ﻁﺮﻔﻳﻭ
ﻦﻜﳝﻭ
ﺴﳌﺍﻭ
ﺪﻗﻭ ﻳ
ﻋﻠﻰ ﻫﺬ
)١( ﲑﺴﻔﺗ
)٢(
ﻪﺟﺮﺧﺃ
٥/٦٩
)٣(
ﻪﺟﺮﺧﺃ
)٤(
ﺡﺎﻀﻳﺇ
)٥(
ﺢﻴﺤﺻ ﺮﻌ(،
)٥( ﻮﻬﻓ
ﻛﺘﺎﺏ ﺍﻟﺸﻌﺮ
:
ﺍﻹﻧﺴﺎﻥ ﺍﻟﺸﻌ
ﺢﻴﺤﺻﻭ ﻢﻠﺴﻣ
:
( . ﺍ ﻟﻐﺎﻟﺐ ﻋﻠﻰ
ﺏﺍﺩ
ﻭ ٤٧/٤:
/١٤٠
. . ١
ﷲ ﻭﺍﻟﻌﻠﻢ ﻭﺍﻟﻘﺮﺁﻥ
ﻩ ﺃﻥ ﻳﻜﻮﻥ ﺍ
ﻒ :١/ ١٠٢
. ﺨﺎﺭﻱ: ﻛﺘﺎﺏ ﺍﻵ
ﺔﻔﲢ ﺍ
ﻷ
ﻱﺫﻮﺣ
٨: /
ﺍﳊﺪﻳﺚ ﺍﻟﺴﺎﺑﻖ
. ﱯﻃﺮﻘﻟﺍ:١٣ /٥٣
ﻳﺼﺪﻩ ﻋﻦ ﺫﻛﺮ ﺍﷲ
)ﺑﺎﺏ ﻣﺎ ﻳﻜﺮ
٤١ ﻭﺇﻳﻀﺎﺡ ﺍﳌﻮﻗﻒ
ﻠﻢ :ﺻﺤﻴﺢ ﺍﻟﺒﺨ
ﺩﻭ :٤/ ٣٠٤
ﻭﲢ
٧١ ﻭﺗﻜﻤﻠﺘﻪ ﺑﻨﺤﻮ
١-١٠٣
ﺍ ﲑﺴﻔﺗﻭ
٧
ﻪﺘﻠﻤﻜﺗﻭ
ﺣﱴ )
ﻩ
ﺍﳊﺪﻳﺚ) :
ﱯﻃﺮﻘﻟﺍ
٦٤/ ٣١ :
ﺟﻪ
ﻱﺭﺎﺨﺒﻟﺍ
ﻠﺴﻣﻭ
١٧ ﻦﻨﺳﻭ
ﰊﺃ ﻭﺍﺩ
ﻪ
ﻢﻠﺴﻣ
٩٦٧ /٥:
ﺡ ﻟﺍ
ﻮ
ﻗﻒ
٢٠/١:
ﺢ
ﻱﺭﺎﺨﺒﻟﺍ
:٤/٧٤
ﻋﻠﻰ ﻫﺬ
)١( ﲑﺴﻔﺗ
)٢(
ﻪﺟﺮﺧﺃ
٥/٦٩
)٣(
ﻪﺟﺮﺧﺃ
)٤(
ﺡﺎﻀﻳﺇ
)٥(
ﺢﻴﺤﺻ )ﺚﺑﺎ) : ﻛﺘﺎﺏ ﺍﻟﺸﻌﺮ
:
ﺢﻴﺤﺻﻭ ﻢﻠﺴﻣ
:
( . ﺏﺍﺩ
ﻭ ٤٧/٤:
/١٤٠
. . ١
ﷲ ﻭﺍﻟﻌﻠﻢ ﻭﺍﻟﻘﺮﺁﻥ
ﻒ :١/ ١٠٢
. ﺨﺎﺭﻱ: ﻛﺘﺎﺏ ﺍﻵ
ﺔﻔﲢ ﺍ
ﻷ
ﻱﺫﻮﺣ
٨: /
ﺍﳊﺪﻳﺚ ﺍﻟﺴﺎﺑﻖ
. ﱯﻃﺮﻘﻟﺍ:١٣ /٥٣
ﻳﺼﺪﻩ ﻋﻦ ﺫﻛﺮ ﺍﷲ
٤١ ﻭﺇﻳﻀﺎﺡ ﺍﳌﻮﻗﻒ
ﻠﻢ :ﺻﺤﻴﺢ ﺍﻟﺒﺨ
ﺩﻭ :٤/ ٣٠٤
ﻭﲢ
٧١ ﻭﺗﻜﻤﻠﺘﻪ ﺑﻨﺤﻮ
١-١٠٣
ﺍ ﲑﺴﻔﺗﻭ
٧
ﻪﺘﻠﻤﻜﺗﻭ
ﺣﱴ )
ﱯﻃﺮﻘﻟﺍ
٦٤/ ٣١ :
ﺟﻪ
ﻱﺭﺎﺨﺒﻟﺍ
ﻠﺴﻣﻭ
١٧ ﻦﻨﺳﻭ
ﰊﺃ ﻭﺍﺩ
ﻪ
ﻢﻠﺴﻣ
٩٦٧ /٥:
ﺡ ﻟﺍ
ﻮ
ﻗﻒ
٢٠/١:
ﺢ
ﻱﺭﺎﺨﺒﻟﺍ
:٤/٧٤
)١( ﲑﺴﻔﺗ
)٢(
ﻪﺟﺮﺧﺃ
٥/٦٩
)٣(
ﻪﺟﺮﺧﺃ
)٤(
ﺡﺎﻀﻳﺇ
)٥(
ﺢﻴﺤﺻ
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bèj’ ﻝﻮ
ﷲﺍ
ﺱﺪﻘﻟﺍ«.)٢(
ﺫﺍﺑﺎ ﻋﻦ
ﻮﺳﺭ
ﺍ ﺃﻳﺪﻩ ﺑﺮﻭﺡ
ﺍﳌﺸﺮﻛﲔ ﻭﺫ
ﺃ ﺍﷲ، ﺍﻟﻠﻬﻢ
ﺭﺍﺩﺍ ﺑﻪ ﻋﻠﻰ
ﻋﻦ ﺭﺳﻮﻝ
ﺭ ﺸﺪ ﺍﻟﺸﻌﺮ
ﺴﺎﻥ :ﺃﺟﺐ
ﺴﺎﻥ ﻭﻫﻮ ﻳﻨﺸ
ﲔ :
»
ﺎﻳ ﺴﺣ
ﺴﳊ
ﲔﻤ . (٤)ﺴﻨﻪ ﺩ ﺍﳋﺎﻣﺲ
ﲑﻩ ﺑﺎﻟﺘﻮﻗﻒ
ﺤﺎﺑﺔ ﺃﻭ ﻣﻦ
ﺮﻈﻨﻳﻭ
:
ﺳﻨﻦ ﺃﰊ
ﻱ ﺃﻥ ﺍﻟﺸﺎﻋﺮ
ﻮﻫ
ﺭﻗﻢ )٥٢٢
(.
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bèj’ ﺍﻟﻌﺪﺩ
ﻣﻮﻥ ﰲ ﺗﻔﺴﲑ
ﱰﻳﻞ ﻣﻦ ﺍﻟﺼﺤ
ﱯﻃﺮ:١٣ / ١٥٣
ﺐ ﺻﺤﻴﺢ، ﻭﺭﻭﻱ
ﻢﻠﺴﻣ :٥/
١٧٦٧
ﺭ
ﺔ
ﺔﻋﺎﲨ
ﻳﻣﺰﻠ
ﻤﻦ ﺷﺎﻫﺪ ﺍﻟﺘﱰ
ﻋﺒﻴﺪ، ﻭﺗﻔﺴﲑ ﺍﻟﻘﺮ
ﺣﺪﻳﺚ ﺣﺴﻦ ﻏﺮﻳﺐ
ﺮﻈﻨﻳﻭ
:
ﻣ ﺢﻴﺤﺻ
ﺣﻮﻟﻴﺔ
ﲟﻘﺘﻀﻰ ﺍﻟﻠﻐﺔ
،
ﻭﺃﻤﻋ
ﻋ ﺍﻟﺘﻮﺟﻴﻪ ﻋﻦ ﺃﰊ
:٨/ ١٣٩
ﺣ ﻝﺎﻗﻭ
ﱯﻃﺮﻘﻟ
:٣/ ١٥٣ ،
•ﺣ
ﻔﺴﲑ ﺍﻟﻘﺮﺁﻥ
ﻒ ﻋﻦ ﺍﻟﻨﱯ
-١٠٣
ﻭﻧﻘﻞ ﻫﺬﺍ
. ٧
:
ﲢﻔﺔ ﺍﻷﺣﻮﺫﻱ
١-١٢٨
ﻭﺗﻔﺴﲑ ﺍ
ﻭﳑﻦ ﻣﻨﻊ ﺗﻔ
ﻉﻮﻤﺴ،
ﺑﺘﻮﻗﻴﻒ
ﺍﻟﻮﻗﻒ
:١/ ١٠٢-
٤/٣٠٤
. ﺢ
ﻱﺭﺎﺨﺒﻟﺍ
٤/٤:
ﻱﺬﻣﺮﺘﻟ ﻲﺋﺎﺴﻨﻟﺍﻭ:
ﺑﻦ ﻣﺎﻟﻚ
. ﻱﱪﻄﻟﺍ
:١٩ / ١٢٧
ﺎﺜﻟﺎﺛ:
ﻋﻠﻰ ﺍﳌﺴ
)١(
ﺡﺎﻀﻳﺇ
ﺩﻭﺍﺩ
:٤
)٢(
ﺢﻴﺤﺻ
)٣(
ﺍﻟﺭﻭﺍﻩ
ﻛﻌﺐ
)٤( ﲑﺴﻔﺗ ﺮﻈﻨﻳﻭ
:
ﺳﻨﻦ ﺃﰊ
ﻱ ﺃﻥ ﺍﻟﺸﺎﻋﺮ
ﻮﻫ
ﺭﻗﻢ )٥٢٢
(. ﱯﻃﺮ:١٣ / ١٥٣
ﺐ ﺻﺤﻴﺢ، ﻭﺭﻭﻱ
ﻢﻠﺴﻣ :٥/
١٧٦٧
ﺭ
ﻋﺒﻴﺪ، ﻭﺗﻔﺴﲑ ﺍﻟﻘﺮ
ﺣﺪﻳﺚ ﺣﺴﻦ ﻏﺮﻳﺐ
ﺮﻈﻨﻳﻭ
:
ﻣ ﺢﻴﺤﺻ
ﻋ ﺍﻟﺘﻮﺟﻴﻪ ﻋﻦ ﺃﰊ
:٨/ ١٣٩
ﺣ ﻝﺎﻗﻭ
ﱯﻃﺮﻘﻟ
:٣/ ١٥٣ ،
-١٠٣
ﻭﻧﻘﻞ ﻫﺬﺍ
. ٧
:
ﲢﻔﺔ ﺍﻷﺣﻮﺫﻱ
١-١٢٨
ﻭﺗﻔﺴﲑ ﺍ
ﺍﻟﻮﻗﻒ
:١/ ١٠٢-
٤/٣٠٤
. ﺢ
ﻱﺭﺎﺨﺒﻟﺍ
٤/٤:
ﻱﺬﻣﺮﺘﻟ ﻲﺋﺎﺴﻨﻟﺍﻭ:
ﺑﻦ ﻣﺎﻟﻚ
. ﻱﱪﻄﻟﺍ
:١٩ / ١٢٧
)١(
ﺡﺎﻀﻳﺇ
ﺩﻭﺍﺩ
:٤
)٢(
ﺢﻴﺤﺻ
)٣(
ﺍﻟﺭﻭﺍﻩ
ﻛﻌﺐ
)٤( ﲑﺴﻔﺗ
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bèj’ ﻔﺴﲑ ﺑﺎﻟﺮﺃﻱ
ﻞﺟﻭ
ﻪﻳﺃﺮﺑ
ﻋﻦ ﻣﺜﻠﻪ ﻋﻦ
ﺿﻲ ﺍﷲ ﻋﻨﻪ
٨٥
، ﻓﻘﺎﻝ
:
ﻢ«.)٣( ﺎﻤﻛ
ﻋﺒﺲ :١٣
، ﺇﺫ
ﻊ ﺇﱃ
ﻪﺴﻔﻧ
ﺱ ﻭﻋﻤﺮ ﺑﻦ
ﻋﻦ ﺍﻟﺘﻔﺴﲑ
ﻔﺴﺮ ﻟﻪ ﺇﱃ
ﻴﻪ، ﻻﺣﺘﻴﺎﺝ
ﻣﻦ ﻗﺒﻴﻞ ﺍﻟﺘﻔ
ﻛﺘﺎﺏ ﺍﷲ ﻋﺰ
ﻋ ﺃﻭ ﺍﻻﻣﺘﻨﺎﻉ
ﻦ ﺃﰊ ﺑﻜﺮ ﺭﺿ
Ï
n
ﺀﺎﺴﻨﻟﺍ
:
ﷲ ﻣﺎ ﱂ ﺃﻋﻠﻢ
Á
Â
n
ﺒﻋ
ﻷﺏ؟ ﰒ ﺭﺟﻊ
ﺸﺔ ﻭﺍﺑﻦ ﻋﺒﺎﺱ
ﺩﺭ
ﺓ
ﰲ ﺍﻟﻨﻬﻲ
ﻻﺣﺘﻴﺎﺝ ﺍﳌﻔ
ﻰ ﺃﺣﺪ ﻣﻌﻨﻴﻴﻪ
ﺍﻟﻠﻐﻮﻱ ﻫﻮ
ﻣﻦ ﻛ ﻗﺎﻝ ﰲ
ﺃ ﺩ ﺍﻟﻨﻬﻲ ﻋﻨﻪ
ﻣﺎ ﺭﻭﻱ ﻋﻦ
Ë
Ì
Í
Î
Ï
ﰲ ﻛﺘﺎﺏ ﺍﷲ
ﻮﻟﻪ ﺗﻌﺎﱃ: o
Á
ﻓﻨﺎﻫﺎ، ﻓﻤﺎ ﺍﻷ
ﻭﻱ ﻋﻦ ﻋﺎﺋﺸ
ﻭﺍﻵﺛﺎﺭ ﺍﻟﻮﺍﺭ
ﻔﺴﲑ ﺍﻟﻠﻔﻆ
ﻞ ﺍﻟﻠﻔﻆ ﻋﻠﻰ
ﻛﺎﻥ ﺍﻟﺘﻔﺴﲑ
ﻪﻟ
: »ﻣ
ﺍﺬ .
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bèj’ ﺎﻤﻛﻭ
ﺩﺭ
ﺫﻟﻚ ﻣﻦﻒ،
: ﱃo
Ê
Ë
ﻘﻠﲏ ﺇﻥ ﻗﻠﺖ
ﲑ)ﺃﺑ
( ﺎ
ﻣﻦ ﻗﻮ
ﻛﻬﺔ ﻗﺪ ﻋﺮﻓ
٤( ﺍﺬﻜﻫﻭ
ﻭﺭ
ﻩ ﺍﻷﺣﺎﺩﻳﺚ
ﲑ ﺗﻔ: ﺎﺃﺣﺪﳘ
ﺎﻤﻬﻴﻧﺎﺛﻭ :ﻞﲪ
ﲔ.)١( ﺎﳌﻭ ﻛ
ﻨﻬﻲ ﻋﻨﻪ
ﻟﻮﻘﺑ
،)٢( ﻮﳓﻭ
ﺬﻫ
ﺤﺎﺑﺔ ﻭﺍﻟﺴﻠﻒ
ﻣﻦ ﻗﻮﻟﻪ ﺗﻌﺎ
ﺃﻱ ﺃﺭﺿﻲ
ﻘﺗ
ﻋﻪ ﻋﻦ ﺗﻔﺴﲑ
ﻝ :ﻫﺬﻩ ﺍﻟﻔﺎﻛ
ﻒ ﻳﺎ ﻋﻤﺮ.)٤
)٥( ﺍﻮﻟﱰﻓ
ﺬﻫ
ﻦﻣ
ﲑﺴﻔﺘﻟﺍ
ﺎﻥ ﺍﻟﻌﺮﺏ،
ﻭ
ﻬﻢ ﻣﻦ ﺍﻟﺘﺎﺑﻌﲔ
ﻮﻉ ﻟﻮﺭﻭﺩ ﺍﻟﻨﻬ
ﻓﻘﺪ ﺃﺧﻄﺄ
«
ﻦ ﻛﺒﺎﺭ ﺍﻟﺼﺤ
ﻦﻋ
) ﺎﺘﻴﻘﻣ
(
ﲰﺎﺀ ﺗﻈﻠﲏ، ﻭﺃ
ﻦ ﻋﻤﺮ ﺍﻣﺘﻨﺎﻋ
ﻠﻰ ﺍﳌﻨﱪ ﻓﻘﺎﻝ
ﻥ ﻫﺬﺍ ﺍﻟﺘﻜﻠﻒ
ﺰﻳﺰ ﻭﻏﲑﻫﻢ.)
ﻋﻠﻰ ﻗﺴﻤﲔ
ﰲ ﻣﻌﺮﻓﺔ ﻟﺴﺎ
ﺃﺧﺬ ﻋﻨﻬ
ﻓﻬﻮ ﳑﻨﻮ
ﺏﺎﺻﺄﻓ
ﲨﺎﻋﺔ ﻣﻦ
ﺃ
ﻧﻪ ﺳﺌﻞ
»ﻱﺃ ﺎﲰ
ﺭﻭﻱ ﻋﻦ
ﻗﺮﺃﻫﺎ ﻋﻠ
ﻝﺎﻘﻓ
ﺇﻥ:
ﻋﺒﺪ ﺍﻟﻌﺰ
ﻋ ﻱﺃﺮﻟﺎﺑ
ﰲ ﺮﺤﺒﺘﻟﺍ ﺩ ﺍﳋﺎﻣﺲ
،ﻦﻨﺳ
ﺃﰊ ﺩﺍﻭﺩ
:
ﺮﺳﻼ ﺑﻠﻔﻆ ﺁﺧﺮ . ﺍﻟﻌﺪﺩ
ﰊ ﺣﺰﻡ ﺗﻜﻠﻤﻮﺍ ﻓﻴﻪ
ﺩ. ﻜﺔ ﻋﻦ ﺃﰊ ﺑﻜﺮ ﻣﺮ
ﻭﻓﻴﻪ ﺳﻬﻴﻞ ﺑﻦ ﺃﰊ
ﻆﻔﻠﻟﺍﻭ
ﻷﰊ ﺩﺍﻭﺩ
ﺭﻭﺍﻩ ﺍﺑﻦ ﺃﰊ
ﺔﻜﻴﻠﻣ
ﲑﺴ :١٠٣
. ﺣﻮﻟﻴﺔ
ﻟﺘﺮﻣﺬﻱ :ﻭ ،ﻏﺮﻳﺐ
ﺪﺋﺍﻮﻔﻟﺍ
:٢/ ١٦٢
. ٢٢٠٠ (ﻝﺎﻗﻭ
ﻭﺭ:
ﻣﺔ ﰲ ﺃﺻﻮﻝ ﺍﻟﺘﻔﺴ
•ﺣ
:٢/ ١٦٤
. ﻭﺍﻟﻨﺴﺎﺋﻲ، ﻗﺎﻝ ﺍﻟﺘ
ﻱ:٨/ ٢٧٩
ﻊﲨﻭ
٥/ ٢٩٢
ﺑﺮﻗﻢ
)٠
١/٣٧ -٣٨ ﺪﻘﻣﻭ
١٨ . . ﻥﰲ
ﻋﻠﻮﻡ ﺍﻟﻘﺮﺁﻥ
ﺑﻮ ﺩﺍﻭﺩ ﻭﺍﻟﺘﺮﻣﺬﻱ
٣
، ﲢﻔﺔ ﺍﻷﺣﻮﺫﻱ
ﻥﺎﳝﻹﺍ
:
ﻲﻘﻬﻴﺒﻟﺍ
:
:ﺗﻔﺴﲑ ﺍﻟﻄﱪﻱ
:
ﺏﺎﺘﻛ
ﺍﳌﺒﺎﱐ
:٨٤
ﺭ
ﻖﺑﺎﺴﻟﺍ
٢٠٢ :
. )١(
ﻥﺎﻫﱪﻟﺍ
)٢(
ﺃﺑﺭﻭﺍﻩ
٣/١٩
ﺷﻌﺐ (٣)
ﺮﻈﻨﻳﻭ:
)٤( ﺔﻣﺪﻘﻣ
)٥(
ﺭﺪﺼﳌﺍ ،ﻦﻨﺳ
ﺃﰊ ﺩﺍﻭﺩ
:
ﺮﺳﻼ ﺑﻠﻔﻆ ﺁﺧﺮ . ﰊ ﺣﺰﻡ ﺗﻜﻠﻤﻮﺍ ﻓﻴﻪ
ﺩ. ﻜﺔ ﻋﻦ ﺃﰊ ﺑﻜﺮ ﻣﺮ
ﻭﻓﻴﻪ ﺳﻬﻴﻞ ﺑﻦ ﺃﰊ
ﻆﻔﻠﻟﺍﻭ
ﻷﰊ ﺩﺍﻭﺩ
ﺭﻭﺍﻩ ﺍﺑﻦ ﺃﰊ
ﺔﻜﻴﻠﻣ
ﲑﺴ :١٠٣
. ﻟﺘﺮﻣﺬﻱ :ﻭ ،ﻏﺮﻳﺐ
ﺪﺋﺍﻮﻔﻟﺍ
:٢/ ١٦٢
. ٢٢٠٠ (ﻝﺎﻗﻭ
ﻭﺭ:
ﻣﺔ ﰲ ﺃﺻﻮﻝ ﺍﻟﺘﻔﺴ
:٢/ ١٦٤
. ﻭﺍﻟﻨﺴﺎﺋﻲ، ﻗﺎﻝ ﺍﻟﺘ
ﻱ:٨/ ٢٧٩
ﻊﲨﻭ
٥/ ٢٩٢
ﺑﺮﻗﻢ
)٠
١/٣٧ -٣٨ ﺪﻘﻣﻭ
١٨ . . ﻥﰲ
ﻋﻠﻮﻡ ﺍﻟﻘﺮﺁﻥ
ﺑﻮ ﺩﺍﻭﺩ ﻭﺍﻟﺘﺮﻣﺬﻱ
٣
، ﲢﻔﺔ ﺍﻷﺣﻮﺫﻱ
ﻥﺎﳝﻹﺍ
:
ﻲﻘﻬﻴﺒﻟﺍ
:
:ﺗﻔﺴﲑ ﺍﻟﻄﱪﻱ
:
ﺏﺎﺘﻛ
ﺍﳌﺒﺎﱐ
:٨٤
ﺭ
ﻖﺑﺎﺴﻟﺍ
٢٠٢ :
. )١(
ﻥﺎﻫﱪﻟﺍ
)٢(
ﺃﺑﺭﻭﺍﻩ
٣/١٩
ﺷﻌﺐ (٣)
ﺮﻈﻨﻳﻭ:
)٤( ﺔﻣﺪﻘﻣ
)٥(
ﺭﺪﺼﳌﺍ ،ﻦﻨﺳ
ﺃﰊ ﺩﺍﻭﺩ
:
ﺮﺳﻼ ﺑﻠﻔﻆ ﺁﺧﺮ . ﰊ ﺣﺰﻡ ﺗﻜﻠﻤﻮﺍ ﻓﻴﻪ
ﺩ. ﻜﺔ ﻋﻦ ﺃﰊ ﺑﻜﺮ ﻣﺮ
ﻭﻓﻴﻪ ﺳﻬﻴﻞ ﺑﻦ ﺃﰊ
ﻆﻔﻠﻟﺍﻭ
ﻷﰊ ﺩﺍﻭﺩ
ﺭﻭﺍﻩ ﺍﺑﻦ ﺃﰊ
ﺔﻜﻴﻠﻣ
ﲑﺴ :١٠٣
. ﻟﺘﺮﻣﺬﻱ :ﻭ ،ﻏﺮﻳﺐ
ﺪﺋﺍﻮﻔﻟﺍ
:٢/ ١٦٢
. ٢٢٠٠ (ﻝﺎﻗﻭ
ﻭﺭ:
ﻣﺔ ﰲ ﺃﺻﻮﻝ ﺍﻟﺘﻔﺴ
:٢/ ١٦٤
. ﻭﺍﻟﻨﺴﺎﺋﻲ، ﻗﺎﻝ ﺍﻟﺘ
ﻱ:٨/ ٢٧٩
ﻊﲨﻭ
٥/ ٢٩٢
ﺑﺮﻗﻢ
)٠
١/٣٧ -٣٨ ﺪﻘﻣﻭ
١٨ . .
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bèj’ ﻥﰲ
ﻋﻠﻮﻡ ﺍﻟﻘﺮﺁﻥ
ﺑﻮ ﺩﺍﻭﺩ ﻭﺍﻟﺘﺮﻣﺬﻱ
٣
، ﲢﻔﺔ ﺍﻷﺣﻮﺫﻱ
ﻥﺎﳝﻹﺍ
:
ﻲﻘﻬﻴﺒﻟﺍ
:
:ﺗﻔﺴﲑ ﺍﻟﻄﱪﻱ
:
ﺏﺎﺘﻛ
ﺍﳌﺒﺎﱐ
:٨٤
ﺭ
ﻖﺑﺎﺴﻟﺍ
٢٠٢ :
. )١(
ﻥﺎﻫﱪﻟﺍ
)٢(
ﺃﺑﺭﻭﺍﻩ
٣/١٩
ﺷﻌﺐ (٣)
ﺮﻈﻨﻳﻭ:
)٤( ﺔﻣﺪﻘﻣ
)٥(
ﺭﺪﺼﳌﺍ ﻋﻪ.) (١
ﻋ ﻥﻭ.)٢( ﺩ ﺍﳋﺎﻣﺲ
ﺍﻟﻌﺪﺩ
١٦ﻭ١٦٨
. ﺮﻈ :ﻥﺎﻫﱪﻟﺍ
:٢/٢
ﺣﻮﻟﻴﺔ
ﻥﻭ:١/ ٢٥٧
، ﻭﻳﻨﻈ
•ﺣ
:٢/ ١٦٨
. ﻭﺍﻟﺘﻔﺴﲑ ﻭﺍﳌﻔﺴﺮ
٢ . . ﻥﰲ
ﻋﻠﻮﻡ ﺍﻟﻘﺮﺁﻥ
ﻁﺎﺒﻨﺘﺳﻻﺍ
٢٤١ :
ﻦﺑﺍ
ﻋﻄﻴﺔ
٣٦٢ :
ﱯﻃﺮﻘﻟﺍ
٣٣/١:
)١(
ﻥﺎﻫﱪﻟﺍ
)٢( ﻝﻮﺻﺃ
)٣( ﺔﻣﺪﻘﻣ
)٤( ﲑﺴﻔﺗ
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bèj’ ﺩ ﺍﳋﺎﻣﺲ
ﺍﻟﻌﺪﺩ
ﺴﲑ ﺑﺎﻟﻈﻦ .(
ﺣﻮﻟﻴﺔ
ﺼﻞ ﰲ )ﺗﺮﻙ ﺍﻟﺘﻔﺴ
•ﺣ
ﻢﻗﺮﺑ
)
٢١٩٩
ﻓﺼ (
٢/ ١٦٢
. ﻥﺎﳝﻹﺍ
٢٩٢ /٥:
ﰲﻥ
ﻋﻠﻮﻡ ﺍﻟﻘﺮﺁﻥ
:
)ﺷﻌﺐ (١
)٢(
ﻥﺎﻫﱪﻟﺍ ﺩ ﺍﳋﺎﻣﺲ
ﺍﻟﻌﺪﺩ
ﺴﲑ ﺑﺎﻟﻈﻦ .(
ﺣﻮﻟﻴﺔ
ﺼﻞ ﰲ )ﺗﺮﻙ ﺍﻟﺘﻔﺴ
•ﺣ
ﻢﻗﺮﺑ
)
٢١٩٩
ﻓﺼ (
٢/ ١٦٢
. ﻥﺎﳝﻹﺍ
٢٩٢ /٥:
ﰲﻥ
ﻋﻠﻮﻡ ﺍﻟﻘﺮﺁﻥ
:
)ﺷﻌﺐ (١
)٢(
ﻥﺎﻫﱪﻟﺍ
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bèj’ ﺩ ﺍﳋﺎﻣﺲ
ﺣﺪ ﻣﻦ ﺃﻫﻞ
ﻄﻒ ﻣﻨﻪ ﻣﺎ
ﻻ ﳜﻠﻮ ﺇﻣﺎ
ﻃﻞ ﻗﻄﻌﺎ ﺃﻥ
ﺎﻫﺪﺣﺃ
ﺃﻧﻪ:
ﻭﺫﻟﻚ ﻻ،
ﻦﻣ
ﻢﻬﺴﻔﻧﺃ
ﺔ ﻭﺍﳌﻔﺴﺮﻳﻦ
ﳉﻤﻊ ﺑﻴﻨﻬ،ﺎ
ﻣﺎ ﺍﻟﻨﻬﻲ ﻓﺈﻧﻪ
ﻓﻴﺘﺄﻭﻟﻪ ﻋﻠﻰ
ﻻﺡ ﻟﻪ ﻣﻦ
ﺭﺓ ﻳﻜﻮﻥ ﻣﻊ
،ﺍﻓﻖ ﻏﺮﺿﻪ
ﺍﻟﻌﺪﺩ
ﻻ ﳚﻬﻠﻪ ﺃﺣ
ﻡ ﻧﻔﻴﺲ ﻧﻘﺘﻄ
ﻥﺁﺮﻘ ﻱﺃﺮﻟﺎﺑ
ﺮ ﺁﺧﺮ، ﻭﺑﺎﻃ
ﻤﻌﻪ ،ﻟﻮﺟﻮﻩ
ﺪﻨﺴﻣﻭ
،ﻪﻴﻟﺇ
ﺍﺑﻦ ﻣﺴﻌﻮﺩ
ﺃﻥ ﺍﻟﺼﺤﺎﺑﺔ
ﻔﺔ ﻻ ﳝﻜﻦ ﺍﳉ
ﻇﻬﺮ ﻟﻪ
ﻭﺃﻣ . ﻪﻌﺒﻃ
،ﻩﺍﻮﻫﻭ
ﻚ ﺍﳍﻮﻯ
ﺎﳌ
ﻪﻤﺼﺧ. ﺭﺎﺗﻭ
ﺟﻪ ﺍﻟﺬﻱ ﻳﻮ
ﺐ ﻫﺬﺍ ﺍﻟﻘﻮﻝ . ﺀ:١/ ٢٨٨
-١
ﺀ : . ٧٣/١
)١(
ﺀﺎﻴﺣﻹﺍ
)٢(
ﺀﺎﻴﺣﻹﺍ
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bèj’ ،ﺎ ﻓﺈﻥ ﺫﻟﻚ
ﳊﺪﻳﺚ ﻡﻛﻼ
ﻋﻦ ﺗﻔﺴﲑ
ﻘﻟﺍ
ﺍﳌﺮﺍﺩ ﺑﻪ
ﺮﻣﺃ
ﺇﻻ ﲟﺎ ﻳﺴﻤ
ﻝ ﺍﷲ
ﺍﺑﻦ ﻋﺒﺎﺱ ﻭﺍ
،
ﱐﺎﺜﻟﺍﻭ
:
ﻞﻳﻭﺎﻗﺃ
ﻔﻠﺘﳐ
ﰲ ﺍﳌﻌﲎ
ﺎﲟ
ﻴﻪ ﻣﻴﻞ ﻣﻦ
ﻃ
ﺿﻪ، ﻭﻟﻮﻻ ﺫﻟﻚ
ﺧ ﺗﻠﺒﻴﺴﺎ ﻋﻠﻰ
ﻬﻤﻪ ﺇﱃ ﺍﻟﻮﺟ
ﱂ ﻳﺸﺮ ﺇﱃ ﺻﺎﺣﺐ
ﺣﻮﻟﻴﺔ
ﻭﻥ ﻣﺎ
ﺎﻫﺍﻮﺳ
ﻬﻲ ﺍﻟﻮﺍﺭﺩ
ﳊﺎﺑ
:
ﻋ ﺇﻥ ﺍﻟﻨﻬﻲ
ﻰ ﺍﻟﻨﻘﻞ، ﺃﻭ
ﺪ ﰲ ﺍﻟﻘﺮﺁﻥ
ﻋﺎ ﻣﻦ ﺭﺳﻮ
ﺍ ﻣﺎ ﻣﺎ ﻳﻘﻮﻟﻪ
ﺴﻤﻌﻮﻩ ﻣﻨﻪ
ﻓﻘﺎﻟﻮﺍ ﻓﻴﻬﺎ
ﻞ ﻣﻔﺴﺮ ﻗﺎﻝ
ﻲﺀ ﺭﺃﻱ، ﻭﺇﻟﻴ
ﺗﺼﺤﻴﺢ ﻏﺮﺿ
ﻋﻠﻰ ﻭﺟﻮﻩ:
ﺎﺤ
ﻪﺘﻋﺪﺒﻟ
ﺗﻭ
ﳏﺘﻤﻠﺔ ﳝﻴﻞ
ﻬﻓ
ﻥﺁﺮﻘ:٢/ ١٧٠
ﱂﻭ
•ﺣ
ﺎ ﺍﳋﺎﺻﺔ ﺩﻭ
ﰲ ﺗﻮﺟﻴﻪ ﺍﻟﻨﻬ
ﺚ ﺑﺎﺧﺘﺼﺎﺭ
ﻻﻗﺘﺼﺎﺭ ﻋﻠﻰ
ﻻ ﻳﺘﻜﻠﻢ ﺃﺣﺪ
ﻟﺫ
ﻚ ﻣﺴﻤﻮﻋ
ﺾ ﺍﻟﻘﺮﺁﻥ، ﺃﻣ
ﻢ ﱂ ﻳﺴ ﻷ
ﻌﺾ ﺍﻵﻳﺎﺕ،
ﻓﺜﺒﺖ ﺃﻥ ﻛﻞ
ﲔ:
ﻥ ﻟﻪ ﰲ ﺍﻟﺸﻲ
ﺗﺼ ﺘﺞ ﺑﻪ ﻋﻠﻰ
ﻋ ﻭﻫﺬﺍ ﳚﺮﻱ
ﻟﻌﻠﻢ، ﺗﺼﺤﻴﺤ
ﳏ ﻛﺎﻧﺖ ﺍﻵﻳﺔ
ﻟﱪﻫﺎﻥ ﰲ ﻋﻠﻮﻡ ﺍﻟﻘ
ﺎﻓﺎﺕ ﺑﺼﻔﺎ
ﻠﺴﺎﻥ.) (١
ﻡﺎﻣ ﰲ ﱄﺍﺰﻐﻟﺍ
ﻮﺿﻮﻉ ﺍﻟﺒﺤﺚ
ﻥ ﺍﳌﺮﺍﺩ ﺑﻪ ﺍ
ﻻ ﳌﺮﺍﺩ ﺑﻪ ﺃﻥ
ﺫ ﺃﻥ ﻳﻜﻮﻥ
ﺇﻻ ﰲ ﺑﻌﺾ
ﺴﲑ ﺑﺎﻟﺮﺃﻱ،
ﰲ ﺗﻔﺴﲑ ﺑﻌ
ﲨﻴﻌﻬﺎ ﳏﺎﻝ،
ﻰ ﺃﺣﺪ ﻭﺟﻬﲔ
ﳘﺎ :ﺃﻥ ﻳﻜﻮﻥ
ﻪ ﻭﻫﻮﺍﻩ ﻟﻴﺤﺘ
ﺫﻟﻚ ﺍﳌﻌﲎ
ﻭ ،
ﻥﻮﻜﻳ
ﻣ
ﻊ
ﻌﻟﺍ
ﻛ ﻭﻟﻜﻦ ﺇﺫﺍ
ﻱﱪﻄﻟﺍ
:١/٣٣ ﺍﻭ
ﻮﺻﻮﳌﺍﻭ
ﺍﻟﻌﻠﻢ ﺑﺎﻟﻠ
ﺎﻣﻺﻟﻭ
ﻳﺘﻌﻠﻖ ﲟﻮ
ﺃﻥ ﻳﻜﻮﻥ
ﻳﻜﻮﻥ ﺍﳌ
ﻁﺮﺘﺸﻳ
ﻳﺼﺎﺩﻑ
ﻓﻬﻮ ﺗﻔﺴ
ﺍﻮﻔﻠﺘﺧﺍ
ﲨ ﻉﺎﲰﻭ
ﻳﱰﻝ ﻋﻠﻰ
ﳘﺪﺣﺃ
ﻭﻓﻖ ﺭﺃﻳﻪ
ﺫ ﻥﺁﺮﻘﻟﺍ
ﻓﺓﺭﺎﺘ
،ﻞﻬﳉﺍ
)١( ﲑﺴﻔﺗ ﷲ
،ﻪ ¡
¢
£
¤
ﻝ:
o
¡ ﺩ ﺍﳋﺎﻣﺲ
ﻊﲨ
،ﻥﺁﺮﻘﻟﺍ
ﺪ ﻻ ﻳﺼﻴﺐ
ﻭﻫﻮ ﻻ ﳝﻨﻊ
ﻜﺮ ﺭﺿﻲ ﺍﷲ
، ﺃﻭﻝ ﺍﻷﻣﺮ
ﻌﻤﺮ ﺃﻳﻀﺎ ﺇﺫ
ﻋﻦ ﺗﻔﺴﲑﻫﺎ
ﺎﻳﻮﻐ .ﺮﺴﻓﻭ
ﻞ ﻣﺎ ﺻﺎﺩﻑ
ﲑﻩ ﺃﻧﻪ ﺍﻟﺘﺒﺲ
P
Q
n
ﻣﻦ ﺃﺻﺤﺎﺏ
، ﻣﻦ ﻛﻨﺎﻧﺔ
ﻣﺎ ﺍﳊﺮﺟﺔ؟
ﺍﻟﻌﺪﺩ
ﻝ ﺍﻷﻣﺮ ﻋﻦ
ﲨ
ﺍﻟﺘﻔﺴﲑ، ﻭﻗﺪ
ﺎ ﺳﺌﻞ ﻋﻨﻪ،
ﺻﺎﺭ ﺃﺑﻮ ﺑﻜ
ﻋﻦ ﺗﻔﺴﲑﻫﺎ
ﻛﻤﺎ ﺣﺼﻞ ﻟﻌ
ﻋ ﻋﻠﻴﻪ ﻭﺍﻣﺘﻨﻊ
ﺃﻧﻪ ﻓﺴﺮﻫﺎ ﻟﻐ
،ﺏﺮﻌﻟﺍ
ﻞﺜﻣ
ﺍﻟﻄﱪﻱ ﻭﻏﲑ
N
O
P
ﻣ ﻛﺎﻥ ﻋﻨﺪﻩ
ﺑﻐﻮﱐ ﺭﺟﻼ
ﻋﻤﺮ :ﻳﺎ ﻓﱴ
ﻦ ﺗﺮﺩﺩﻩ ﺃﻭﻝ
ﻰ ﻣﺬﻫﺒﻪ ﰲ
ﻪﻤﻠﻋ
ﻫﻮ ﲟﺎ
ﺏ.)١( ﺍﺬﳍﻭ
ﻋ ﻪ ﺍﻻﻣﺘﻨﺎﻉ
ﻨﺎ ﻟﺬﻟﻚ .ﻭﻛ
ﻱ
ﻋ ﻞﻜﺷﺃ
،ﻚ ﻣﻨﻪ
ﻊﻣ
ﺘﺠﺎ ﻟﻪ ﺑﻜﻼﻡ
ﺍ ﺝ ﺍﺑﻦ ﺟﺮﻳﺮ
K
L
M
N
ﻛ ﺃ ﺑﻌﺾ ﻣﻦ
ﻝ ﻋﻤﺮ :
»ﺃ
ﻋ ﻪ، ﻓﻘﺎﻝ ﻟﻪ
١٠٧ .
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bèj’ ﺣﻮﻟﻴﺔ
ﻷﰊ ﺑﻜﺮ ﻣﻦ
ﻣﺎﻣﺎ ﻳﺒﲎ
ﻰﻠﻋ
ﻨﻊ ﻣﻨﻪ ﻟﻌﺪﻡ
ﻫﺎ ﻣﻦ ﺍﻷﺳﺒﺎ
ﺃﻥ ﺭﻭﻱ ﻋﻨﻪ
ﺎﻫﲑ
ﺎﻤﻛ ﺎﻨﻠﺜﻣ
o
Â
n
ﺬﻟﺍ
ﺍﺳﺘﺤﺴﻦ ﺫﻟﻚ
ﺎ ﻳﻈﻬﺮ ﻟﻪ ﳏﺘﺠ
،
ﻓﻘﺪ ﺃﺧﺮﺝ
ﱃ: o
J
K
ﺢ ﺍﻟﺮﺍﺀ، ﻭﻗﺮﺃ
ﺍﻟﺮﺍﺀ ، ﻓﻘﺎﻝ
ﻗﺎﻝ :ﻓﺄﺗﻮﻩ ﺑﻪ
ﲑﺴﻔﺘﻟ :١٠٦
-٧
•ﺣ
ﺜﺎﻝ ﻣﺎ ﺍﺗﻔﻖ
ﻋﻦ ﺍﻟﻔﺘﻴﺎ . ﻲ ﻻ ﳚﻌﻞ ﺇ
ﳝﺘﻨﻊ ﻣﻦ ﺍﻣﺘﻨ
ﺑﻪ، ﺃﻭ ﻟﻨﺤﻮﻫ
،ﺔﻟﻼ)٢( ﺪﻌﺑ ﺃ
ﻅ ﺃﺧﺮﻯ ﻏﲑ
ﻣﻌﲎ ﻟﻔﻈﺔ
:
ﺍ ﺍﺑﻦ ﻋﺒﺎﺱ
ﻇﺎﻔﻟﹰ
ﺎ
ﺃﺧﺮ ﲟﺎ
ﺟ
ﲣ ، ﻭﺎ(ﻑﻮ،
ﻣﻦ ﻗﻮﻟﻪ ﺗﻌﺎ
ﻫﺎ ﻋﻤﺮ ﺑﻔﺘﺢ
ﺎﺟﹺ
ﺮ (ﺮﺴﻜﺑ
ﻗ ،ﻜﻦ ﻣﺪﳉﻴﺎ
ﻣﻘﺪﻣﺔ ﰲ ﺃﺻﻮﻝ ﺍﻟ
٢ . ﻣﻨﻪ، ﻣﺜﺎﻟﻪ ﻣﺜ
ﻋ ﺮﺩﺩ ﺑﻌﻀﻬﻢ
ﺭﻏﺒﺔ ﻣﻨﻪ ﻟﻜﻲ
ﺮﺍﺩ .ﻭﺇﻣﺎ ﺃﻥ
ﻥ
ﻏﲑﻩ ﻋﺎﳌﺎ
ﺗﻔﺴﲑ ﺍﻟﻜﻼ
ﰲ ﺗﻔﺴﲑ ﺃﻟﻔﺎﻅ
ﻦ ﻋﺒﺎﺱ ﻋﻦ
ﻭﳌﺎ ﻓﺴﺮﻫﺎ ﻟﻪ
ﺿﻲ ﺍﷲ ﻋﻨﻪ ﺃﻟ
ﱵﻤﻠﻛ)ﺟﺮﺣ
ﲎ )ﺍﳊﺮﺝ
(
١ ، ﻓﻘﺪ ﻗﺮﺃﻫ
)ﹺ
ﹰ ﺣﺮ
ﺎﻘﻴﺿ
ﻩ ﺭﺍﻋﻴﺎ، ﻭﻟﻴﻜ
ﻱﱪﻄﻟﺍ
:١/٣٩
ﻣﻭ
ﻱﱪﻄﻟﺍ
٤٧٢ /١:
ﳌﺍ
ﻞﻜﺸ
ﻭﻣﺜﺎﻝ ﺗﺮ
ﻭﺇﻣﺎ
ﺍﳊﻖ ﺍﳌﺮ
ﺃﻥ ﻳﻜﻮﻥ
ﻋﻨﻪ ﺇﱃ
ﰲ ﺍﺬﻛﻭ
ﺳﺄﻝ ﺍﺑﻦ
ﻭ ،ﻪﻳﺃﺮﺑ
ﻋﻤﺮ ﺭﺿ
ﻛ ﻟﻪ ﻣﻊ
ﻋﻠﻴﻪ ﻣﻌﲎ
ﻡﺎﻌﻧﻷﺍ
: ١٢٥
ﺍﻟﻨﱯ
ﻩﻮﻠﻌﺟﺍﻭ
)١( ﲑﺴﻔﺗ
)٢( ﲑﺴﻔﺗ ﺎ
ﻋ
ﻛ
ﻠﻋ
ﻪﻤﻠﻋ
ﻫﻮ ﲟﺎ
ﺏ.)١( ﺍﺬﳍﻭ
ﻋ ﻪ ﺍﻻﻣﺘﻨﺎﻉ
ﻨﺎ ﻟﺬﻟﻚ .ﻭﻛ
ﻱ
ﻋ ﻞﻜﺷﺃ
ﻨﻊ ﻣﻨﻪ ﻟﻌﺪﻡ
ﻫﺎ ﻣﻦ ﺍﻷﺳﺒﺎ
ﺃﻥ ﺭﻭﻱ ﻋﻨﻪ
ﺎﻫﲑ
ﺎﻤﻛ ﺎﻨﻠﺜﻣ
o
Â
n
ﺬﻟﺍ
ﳝﺘﻨﻊ ﻣﻦ ﺍﻣﺘﻨ
ﺑﻪ، ﺃﻭ ﻟﻨﺤﻮﻫ
،ﺔﻟﻼ)٢( ﺪﻌﺑ ﺃ
ﻅ ﺃﺧﺮﻯ ﻏﲑ
ﻣﻌﲎ ﻟﻔﻈﺔ
:
ﻥﺃ
ﺎﳌﺎ
ﻼﻜﻟ
ﻅﺎﻔﻟ
ﻦﻋ o
Â
n
ﺍ
ﺔﻈ:
òÌÜÛa
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À
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ÐnÛa
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òîvy
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bèj’ ١٥٤
ﺩ ﺍﳋﺎﻣﺲ
ﺭﺍﻋﻴﺔ، ﻭﻻ
ﻴﻪ ﺷﻲﺀ ﻣﻦ
ﻠﻰ ﺍﳌﻨﱪ ﻋﻦ
،ﺦ ﻣﻦ ﻫﺬﻳﻞ
ﺍﻟﻌﺮﺏ ﺫﻟﻚ
ﻦﹶ
ﻔﺴﻟ
ﻓﻴﻪ ﺗﻔﺴﲑ
١١-١١١
، ﻭﰲ
ﻮ ﰲ ﻟﺴﺎﻥ ﺍﻟﻌﺮﺏ
ﻧﻘﻼ ﻋﻦ ﺻﺤﺎﺡ
ﻟ
ﻌﺠﻼﻥ ﺍﻟﻨﻬﺪﻱ
. ﰲ ﹺﻱ
ﻭﺎﻀﻴﺒﻟﺍ ﻩﺍﺰ
ﺝ ﺍﻟﻌﺮﻭﺱ :
ﺓﺩﺎﻣ
ﻑ ﺍﻟﺴﲑ(، ﻳﻨﻈﺮ
:
ﻲِ
ﺴﺎ ﺍﻟﻘ ﺩﺮﺒﺗ . ﻲﺴﻘﻟﺍ ﺐ . ﻥﺎﺴﻟ
ﺍﻟﻌﺪﺩ
ﻳﺼﻞ ﺇﻟﻴﻬﺎ
، ﻻ ﻳﺼﻞ ﺇﻟﻴ
ﺳﺄﻝ ﻭﻫﻮ ﻋﻠ
٤
، ﻓﻘﺎﻡ ﺷﻴﺦ
ﺍ ﻓﻬﻞ ﺗﻌﺮﻑ
ﻧﺎﻗﺘﻪﻒ
:
ﻟﺍ ﺔﻌﺒﻨﻟﺍ ﺩﻮﻋ
ﳉﺎﻫﻠﻴﺔ، ﻓﺈﻥ
ﺎﳘ . ﲑ ﺍﻟﻘﺮﻃﱯ:٠/ ٠١
ﺍﻟﺒﻴﺖ ﻟﺰﻫﲑ، ﻭﻫﻮ
ﻧﻘ ﻋﺰﺍﻩ ﻟﺬﻱ ﺍﻟﺮﻣﺔ
ﻭ ﻋﺒﺪ ﺍﻟﻠﻴﺚ
ﻦﺑﻟﺍ
ﺎﻝ ﺍﻟﺰﺑﻴﺪﻱ :
»ﺰﻋﻭ
ﻱﹺ
ﻫﺬﺍ ﺍﻟﺮﻭ
«.
òÌÜÛa
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bèj’ ﻥﺎﺴﻟ
ﺍﻟﻌﺪﺩ
ﺳﺄﻝ ﻭﻫﻮ ﻋﻠ
٤
، ﻓﻘﺎﻡ ﺷﻴﺦ
ﺍ ﻓﻬﻞ ﺗﻌﺮﻑ
ﻧﺎﻗﺘﻪﻒ
:
ﻟﺍ ﺔﻌﺒﻨﻟﺍ ﺩﻮﻋ
ﳉﺎﻫﻠﻴﺔ، ﻓﺈﻥ
ﺎﳘ . ﲑ ﺍﻟﻘﺮﻃﱯ:٠/ ٠١
ﺍﻟﺒﻴﺖ ﻟﺰﻫﲑ، ﻭﻫﻮ
ﻧﻘ ﻋﺰﺍﻩ ﻟﺬﻱ ﺍﻟﺮﻣﺔ
ﻭ ﻋﺒﺪ ﺍﻟﻠﻴﺚ
ﻦﺑﻟﺍ
ﺎﻝ ﺍﻟﺰﺑﻴﺪﻱ :
»ﺰﻋﻭ
ﻱﹺ
ﻫﺬﺍ ﺍﻟﺮﻭ
«. ﺝﺎﺗ
ﺃﻳﻀﺎ ﺑﻠﻔﻆ
ﲣﻮﻑ )
ﺗ ﺍﳊﺪﻳﺪﺓ ﺍﻟﱵﻦ
ﺐﹸ ﺧﺸ
ﺓﺪﻳﺪﹶ
ﻫﺬﻩ ﺍﳊ
ﺍﷲ ﻋﻨﻪ
» :
|
n
ﻞﺤﻨﻟﺍ
٧:
ﺎﻝ ﻟﻪ ﻋﻤﺮ:
ﺍﳍﺬﱄ ﻳﺼﻒ
ﻑﻮﺨﻛﻤﺎ ﺗ
ﻧﻜﻢ ﺷﻌﺮ ﺍﳉ
ﻜﻲ ﻭﺍﻟﺜﻌﻠﱯ ﻭﻏﲑﳘ
ﻑ:٢/ ٤١١
ﲑﺴﻔﺗﻭ
ﻭﻧﺴﺐ ﺍﻟﺰﳐﺸﺮﻱ
ﻋ ﻭﰲ ﻣﻮﺿﻊ ﻣﻨﻪ
ﺎ،
ﻭﻫﻮ ﻟﻌﺒﺪ ﺍﷲ
ﻭﺃ
ﻲﻟﺎﻤﱡ
ﹴ ﺍﻟﺜ
ﻢﺣﺍﺰﻣ .ﻗ
ﺎ
ﹰ ﻋﻠﻰ
ﹴ ﻟﻪ ﻗﺼﻴﺪﺓ
ﻞ
ﻦ(. ﻭﺭﻭﻱ ﺍﻟﺒﻴﺖ
ﺓ)ﺧﻮﻑ
(. ﻭﻦﹶ
ﻔﺴﻟﺍ
ﻫ ﹸ
ﻞﻛﹾ
ﻛﻤﺎ ﺗﺄﱠﺺ
ﻘ
ﺣﻮﻟﻴﺔ
ﻥ ﻋﻤﺮ ﺭﺿﻲ
y
z
{
|
ﺍﻟﺘﻨﻘﺺ، ﻓﻘﺎ
ﺮﻧﺎ ﺃﺑﻮ ﻛﺒﲑ
ﹰ
ﺍﺩﹺ
ﺮﹶ
ﹰ ﻗ
ﺎﻜ ...ﻛ
ﻋﻠﻴﻜﻢ ﺑﺪﻳﻮ
ﺍ
ﻜﻧ
٨١
ﻭﻧﻘﻠﻪ ﻋﻦ ﻣﻜ
ﺔﻴ:٢٧١
، ﺍﻟﻜﺸﺎﻑ
ﻛﺘﺎﺏ ﺍﻟﺘﻔﺴﲑ
. ﻭ
ﻭ ،ﻫﺮﻱ ﻭﺍﻟﺰﺟﺎﺝ
ﻩ ﳍﻤﺎ :ﺎﻤﹶﻬ
ﻭﻟﻴﺲ ﻟ
ﺮﻟﺍ ﻣ ﹺ
ﻦﻪ ﻻﺑﹶﻧ
ﺃ ﺔﻳﹺ
ﻭﺍ
ﹴ
ﻞﻳﹶ
ﺬﹺ ﻫ
ﺮﻌ ﺩﻳﻮﺍﻥ ﺷ
ﻣﺶ، ﻣﺎﺩﺓ
) ﻦﻔﺳ
ﺔﻏ:١/ ١٢٦
، ﻣﺎﺩﺓ
ﺎ ﺍﻟﻘﺴﻲ
. ﻱﹶ
ﺃ:
ﱠ
ﻘﻨﺗ
•ﺣ
ﻦ ﺍﳌﺴﻴﺐ ﺃﻥ
ﻮﻟﻪ ﺗﻌﺎﱃ:
o
ﺨﻮﻑ ﻋﻨﺪﻧﺎ:
ﻌﻢ، ﻗﺎﻝ ﺷﺎﻋ
ﺮ
ﻜﹸ ﻣﻨﻬﺎ ﺗﺎﻣ
ﻞﺣ
ﻋ ﻳﻬﺎ ﺍﻟﻨﺎﺱ
ﻢﻬﻣﻼ«. )٢(
ﺗﻔﺴﲑ ﺍﻟﻘﺮﻃﱯ:/٧
١
، ﻣﻘﺪﻣﺔ ﺍﺑﻦ ﻋﻄﻴ
ﻱﺭﺎﺨﺒﻟﺍ
: ٣/٩٩
ﺒﻞ ﻧﻘﻠﻪ ﻋﻦ ﺍﻷﺯﻫ
ﻲ ﺑﻌﺪ ﺃﻥ ﻧﻘﻞ ﻋﺰﻩ
ﺎﺩﻤ ﺣﺔﻤﺟﺮ ﰲ ﺗ
ﰲ ﺪﹺ
ﺟﹶ
ﻭﱂ ﺃﻲﹶﻟ
ﺬ
ﺏ :/ ٣١
٢٠٩
ﺎﻫ
ﺮﻱ :ﺃﺳﺎﺱ ﺍﻟﺒﻼﻏ
ﺎﺮ ﻳﺼﻨﻊ ﻣﻦ ﻋﻴﺪﺍ
ﺳ ﻦﻋ
ﻌﻴﺪ ﺑﻦ
ﲣﻮﻑ (ﻣﻦ ﻗﻮ
ﺬﻩ ﻟﻐﺘﻨﺎ، ﺍﻟﺘﺨ
ﺭﻫﺎ؟، ﻗﺎﻝ:ﻧﻌ
ﺣﺮﻟﺍ ﻑﻮﺨﺗ
ﺮﻤﻋ
ﻳﺎ ﺃﻳ :
ﻭﻣﻌﺎﱐ ﻛﻼ،
ﻱﱪﻄﻟﺍ
:٨/٢٨ ﺗﻭ
ﻱﱪﻄﻟﺍ
:١٤ / ١١٣
ﻚ ﻳﻨﻈﺮ :ﺻﺤﻴﺢ
)ﺧﻮﻑ
(
ﻻﺑﻦ ﻣﻘﺒ
،ﻱﺮ
ﻲﹺ
ﺎﻏﺎﻧﻗﺎﻝ ﺍﻟﺼ
ﻲﹺ
ﻧﺎﹶ
ﻏَ
ﻷﺍ ﺐﺣﺎﺻ
ﺬﹸ
ﹴ ﺍﳍ
ﲑﹺ
ﺒﹶ
ﹶﰊ ﻛ
ﹺﱃ ﺃ
ﻩ ﺇ
ﻑ .(ﻭﻟﺴﺎﻥ ﺍﻟﻌﺮﺏ
ﺔﻏﻼﺒﻟﺍ
:
ﺮﺸﳐﺰﻟﺍ
:
ﻧﻮﻉ ﻣﻦ ﺍﻟﺸﺠﺮ
. ٩٩ / ٩ :
ﺀﺎﺟﻭ
ﻣﻌﲎ
)ﲣ
ﻝﺎﻘﻓ
:ﺬﻫ
ﰲ ﺃﺷﻌﺎﺭ
ﻝﺎﻘﻓ
،ﻢﻜﺑﺎﺘﻛ
)١( ﲑﺴﻔﺗ
)٢( ﲑﺴﻔﺗ
ﳓﻮ ﺫﻟﻚ
ﻣﺎﺩﺓ )
ﺮﻫﻮﳉﺍ
ﻯﻭﺭﻭ
ﻩﹺ
ﲑِ
ﺴﹾ
ﻔﺗ
ﺧﻮﻑ )
ﺱﺎﺳﺃ
ﺔﻌﺒﻨﻟﺍﻭ
ﺏﺮﻌﻟﺍ ١١-١١١
، ﻭﰲ
ﻮ ﰲ ﻟﺴﺎﻥ ﺍﻟﻌﺮﺏ
ﻧﻘﻼ ﻋﻦ ﺻﺤﺎﺡ
ﻟ
ﻌﺠﻼﻥ ﺍﻟﻨﻬﺪﻱ
. ﰲ ﹺﻱ
ﻭﺎﻀﻴﺒﻟﺍ ﻩﺍﺰ
ﺝ ﺍﻟﻌﺮﻭﺱ :
ﺓﺩﺎﻣ
ﻑ ﺍﻟﺴﲑ(، ﻳﻨﻈﺮ
:
ﻲِ
ﺴﺎ ﺍﻟﻘ ﺩﺮﺒﺗ . ﻲﺴﻘﻟﺍ ﺐ . ﻥﺎﺴﻟ
ﺎﳘ .
òÌÜÛa
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bèj’ ﺝﺎﺗ
ﺃﻳﻀﺎ ﺑﻠﻔﻆ
ﲣﻮﻑ )
ﺗ ﺍﳊﺪﻳﺪﺓ ﺍﻟﱵﻦ
ﺐﹸ ﺧﺸ
ﺓﺪﻳﺪﹶ
ﻫﺬﻩ ﺍﳊ
ﺠﺎﺭ ﺍﻟﱵ ﻻ
ﻗﻠﺐ ﺍﳌﻨﺎﻓﻖ
ﺍﷲ ﻋﻨﻪ
» :
|
n
ﻞﺤﻨﻟﺍ
٧:
ﺎﻝ ﻟﻪ ﻋﻤﺮ:
ﺍﳍﺬﱄ ﻳﺼﻒ
ﻑﻮﺨﻛﻤﺎ ﺗ
ﻧﻜﻢ ﺷﻌﺮ ﺍﳉ
ﻜﻲ ﻭﺍﻟﺜﻌﻠﱯ ﻭﻏﲑﳘ
ﻑ:٢/ ٤١١
ﲑﺴﻔﺗﻭ
ﻭﻧﺴﺐ ﺍﻟﺰﳐﺸﺮﻱ
ﻋ ﻭﰲ ﻣﻮﺿﻊ ﻣﻨﻪ
ﺎ،
ﻭﻫﻮ ﻟﻌﺒﺪ ﺍﷲ
ﻭﺃ
ﻲﻟﺎﻤﱡ
ﹴ ﺍﻟﺜ
ﻢﺣﺍﺰﻣ .ﻗ
ﺎ
ﹰ ﻋﻠﻰ
ﹴ ﻟﻪ ﻗﺼﻴﺪﺓ
ﻞ
ﻦ(. ﻭﺭﻭﻱ ﺍﻟﺒﻴﺖ
ﺓ)ﺧﻮﻑ
(. ﻭﻦﹶ
ﻔﺴﻟﺍ
ﻫ ﹸ
ﻞﻛﹾ
ﻛﻤﺎ ﺗﺄﱠﺺ
ﻘ
ﺣﻮﻟﻴﺔ
ﻮﻥ ﺑﲔ ﺍﻷﺷﺠ
ﺮﻤﻋ :ﻚﻟﺬﻛ
ﻥ ﻋﻤﺮ ﺭﺿﻲ
y
z
{
|
ﺍﻟﺘﻨﻘﺺ، ﻓﻘﺎ
ﺮﻧﺎ ﺃﺑﻮ ﻛﺒﲑ
ﹰ
ﺍﺩﹺ
ﺮﹶ
ﹰ ﻗ
ﺎﻜ ...ﻛ
ﻋﻠﻴﻜﻢ ﺑﺪﻳﻮ
ﺍ
ﻜﻧ
٨١
ﻭﻧﻘﻠﻪ ﻋﻦ ﻣﻜ
ﺔﻴ:٢٧١
، ﺍﻟﻜﺸﺎﻑ
ﻛﺘﺎﺏ ﺍﻟﺘﻔﺴﲑ
. ﻭ
ﻭ ،ﻫﺮﻱ ﻭﺍﻟﺰﺟﺎﺝ
ﻩ ﳍﻤﺎ :ﺎﻤﹶﻬ
ﻭﻟﻴﺲ ﻟ
ﺮﻟﺍ ﻣ ﹺ
ﻦﻪ ﻻﺑﹶﻧ
ﺃ ﺔﻳﹺ
ﻭﺍ
ﹴ
ﻞﻳﹶ
ﺬﹺ ﻫ
ﺮﻌ ﺩﻳﻮﺍﻥ ﺷ
ﻣﺶ، ﻣﺎﺩﺓ
) ﻦﻔﺳ
ﺔﻏ:١/ ١٢٦
، ﻣﺎﺩﺓ
ﺎ ﺍﻟﻘﺴﻲ
. ﻱﹶ
ﺃ:
ﱠ
ﻘﻨﺗ
•ﺣ
À
ﻟﺸﺠﺮﺓ ﺗﻜﻮ
ﻝﺎﻗ
:
ﻋ ﻝﺎﻘﻓ
ﻦ ﺍﳌﺴﻴﺐ ﺃﻥ
ﻮﻟﻪ ﺗﻌﺎﱃ:
o
ﺨﻮﻑ ﻋﻨﺪﻧﺎ:
ﻌﻢ، ﻗﺎﻝ ﺷﺎﻋ
ﺮ
ﻜﹸ ﻣﻨﻬﺎ ﺗﺎﻣ
ﻞﺣ
ﻋ ﻳﻬﺎ ﺍﻟﻨﺎﺱ
ﻢﻬﻣﻼ«. )٢(
ﺗﻔﺴﲑ ﺍﻟﻘﺮﻃﱯ:/٧
١
، ﻣﻘﺪﻣﺔ ﺍﺑﻦ ﻋﻄﻴ
ﻱﺭﺎﺨﺒﻟﺍ
: ٣/٩٩
ﺒﻞ ﻧﻘﻠﻪ ﻋﻦ ﺍﻷﺯﻫ
ﻲ ﺑﻌﺪ ﺃﻥ ﻧﻘﻞ ﻋﺰﻩ
ﺎﺩﻤ ﺣﺔﻤﺟﺮ ﰲ ﺗ
ﰲ ﺪﹺ
ﺟﹶ
ﻭﱂ ﺃﻲﹶﻟ
ﺬ
ﺏ :/ ٣١
٢٠٩
ﺎﻫ
ﺮﻱ :ﺃﺳﺎﺱ ﺍﻟﺒﻼﻏ
ﺎﺮ ﻳﺼﻨﻊ ﻣﻦ ﻋﻴﺪﺍ
ﳊﺮﺟﺔ ﻓﻴﻨﺎ :ﺍ
ﻭﻻ ﺷﻲﺀ،
) (١
ﺳ ﻦﻋ
ﻌﻴﺪ ﺑﻦ
ﲣﻮﻑ (ﻣﻦ ﻗﻮ
ﺬﻩ ﻟﻐﺘﻨﺎ، ﺍﻟﺘﺨ
ﺭﻫﺎ؟، ﻗﺎﻝ:ﻧﻌ
ﺣﺮﻟﺍ ﻑﻮﺨﺗ
ﺮﻤﻋ
ﻳﺎ ﺃﻳ :
ﻭﻣﻌﺎﱐ ﻛﻼ،
ﻱﱪﻄﻟﺍ
:٨/٢٨ ﺗﻭ
ﻱﱪﻄﻟﺍ
:١٤ / ١١٣
ﻚ ﻳﻨﻈﺮ :ﺻﺤﻴﺢ
)ﺧﻮﻑ
(
ﻻﺑﻦ ﻣﻘﺒ
،ﻱﺮ
ﻲﹺ
ﺎﻏﺎﻧﻗﺎﻝ ﺍﻟﺼ
ﻲﹺ
ﻧﺎﹶ
ﻏَ
ﻷﺍ ﺐﺣﺎﺻ
ﺬﹸ
ﹴ ﺍﳍ
ﲑﹺ
ﺒﹶ
ﹶﰊ ﻛ
ﹺﱃ ﺃ
ﻩ ﺇ
ﻑ .(ﻭﻟﺴﺎﻥ ﺍﻟﻌﺮﺏ
ﺔﻏﻼﺒﻟﺍ
:
ﺮﺸﳐﺰﻟﺍ
:
ﻧﻮﻉ ﻣﻦ ﺍﻟﺸﺠﺮ
. ٩٩ / ٩ :
è
ﻝﺎﻗ
ﺍﳊ :
،ﺔﻴﺸﺣﻭ
ﲑﳋﺍ
«.)
ﺀﺎﺟﻭ
ﻣﻌﲎ
)ﲣ
ﻝﺎﻘﻓ
:ﺬﻫ
ﰲ ﺃﺷﻌﺎﺭ
ﻝﺎﻘﻓ
،ﻢﻜﺑﺎﺘﻛ
)١( ﲑﺴﻔﺗ
)٢( ﲑﺴﻔﺗ
ﳓﻮ ﺫﻟﻚ
ﻣﺎﺩﺓ )
ﺮﻫﻮﳉﺍ
ﻯﻭﺭﻭ
ﻩﹺ
ﲑِ
ﺴﹾ
ﻔﺗ
ﺧﻮﻑ )
ﺱﺎﺳﺃ
ﺔﻌﺒﻨﻟﺍﻭ
ﺏﺮﻌﻟﺍ ﺩ ﺍﳋﺎﻣﺲ
ﻠﻰ ﺍﳌﻨﱪ ﻋﻦ
،ﺦ ﻣﻦ ﻫﺬﻳﻞ
ﺍﻟﻌﺮﺏ ﺫﻟﻚ
ﻦﹶ
ﻔﺴﻟ
ﻓﻴﻪ ﺗﻔﺴﲑ
١١-١١١
، ﻭﰲ
ﻮ ﰲ ﻟﺴﺎﻥ ﺍﻟﻌﺮﺏ
ﻧﻘﻼ ﻋﻦ ﺻﺤﺎﺡ
ﻟ
ﻌﺠﻼﻥ ﺍﻟﻨﻬﺪﻱ
. ﰲ ﹺﻱ
ﻭﺎﻀﻴﺒﻟﺍ ﻩﺍﺰ
ﺝ ﺍﻟﻌﺮﻭﺱ :
ﺓﺩﺎﻣ
ﻑ ﺍﻟﺴﲑ(، ﻳﻨﻈﺮ
:
ﻲِ
ﺴﺎ ﺍﻟﻘ ﺩﺮﺒﺗ . ﻲﺴﻘﻟﺍ ﺐ .
òÌÜÛa
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bèj’ ﲑ ﺍﻟﻘﺮﻃﱯ:٠/ ٠١
ﺍﻟﺒﻴﺖ ﻟﺰﻫﲑ، ﻭﻫﻮ
ﻧﻘ ﻋﺰﺍﻩ ﻟﺬﻱ ﺍﻟﺮﻣﺔ
ﻭ ﻋﺒﺪ ﺍﻟﻠﻴﺚ
ﻦﺑﻟﺍ
ﺎﻝ ﺍﻟﺰﺑﻴﺪﻱ :
»ﺰﻋﻭ
ﻱﹺ
ﻫﺬﺍ ﺍﻟﺮﻭ
«. ﺝﺎﺗ
ﺃﻳﻀﺎ ﺑﻠﻔﻆ
ﲣﻮﻑ )
ﺗ ﺍﳊﺪﻳﺪﺓ ﺍﻟﱵﻦ
ﺐﹸ ﺧﺸ
ﺓﺪﻳﺪﹶ
ﻫﺬﻩ ﺍﳊ
ﻜﻲ ﻭﺍﻟﺜﻌﻠﱯ ﻭﻏﲑﳘ
ﻑ:٢/ ٤١١
ﲑﺴﻔﺗﻭ
ﻭﻧﺴﺐ ﺍﻟﺰﳐﺸﺮﻱ
ﻋ ﻭﰲ ﻣﻮﺿﻊ ﻣﻨﻪ
ﺎ،
ﻭﻫﻮ ﻟﻌﺒﺪ ﺍﷲ
ﻭﺃ
ﻲﻟﺎﻤﱡ
ﹴ ﺍﻟﺜ
ﻢﺣﺍﺰﻣ .ﻗ
ﺎ
ﹰ ﻋﻠﻰ
ﹴ ﻟﻪ ﻗﺼﻴﺪﺓ
ﻞ
ﻦ(. ﻭﺭﻭﻱ ﺍﻟﺒﻴﺖ
ﺓ)ﺧﻮﻑ
(. ﻭﻦﹶ
ﻔﺴﻟﺍ
ﻫ ﹸ
ﻞﻛﹾ
ﻛﻤﺎ ﺗﺄﱠﺺ
ﻘ
٨١
ﻭﻧﻘﻠﻪ ﻋﻦ ﻣﻜ
ﺔﻴ:٢٧١
، ﺍﻟﻜﺸﺎﻑ
ﻛﺘﺎﺏ ﺍﻟﺘﻔﺴﲑ
. ﻭ
ﻭ ،ﻫﺮﻱ ﻭﺍﻟﺰﺟﺎﺝ
ﻩ ﳍﻤﺎ :ﺎﻤﹶﻬ
ﻭﻟﻴﺲ ﻟ
ﺮﻟﺍ ﻣ ﹺ
ﻦﻪ ﻻﺑﹶﻧ
ﺃ ﺔﻳﹺ
ﻭﺍ
ﹴ
ﻞﻳﹶ
ﺬﹺ ﻫ
ﺮﻌ ﺩﻳﻮﺍﻥ ﺷ
ﻣﺶ، ﻣﺎﺩﺓ
) ﻦﻔﺳ
ﺔﻏ:١/ ١٢٦
، ﻣﺎﺩﺓ
ﺎ ﺍﻟﻘﺴﻲ
. ﻱﹶ
ﺃ:
ﱠ
ﻘﻨﺗ
ﺗﻔﺴﲑ ﺍﻟﻘﺮﻃﱯ:/٧
١
، ﻣﻘﺪﻣﺔ ﺍﺑﻦ ﻋﻄﻴ
ﻱﺭﺎﺨﺒﻟﺍ
: ٣/٩٩
ﺒﻞ ﻧﻘﻠﻪ ﻋﻦ ﺍﻷﺯﻫ
ﻲ ﺑﻌﺪ ﺃﻥ ﻧﻘﻞ ﻋﺰﻩ
ﺎﺩﻤ ﺣﺔﻤﺟﺮ ﰲ ﺗ
ﰲ ﺪﹺ
ﺟﹶ
ﻭﱂ ﺃﻲﹶﻟ
ﺬ
ﺏ :/ ٣١
٢٠٩
ﺎﻫ
ﺮﻱ :ﺃﺳﺎﺱ ﺍﻟﺒﻼﻏ
ﺎﺮ ﻳﺼﻨﻊ ﻣﻦ ﻋﻴﺪﺍ
ﻱﱪﻄﻟﺍ
:٨/٢٨ ﺗﻭ
ﻱﱪﻄﻟﺍ
:١٤ / ١١٣
ﻚ ﻳﻨﻈﺮ :ﺻﺤﻴﺢ
)ﺧﻮﻑ
(
ﻻﺑﻦ ﻣﻘﺒ
،ﻱﺮ
ﻲﹺ
ﺎﻏﺎﻧﻗﺎﻝ ﺍﻟﺼ
ﻲﹺ
ﻧﺎﹶ
ﻏَ
ﻷﺍ ﺐﺣﺎﺻ
ﺬﹸ
ﹴ ﺍﳍ
ﲑﹺ
ﺒﹶ
ﹶﰊ ﻛ
ﹺﱃ ﺃ
ﻩ ﺇ
ﻑ .(ﻭﻟﺴﺎﻥ ﺍﻟﻌﺮﺏ
ﺔﻏﻼﺒﻟﺍ
:
ﺮﺸﳐﺰﻟﺍ
:
ﻧﻮﻉ ﻣﻦ ﺍﻟﺸﺠﺮ
. ٩٩ / ٩ :
)١( ﲑﺴﻔﺗ
)٢( ﲑﺴﻔﺗ
ﳓﻮ ﺫﻟﻚ
ﻣﺎﺩﺓ )
ﺮﻫﻮﳉﺍ
ﻯﻭﺭﻭ
ﻩﹺ
ﲑِ
ﺴﹾ
ﻔﺗ
ﺧﻮﻑ )
ﺱﺎﺳﺃ
ﺔﻌﺒﻨﻟﺍﻭ
ﺏﺮﻌﻟﺍ ١( ﺔﺸ)١ ﻫﻭ
(٢)
،ﻪﺤ
ﻪﺑ . (٢)
ﻪﺑ . ١
•ﺣ
òÌÜÛa
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ﻰ ﻫﺬﻩ ﺍﻟﺮﻭﺍ
ﻣﻦ ﺍﳌﻌﺎﺭﺽ
ﻲ ﱂ ﺗﺴﻠﻢ ﻣﻦ
ﻣ ﻹﻣﺎﻡ ﺃﲪﺪ
ﺗﻔﺴﲑ ﺍﻟﻘﺮﺁﻥ
» ﺯﻮﳚ ﲑﺴﻔﺗ
ﷲﺍ«
.ﻝﺎﻗﻭ
ﻣﺎﻡ ﺃﲪﺪ ﺭﺿ
ﺀ ﺑﺮﻭﺍﻳﺔ ﺍﳌﺮﻭ
ﻠﻘﻬﺎ ﺍﷲ ﺗﻌﺎﱃ
o
²
³
، ﺃﻱ
:
ﻞﻌﻓﺄﺳ
ﻰ ﺍﻟﻠﻐﺔ«. ﺻﻮﻝ ﺍﲪﺪ :٥٨٠
ﻮﻝ ﺃﲪﺪ :٥٨١
. ١
ﺓﺩﻮﺴ :١٧٥-٧٦
pb
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ﻥ ﻣﺎ ﻳﺮﺩ ﻋﻠﻰ
-ﺇﻥ ﺳﻠﻤﺖ
، ﻛﻴﻒ ﻭﻫﻲ
ﻯﻭﺭ
ﻋﻦ ﺍﻹ
)١( » ﺯﻮﳚﻭ
ﻲﻠﺒﻨﳊﺍ
(٢) :
ﺍ ﺃﲪﺪ ﺭﲪﻪ
ﺍﻟﻠﻐﺔ ﻋﻨﺪ ﺍﻹ
ﺪ ﻫﺬﺍ ﻣﺎ ﺟﺎ
ﺎ ﺭﻭﺡ ﺧﻠﺃ
ﻪﻧﺎﺤﺒﺳ
: o
ﹰ
ﻱ ﻋﻠﻴﻚ ﺭﺯﻗﺎ
ﻩ ﻋﻠﻰ ﻣﻘﺘﻀﻰ
ﺓ :١٨٢ / ٢
ﺻﺃﻭ
ﺪ :٢/ ٢٨١
ﻮﺻﺃﻭ
ﺐ
ﲑﻨﳌﺍ
٨٥/٢:
ﺪ :٢/ ٢٨١
ﺴﳌﺍﻭ
ﺪ :٢/ ٢٨١
. @@@@@@@
bèj’
ﻻﺇ
ﻥﺃ
ﺔﻠﺑﺎﻨﳊﺍ
-
ﻕﻼﻃﻹﺍ
ﺪﻘﻓ
ﺓﺩﻮﺴﳌﺍ
:
ﺏﺎﻄﳋﺍ
ﻗﺪ ﻓﺴﺮ
ﻰﻀﺘﻘﲟ
ﺪﻀﻌﻳ
ﺎﻫﺎﻨﻌﻣ
:
ﰲ ﻗﻮﻟﻪ
ﻱﺮﺟﺄﺳ
ﺃ
ﻧﻪ ﻓﺴﺮﻩ
)١(
ﺓﺩﻮﺴﳌﺍ
)٢(
ﺪﻴﻬﻤﺘﻟﺍ
)٣(
ﺍﻟﻜﻮﻛﺐ
)٤(
ﺪﻴﻬﻤﺘﻟﺍ
)٥(
ﺪﻴﻬﻤﺘﻟﺍ
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bèj’ ﻘﺪ ﺟﺎﺀ ﰲ
«
. ﻝﺎﻗﻭ
ﻮﺑﺃ
ﺷﻴﺨﻨﺎ ﻭﻗﺎﻝ:
ﻔﺴﲑ ﺍﻟﻘﺮﺁﻥ
ﷲﺍ
ﺇﳕﺎ» (:
ﺽ ﺍﷲ، ﻭﻗﺎﻝ
ﻮﻝ ﺍﻟﺮﺟﻞ:
٥( »ﻩﺮﻫﺎﻇﻭ
ﻋﻨﻪ ﺃﻭﻻ، ﻓﻘ
ﻛﺮﻩ ﺷﻴﺨﻨﺎ«
ﺷ ﻠﻐﺔ؛ ﺫﻛﺮﻩ
»ﻻﻭ
ﳛﺮﻡ ﺗﻔ
ﻪﺑ«
. ﺎﻝ ﰲ)ﺭﻭﺡ
ﲰﺎﺀ ﺍﷲ ﻭﺃﺭﺽ
ﰲ ﺍﻟﻠﻐﺔ، ﻳﻘﻮ
ﻮ ﺍﳋﻄﺎﺏ:)٥
ﻣﻊ ﺍﳌﻨﻘﻮﻝ
ﻀﻰ ﺍﻟﻠﻐﺔ، ﺫﻛ
ﻰ ﻣﻘﺘﻀﻰ ﺍﻟﻠ
ﻲﺣﻮﺘﻔﻟ:)٣( »
ﺃﻭ
ﻛﺜﺮ ﺃﺻﺤﺎﺑﻪ
ﲪﻪ ﺍﷲ
ﻪﻧﺃ ﺎﻗ
:
ﻋﺒﺪ ﺍﷲ
ﲰﻭ
ﻫﻮ ﺟﺎﺋﺰ ،
«.)٤( ﻝﺎﻗ ﻮﺑﺃ
ﻣﺎ ﻳﺘﻌﺎﺭﺽ
ﻥ ﻋﻠﻰ ﻣﻘﺘﻀ
ﲑ ﺍﻟﻘﺮﺁﻥ ﻋﻠﻰ
ﺍﺑﻦ ﺍﻟﻨﺠﺎﺭ ﺍﻟ
ﻭ ﺿﻲ ﺍﷲ ﻋﻨﻪ
ﻭﺯﻱ ﻋﻨﻪ ﺭﲪ
ﱃ، ﻛﻤﺎ ﻳﻘﺎﻝ
³
n
٦٤ : ﻃﻪ
،
ﹰ
ﻞ ﻟﻚ ﺧﲑﺍ«
ﻣ ﻹﻣﺎﻡ ﺃﲪﺪ
ﺗﻔﺴﲑ ﺍﻟﻘﺮﺁﻥ
» ﺯﻮﳚ ﲑﺴﻔﺗ
ﷲﺍ«
.ﻝﺎﻗﻭ
ﻣﺎﻡ ﺃﲪﺪ ﺭﺿ
ﺀ ﺑﺮﻭﺍﻳﺔ ﺍﳌﺮﻭ
ﻠﻘﻬﺎ ﺍﷲ ﺗﻌﺎﱃ
o
²
³
، ﺃﻱ
:
ﻞﻌﻓﺄﺳ
ﻰ ﺍﻟﻠﻐﺔ«. ﻯﻭﺭ
ﻋﻦ ﺍﻹ
)١( » ﺯﻮﳚﻭ
ﻲﻠﺒﻨﳊﺍ
(٢) :
ﺍ ﺃﲪﺪ ﺭﲪﻪ
ﺍﻟﻠﻐﺔ ﻋﻨﺪ ﺍﻹ
ﺪ ﻫﺬﺍ ﻣﺎ ﺟﺎ
ﺎ ﺭﻭﺡ ﺧﻠﺃ
ﻪﻧﺎﺤﺒﺳ
: o
ﹰ
ﻱ ﻋﻠﻴﻚ ﺭﺯﻗﺎ
ﻩ ﻋﻠﻰ ﻣﻘﺘﻀﻰ
ﺪﻘﻓ
ﺓﺩﻮ:
ﺏﺎﻄ
ﺮﺴﻓ
ﻰﻀ
ﺪﻀﻌ
ﺎﻫﺎ:
ﻪﻟﻮﻗ
ﻱﺮﺟ
ﻩﺮﺴﻓ «
. ﻝﺎﻗﻭ
ﻮﺑﺃ
ﺷﻴﺨﻨﺎ ﻭﻗﺎﻝ:
ﻔﺴﲑ ﺍﻟﻘﺮﺁﻥ
ﷲﺍ
ﺇﳕﺎ» (:
ﺽ ﺍﷲ، ﻭﻗﺎﻝ
ﻮﻝ ﺍﻟﺮﺟﻞ:
٥( »ﻩﺮﻫﺎﻇﻭ
ﻛﺮﻩ ﺷﻴﺨﻨﺎ«
ﺷ ﻠﻐﺔ؛ ﺫﻛﺮﻩ
»ﻻﻭ
ﳛﺮﻡ ﺗﻔ
ﻪﺑ«
. (٢)
ﻪﺑ . ﺩ ﺍﳋﺎﻣﺲ
ﺍﻟﻌﺪﺩ
ﺣﻮﻟﻴﺔ
ﲑ . •ﺣ
٠١ ﻛﺘﺎﺏ ﺍﻟﺘﻔﺴﲑ
. ٠٢
ﺢ ﺍﻟﺒﺨﺎﺭﻱ:١٠/٣
ﺏﺎﺘﻛ
ﺍﳌﺒﺎﱐ
٣:
)١(
ﺢﻴﺤﺻ
)٢( ﺔﻣﺪﻘﻣ
òÌÜÛa
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bèj’
òÌÜÛa
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bèj’ ﻀﺎ. ) (٤ ١ . ﻝﻮﻘ: ١/٢٣ﻭ١٧٧
٨١٤ ﻭﻣﻨﺎﻫﺞ ﺍﻟﻌﻘﻮ
ﻥﻭﺮﺴ :١/ ٢٥٨
. ﺤﻮﻝ : . ٥١
٤١
، ﺍﻟﺘﻠﻮﻳﺢ
: ١/٨
٥
ﻭﺍﻟﺘﻔﺴﲑ ﻭﺍﳌﻔﺴ
٢٩٦
ﻭﺇﺭﺷﺎﺩ ﺍﻟﻔﺤ
ﻡﺎ :ﻦﺑﺍ ﻡﺰﺣ: ٣/
ﻥ :ﲏﻳﻮﳉﺍ
: ١/١٥
ﻝ :ﻕ
/ ١ﺟـ ١
ﻝﻮﻘﻌﻟﺍ
. ٣٢/١:
)١(
ﺎﻜﺣﻹﺍ
)٢(
ﻥﺎﻫﱪﻟﺍ
)٣(
ﻝﻮﺼﶈﺍ
)٤( ﺞﻫﺎﻨﻣ ﻦ.) (١
òÌÜÛa
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bèj’ ﺸﻌﺮ؟ ﻓﻘﺎﻝ:
ﺪ ﰲﻧﻘﻞ ﺍ
ﺘﺎﺏ ﺍﻟﺼﻼﺓ
ﻣﺮ ﺃﻧﻪ ﺃﺑﺪﻱ
ﺑﺸﻲﺀ ﻣﻦ ﺍﻟﺸ
ﻊﻨﳌ«
)ﻭﻧ. (١
ﺍﻟﺘﻤﺎﻡ (ﰲ ﻛﺘ
ﻘﺎ، ﻓﻜﻞ ﺍﻷﻣ
ﺑ ﻞ ﺍﻟﺮﺟﻞ ﻟﻪ
ﺬﺍ ﻳﻘﺘﻀﻲ ﺍﳌﻨ
ﰊ ﻳﻌﻠﻰ ﰲ)ﺍ
ﺪﻡ ﺍﳉﻮﺍﺯ.)(٢
ﲏﻊﻨﳌﺍ
ﻘﻠﻄﻣ
ﺍﻟﻘﺮﺁﻥ ﻳﺘﻤﺜﻞ
:
ﻭﻇﺎﻫﺮ ﻫﺬ
ﺑﻦ ﺍﻟﻘﺎﺿﻲ ﺃﰊ
ﻭﺻﺤﺢ ﻋﺪ
ﻌﺠﺒﲏ (ﻻ ﻳﻌﲏ
ﻪﻧ
ﺳﺌﻞ ﻋﻦ
ﻭﺃﺑﻮ ﺍﻟﻄﻴﺐ
ﺑﻦ ﺍﺑﻦ ﺣﺴﲔ
ﺪﲪ
،ﰲ ﺫﻟﻚ
ﺃﲪﺪ) :ﻻ ﻳﻌ
ﺎﻡ ﺃﲪﺪ: »ﺃ
ﲏ .ﻗﺎﻝ ﻫﻮ
ﻋﻦ
ﻲﺿﺎﻘﻟﺍ
ﻋﻦ ﺍﻹﻣﺎﻡ
ﲪﺃ
ﺮﻫ
ﺃﻥ ﻗﻮﻝﺃ
ﻦ ﺍﻹﻣﺎ
ﻻ
ﲏﺒﺠﻌﻳ
ﺓﺩﻮﺴ
ﻳﺍﻭﲔﺘ
ﺎﻈﻟﺍﻭ ﺩ ﺍﳋﺎﻣﺲ
ﺍﻟﻌﺪﺩ
ﺓﺩﻮﺴ :١٧٥
. ﺣﻮﻟﻴﺔ
ﻰﻠ :٢/ ٦٠٥
ﺴﳌﺍﻭ
. •ﺣ
ﻋﻦ ﺍﻟﻌﺪﺓ ﻷﰊ ﻳﻌﻠ
ﺐ ﺍﳌﻨﲑ :٨٥١ /٢
ﺪ :٢/ ٢٨١
ﻪﻠﻘﻧﻭ
ﺓ :١٧٦
ﻭﺍﻟﻜﻮﻛﺐ
)١(
ﺪﻴﻬﻤﺘﻟﺍ
)٢(
ﺓﺩﻮﺴﳌﺍ ١٥٩
ﺩ ﺍﳋﺎﻣﺲ
ﺍﶈﻘﻘﲔ ﻣﻦ
ﻊ ﻋﻨﺪﻩ ﻋﻠﻰ
ﻘﺪ ﺟﺎﺀ ﰲ
«
. ﻝﺎﻗﻭ
ﻮﺑﺃ
ﺷﻴﺨﻨﺎ ﻭﻗﺎﻝ:
ﻔﺴﲑ ﺍﻟﻘﺮﺁﻥ
ﷲﺍ
ﺇﳕﺎ» (:
ﺽ ﺍﷲ، ﻭﻗﺎﻝ
ﻮﻝ ﺍﻟﺮﺟﻞ:
٥( »ﻩﺮﻫﺎﻇﻭ
ﺍﻟﻌﺪﺩ
ﻈﻬﺮﻩ ﺑﻌﺾ
ﺎ ﺍﳌﻨﻊﺕ ﺇﻓﺎﺩ
ﻋﻨﻪ ﺃﻭﻻ، ﻓﻘ
ﻛﺮﻩ ﺷﻴﺨﻨﺎ«
ﺷ ﻠﻐﺔ؛ ﺫﻛﺮﻩ
»ﻻﻭ
ﳛﺮﻡ ﺗﻔ
ﻪﺑ«
. ﺎﻝ ﰲ)ﺭﻭﺡ
ﲰﺎﺀ ﺍﷲ ﻭﺃﺭﺽ
ﰲ ﺍﻟﻠﻐﺔ، ﻳﻘﻮ
ﻮ ﺍﳋﻄﺎﺏ:)٥
ﻊﻨ
ﺎﻤﻛ ﻈﺘﺳﺍ
ﺴﻠﻢ ﰲ ﺛﺒﻮﺕ
. ﻣﻊ ﺍﳌﻨﻘﻮﻝ
ﻀﻰ ﺍﻟﻠﻐﺔ، ﺫﻛ
ﻰ ﻣﻘﺘﻀﻰ ﺍﻟﻠ
ﻲﺣﻮﺘﻔﻟ:)٣( »
ﺃﻭ
ﻛﺜﺮ ﺃﺻﺤﺎﺑﻪ
ﲪﻪ ﺍﷲ
ﻪﻧﺃ ﺎﻗ
:
ﻋﺒﺪ ﺍﷲ
ﲰﻭ
ﻫﻮ ﺟﺎﺋﺰ ،
«.)٤( ﻝﺎﻗ ﻮﺑﺃ
ﺣﻮﻟﻴﺔ
ﺍﻳﺔ ﺍﳌﻔﻴﺪﺓ ﺍﳌﻨ
ﺽ-
ﺎ ﱂﻓﺈ
ﺴﺗ
ﻦ ﺍﳌﻌﺎﺭﺽ!؟
ﻣﺎ ﻳﺘﻌﺎﺭﺽ
ﻥ ﻋﻠﻰ ﻣﻘﺘﻀ
ﲑ ﺍﻟﻘﺮﺁﻥ ﻋﻠﻰ
ﺍﺑﻦ ﺍﻟﻨﺠﺎﺭ ﺍﻟ
ﻭ ﺿﻲ ﺍﷲ ﻋﻨﻪ
ﻭﺯﻱ ﻋﻨﻪ ﺭﲪ
ﱃ، ﻛﻤﺎ ﻳﻘﺎﻝ
³
n
٦٤ : ﻃﻪ
،
ﹰ
ﻞ ﻟﻚ ﺧﲑﺍ«
١٠ . . . (٢)
ﻪﺑ . ﺎﻝ ﰲ)ﺭﻭﺡ
ﲰﺎﺀ ﺍﷲ ﻭﺃﺭﺽ
ﰲ ﺍﻟﻠﻐﺔ، ﻳﻘﻮ
ﻮ ﺍﳋﻄﺎﺏ:)٥
ﻀﻰ ﺍﻟﻠﻐﺔ، ﺫﻛ
ﻰ ﻣﻘﺘﻀﻰ ﺍﻟﻠ
ﻲﺣﻮﺘﻔﻟ:)٣( »
ﺃﻭ
ﻛﺜﺮ ﺃﺻﺤﺎﺑﻪ
ﲪﻪ ﺍﷲ
ﻪﻧﺃ ﺎﻗ
:
ﻋﺒﺪ ﺍﷲ
ﲰﻭ
ﻫﻮ ﺟﺎﺋﺰ ،
«.)٤( ﻝﺎﻗ ﻮﺑﺃ
ﻥ ﻋﻠﻰ ﻣﻘﺘﻀ
ﲑ ﺍﻟﻘﺮﺁﻥ ﻋﻠﻰ
ﺍﺑﻦ ﺍﻟﻨﺠﺎﺭ ﺍﻟ
ﻭ ﺿﻲ ﺍﷲ ﻋﻨﻪ
ﻭﺯﻱ ﻋﻨﻪ ﺭﲪ
ﱃ، ﻛﻤﺎ ﻳﻘﺎﻝ
³
n
٦٤ : ﻃﻪ
،
ﹰ
ﻞ ﻟﻚ ﺧﲑﺍ«
ﺗﻔﺴﲑ ﺍﻟﻘﺮﺁﻥ
» ﺯﻮﳚ ﲑﺴﻔﺗ
ﷲﺍ«
.ﻝﺎﻗﻭ
ﻣﺎﻡ ﺃﲪﺪ ﺭﺿ
ﺀ ﺑﺮﻭﺍﻳﺔ ﺍﳌﺮﻭ
ﻠﻘﻬﺎ ﺍﷲ ﺗﻌﺎﱃ
o
²
³
، ﺃﻱ
:
ﻞﻌﻓﺄﺳ
ﻰ ﺍﻟﻠﻐﺔ«. )١( » ﺯﻮﳚﻭ
ﻲﻠﺒﻨﳊﺍ
(٢) :
ﺍ ﺃﲪﺪ ﺭﲪﻪ
ﺍﻟﻠﻐﺔ ﻋﻨﺪ ﺍﻹ
ﺪ ﻫﺬﺍ ﻣﺎ ﺟﺎ
ﺎ ﺭﻭﺡ ﺧﻠﺃ
ﻪﻧﺎﺤﺒﺳ
: o
ﹰ
ﻱ ﻋﻠﻴﻚ ﺭﺯﻗﺎ
ﻩ ﻋﻠﻰ ﻣﻘﺘﻀﻰ
ﺓﺩﻮ:
ﺏﺎﻄ
ﺮﺴﻓ
ﻰﻀ
ﺪﻀﻌ
ﺎﻫ:
ﻪﻟﻮﻗ
ﻱﺮﺟ
ﻩﺮﺴﻓ ١٠ . . . ١
ﺻﻮﻝ ﺍﲪﺪ :٥٨٠
ﻮﻝ ﺃﲪﺪ :٥٨١
. ١
ﺓﺩﻮﺴ :١٧٥-٧٦
ﺓ :١٨٢ / ٢
ﺻﺃﻭ
ﺪ :٢/ ٢٨١
ﻮﺻﺃﻭ
ﺐ
ﲑﻨﳌﺍ
٨٥/٢:
ﺪ :٢/ ٢٨١
ﺴﳌﺍﻭ
ﺪ :٢/ ٢٨١
. )١(
ﺓﺩﻮﺴﳌﺍ
)٢(
ﺪﻴﻬﻤﺘﻟﺍ
)٣(
ﺍﻟﻜﻮﻛﺐ
)٤(
ﺪﻴﻬﻤﺘﻟﺍ
)٥(
ﺪﻴﻬﻤﺘﻟﺍ
òÌÜÛa
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À
@
ÐnÛa
pb
@
Þìy
@
òîvy
@a
@@@@@@
bèj’ ﺎ ﻼ ﻳﻜﺘﻔﻰ
ﺍﺟﺤﺔ ﻋﻠﻴﻬﺎ
ﻛﺬﻟﻚ ﻓﻬﻲ
ﻫﻮ ﺍﳌﺮﻭﻱ،
ﺍﻭ
ﻳﺔ ﺍﳉﻮﺍﺯ
(
ﻭﻣﻨﻬﺎ
ﺃﻥ:
ﺍﳉﺒﻞ ﺃﺣﺪ
ﺍﻟﻠﻐﺔ ﻛﺜﲑﺍ،
ﻧﻘﻠﻨﺎ ﻧﺺ
»ﻪﻨﻋﻭ-
ﻦﻋ
ﺍﻟﺘﻌﺎﺭﺽ،
ﻼﻓ
ﻴﺪﺓ ﺍﳉﻮﺍﺯ ﺭﺍ
ﻛ ﺮﻯ ﻟﻴﺴﺖ
،ﻫﺎ ﺩﻟﻴﻞ ﺁﺧﺮ
ﺬﻩ ﺍﻟﺮﻭﺍﻳﺔ)ﺭﻭ
ﻛﺜﺮ ﺍﻟﺴﻠﻒ. ﻝ ﺍﺑﻦ ﻗﺎﺿﻲ
ﺴﲑ ﲟﻘﺘﻀﻰ
ﺏ«.)٣( ﺪﻗﻭ
ﻲﺣﻮﺘﻔﻟﺍ
: »
ﻮﻥ ﰲ ﻣﺮﺗﺒﺔ
ﻭﺍﻳﺔ ﻋﻨﻪ ﺍﳌﻔﻴ
ﳊﻜﻢ، ﻭﺍﻷﺧﺮ
ﳉﻮﺍﺯ ﻳﻌﻀﺪﻫ
ﻱ ﺗﻀﻤﻨﺘﻪ ﻫﺬ
ﺍﻟﻌﻤﻞ ﻋﻨﺪ ﺍﻛ
٢( ﺍﺬﻟﻭ ﻝﻮﻘﻳ
ﺠﺎﺝ ﰲ ﺍﻟﺘﻔﺴ
ﻰ ﻟﻐﺔ ﺍﻟﻌﺮﺏ
ﹰ ﻣﺎ ﻗﺎﻟﻪ
ﺎﻀ
ﺩﻟﻴﻞ«.) (٤
ﰲ ﺍﻟﺮﺗﺒﺔ ﺗﻜﻮ
ﻛﻴﻒ ﻭﺃﻥ ﺍﻟﺮﻭ
ﻟﺘﺼﺮﻳﺢ ﺑﺎﳊ
:ﺃﻥ ﺭﻭﺍﻳﺔ ﺍﳉ
ﺍﳊﻜﻢ ﺍﻟﺬﻱ
ﺮﻑ، ﻭﻋﻠﻴﻪ
ﺭﻮ ﻪﺑﺎﺤﺻﺃ
.)
ﻋﺒﺎﺱ ﺍﻻﺣﺘﺠ
ﺴﲑﻩ ﲟﻘﺘﻀﻰ
ﳑﺎ ﻳﺪﻝ ﻟﻪ ﺃﻳﻀ
ﺩ ﻠﻐﺔ ﻣﻦ ﻏﲑ
ﰲ ﺇﺫﺍ ﺗﺴﺎﻭﺕ
ﺎﺎﺤﺟ.)١( ﻛ
ﳉﻮﺍﺯ ﻓﻴﻬﺎ ﺍ
ﺎﻬﻴﻠﻋ .ﺎﻬﻨﻣﻭ:
.ﺎﻬﻨﻣﻭ
ﺃﻥ:
ﻉ ﻭﺍﻟﻠﻐﺔ
ﺮﻌﻟﺍﻭ
ﺯ ﻋﻠﻴﻬﺎ
ﻮﻬﲨ
ﻮﻝ ﻋ ﻋﻦ ﺍﺑﻦ
ﺠﻴﻓ
ﻮﺯ ﺗﻔﺴ
ﻚ ﺃﻳﻀﺎ، ﻭﳑﺎ
ﲑﻩ ﲟﻘﺘﻀﻰ ﺍﻟﻠ
ﺇ ﻥ ﺍﻟﺮﻭﺍﻳﺎﺕ
ﺘﺪﻝ ﺩﻭﻥ ﺭﺟ
ﻩﻮ :
ﻥﺃ
ﺭﻭﺍﻳﺔ ﺍﳉ
ﻓﺘﻘﺪﻡ ﺗﻠﻚ
ﻋ
ﻋﻤﻠﻪ ﺑﺬﻟﻚ. ﺮﻯ) . (١ (٢)
ﻪﺑ . ﺷﻬﺮ ﰲ ﺍﻟﺸﺮﻉ
ﳌﻔﻴﺪﺓ ﺍﳉﻮﺍﺯ
ﺔﻠﺑﺎﻨ: »ﻮﻘﻨﳌﺍ
ﻘﺮﺁﻥ ﻋﺮﰊ،
ﻲ ﺍﳌﻔﻴﺪ ﻟﺬﻟﻚ
ﻻ ﳚﻮﺯ
ﲑﺴﻔﺗ
ﺃﻳﻪ، ﻻﻥ
ﻦ ﺍﳌﺴﺘ
ﻦ ﻭﺟﻮ
ﺎﻬﻨﻣ:
،ﺔﻠﻤﺘ
ﻨﻪ ﻣﻦ
ﻮ ﺍﻷﺷﻬ
ﺮﻭﺍﻳﺔ ﺍ
ﻤﺔ ﺍﳊﻨ
ﻥﻷ
ﻘﻟﺍ
ﻲﺣﻮﺘﻔ
ﺪﲪ- ﻻ ﺎ ﻼ ﻳﻜﺘﻔﻰ
ﺍﺟﺤﺔ ﻋﻠﻴﻬﺎ
ﻛﺬﻟﻚ ﻓﻬﻲ
ﻫﻮ ﺍﳌﺮﻭﻱ،
ﺍﻭ
ﻳﺔ ﺍﳉﻮﺍﺯ
(
ﻭﻣﻨﻬﺎ
ﺃﻥ:
ﺍﳉﺒﻞ ﺃﺣﺪ
ﺍﻟﻠﻐﺔ ﻛﺜﲑﺍ،
ﻧﻘﻠﻨﺎ ﻧﺺ
»ﻪﻨﻋﻭ-
ﻦﻋ
ﺍﻟﺘﻌﺎﺭﺽ،
ﻼﻓ
ﻴﺪﺓ ﺍﳉﻮﺍﺯ ﺭﺍ
ﻛ ﺮﻯ ﻟﻴﺴﺖ
،ﻫﺎ ﺩﻟﻴﻞ ﺁﺧﺮ
ﺬﻩ ﺍﻟﺮﻭﺍﻳﺔ)ﺭﻭ
ﻛﺜﺮ ﺍﻟﺴﻠﻒ. ﻝ ﺍﺑﻦ ﻗﺎﺿﻲ
ﺴﲑ ﲟﻘﺘﻀﻰ
ﺏ«.)٣( ﺪﻗﻭ
ﻲﺣﻮﺘﻔﻟﺍ
: »
ﻮﻥ ﰲ ﻣﺮﺗﺒﺔ
ﻭﺍﻳﺔ ﻋﻨﻪ ﺍﳌﻔﻴ
ﳊﻜﻢ، ﻭﺍﻷﺧﺮ
ﳉﻮﺍﺯ ﻳﻌﻀﺪﻫ
ﻱ ﺗﻀﻤﻨﺘﻪ ﻫﺬ
ﺍﻟﻌﻤﻞ ﻋﻨﺪ ﺍﻛ
٢( ﺍﺬﻟﻭ ﻝﻮﻘﻳ
ﺠﺎﺝ ﰲ ﺍﻟﺘﻔﺴ
ﻰ ﻟﻐﺔ ﺍﻟﻌﺮﺏ
ﹰ ﻣﺎ ﻗﺎﻟﻪ
ﺎﻀ
ﺩﻟﻴﻞ«.) (٤
ﰲ ﺍﻟﺮﺗﺒﺔ ﺗﻜﻮ
ﻛﻴﻒ ﻭﺃﻥ ﺍﻟﺮﻭ
ﻟﺘﺼﺮﻳﺢ ﺑﺎﳊ
:ﺃﻥ ﺭﻭﺍﻳﺔ ﺍﳉ
ﺍﳊﻜﻢ ﺍﻟﺬﻱ
ﺮﻑ، ﻭﻋﻠﻴﻪ
ﺭﻮ ﻪﺑﺎﺤﺻﺃ
.)
ﻋﺒﺎﺱ ﺍﻻﺣﺘﺠ
ﺴﲑﻩ ﲟﻘﺘﻀﻰ
ﳑﺎ ﻳﺪﻝ ﻟﻪ ﺃﻳﻀ
ﺩ ﻠﻐﺔ ﻣﻦ ﻏﲑ
ﰲ ﺇﺫﺍ ﺗﺴﺎﻭﺕ
ﺎﺎﺤﺟ.)١( ﻛ
ﳉﻮﺍﺯ ﻓﻴﻬﺎ ﺍ
ﺎﻬﻴﻠﻋ .ﺎﻬﻨﻣﻭ:
.ﺎﻬﻨﻣﻭ
ﺃﻥ:
ﻉ ﻭﺍﻟﻠﻐﺔ
ﺮﻌﻟﺍﻭ
ﺯ ﻋﻠﻴﻬﺎ
ﻮﻬﲨ
ﻮﻝ ﻋ ﻋﻦ ﺍﺑﻦ
ﺠﻴﻓ
ﻮﺯ ﺗﻔﺴ
ﻚ ﺃﻳﻀﺎ، ﻭﳑﺎ
ﲑﻩ ﲟﻘﺘﻀﻰ ﺍﻟﻠ
ﺇ ﻥ ﺍﻟﺮﻭﺍﻳﺎﺕ
ﺘﺪﻝ ﺩﻭﻥ ﺭﺟ
ﻩﻮ :
ﻥﺃ
ﺭﻭﺍﻳﺔ ﺍﳉ
ﻓﺘﻘﺪﻡ ﺗﻠﻚ
ﻋ
ﻋﻤﻠﻪ ﺑﺬﻟﻚ. ﺷﻬﺮ ﰲ ﺍﻟﺸﺮﻉ
ﳌﻔﻴﺪﺓ ﺍﳉﻮﺍﺯ
ﺔﻠﺑﺎﻨ: »ﻮﻘﻨﳌﺍ
ﻘﺮﺁﻥ ﻋﺮﰊ،
ﻲ ﺍﳌﻔﻴﺪ ﻟﺬﻟﻚ
ﻻ ﳚﻮﺯ
ﲑﺴﻔﺗ
ﻳﻪ، ﻻﻥ
ﻦ ﺍﳌﺴﺘ
ﻦ ﻭﺟﻮ
ﺎﻬﻨﻣ:
،ﺔﻠﻤﺘ
ﻪ ﻣﻦ
ﻮ ﺍﻷﺷﻬ
ﺮﻭﺍﻳﺔ ﺍ
ﻤﺔ ﺍﳊﻨ
ﻥﻷ
ﻘﻟﺍ
ﻲﺣﻮﺘﻔ
ﺪﲪ- ﻻ ٢(
ﻪﺑ.) . ﻱﺪﻣﻵﺍ
٠٥/٣:
٥١٣
ﻡﺎﻜﺣﻹﺍﻭ
:
-٢٧٩
. . ١
. ١
ﻥ :
ﲏﻳﻮﳉﺍ
٣/١:
ﻟﺍ
ﻔ
ﺤﻮﻝ
٨٧٢ : -
ﺐ
ﲑﻨﳌﺍ
٨٥/٢:
ﺭ
ﻖﺑﺎﺴﻟﺍ
٨٥/٢:
)١(
ﻥﺎﻫﱪﻟﺍ
)٢( ﺩﺎﺷﺭﺇ
)٣(
ﺍﻟﻜﻮﻛﺐ
)٤(
ﺭﺪﺼﳌﺍ
òÌÜÛa
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À
@
ÐnÛa
pb
@
Þìy
@
òîvy
@a
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bèj’ :
»ﻝﻭ «.) (٢
ﺎﻬ« «.)ﺎﻬ« ﻐﻮﻱ ﺃﻥ
ﺓﺪ.)٢( ﻪﺑﺍ.)٣( ﺩ ﺍﳋﺎﻣﺲ
ﺍﻟﻌﺪﺩ
ﻴﻪ ﻓﻴﻪ . ٢١٨ . ﻷﺩﺑﺎﺀ ﻭﱂ ﺍﻗﻒ ﻋﻠﻴﻪ
. ٢٣
ﺭ ﺷﺎﻫﲔ :ﻭ ٧٢١
٨
ﺣﻮﻟﻴﺔ
ﻧﻘﻠﻪ ﻋﻦ ﻣﻌﺠﻢ ﺍﻷ
ﺕ ﺍﳌﻔﺴﺮﻳﻦ :٦/٢
: ﻥ
ﺩ. ﻋﺒﺪ ﺍﻟﺼﺒﻮﺭ
•ﺣ
ﻭﻱ ﺍﳉﻮﻳﲏ: ٥٨
٢ ﺮﻈﻨﻳﻭ
:
ﻃﺒﻘﺎﺕ
٢١١ ﻭﺗﺎﺭﻳﺦ ﺍﻟﻘﺮﺁ
ﰲ ﺍﻟﺘﻔﺴﲑ
:
ﻭﺎﺼﻟﺍ
ﺍﻷﺩﺑﺎﺀ
٥٥/ ٦١ :
ﺳﺖ :ﺍﺑﻦ ﺍﻟﻨﺪﱘ
٢:
)١( ﺞﻫﺎﻨﻣ
)٢( ﻢﺠﻌﻣ
)٣(
ﺳﺮﻬﻔﻟﺍ ﺩ ﺍﳋﺎﻣﺲ
ﺍﻟﻌﺪﺩ
ﻴﻪ ﻓﻴﻪ . ٢١٨ . ﻷﺩﺑﺎﺀ ﻭﱂ ﺍﻗﻒ ﻋﻠﻴﻪ
. ٢٣
ﺭ ﺷﺎﻫﲔ :ﻭ ٧٢١
٨
ﺣﻮﻟﻴﺔ
ﻧﻘﻠﻪ ﻋﻦ ﻣﻌﺠﻢ ﺍﻷ
ﺕ ﺍﳌﻔﺴﺮﻳﻦ :٦/٢
: ﻥ
ﺩ. (٢)
ﻪﺑ . ﻋﺒﺪ ﺍﻟﺼﺒﻮﺭ
•ﺣ
ﻭﻱ ﺍﳉﻮﻳﲏ: ٥٨
٢ ﺮﻈﻨﻳﻭ
:
ﻃﺒﻘﺎﺕ
٢١١ ﻭﺗﺎﺭﻳﺦ ﺍﻟﻘﺮﺁ
ﰲ ﺍﻟﺘﻔﺴﲑ
:
ﻭﺎﺼﻟﺍ
ﺍﻷﺩﺑﺎﺀ
٥٥/ ٦١ :
ﺳﺖ :ﺍﺑﻦ ﺍﻟﻨﺪﱘ
٢:
)١( ﺞﻫﺎﻨﻣ
)٢( ﻢﺠﻌﻣ
)٣(
ﺳﺮﻬﻔﻟﺍ ﺩ ﺍﳋﺎﻣﺲ
ﺎﻡ ﺃﲪﺪ ﻣﻦ
،ﺓ ﻭﺍﻟﻠﻐﻮﻳﲔ
ﺝ ﺑﺎﻟﻠﻐﺔ ﰲ
ﻪ ﻋﻨﺪ ﺗﻮﻓﺮ
ﺗﺄﻟﻴﻔﻪ ﻛﺘﺎﺑﻪ
ﺮﻳﺐ ﺍﻟﻘﺮﺁﻥ
ﺎﻬﺟﺎﺘﳛ ﻢﻬﻓ
ﻜﺘﺎﺑﻪ ﺍﻵﻧﻒ
ﻪ.)٢( ﺩﺪﺷﻭ
ﻲ ﻭﺃﰊ ﺣﺎﰎ
ﺑﺎﻟﺮﺃﻱ ﻣﺜﻞ
ﻈﻦ ﺃﻥ ﺫﻟﻚ
ﺍﻟﻌﺪﺩ
،
ﻓﻤﻨﻌﻪ ﺍﻹﻣﺎ
ﻝﺍﻮﻗﺃ
ﺓﺎﺤﻨﻟﺍ
ﺻﺤﺔ ﺍﻻﺣﺘﺠﺎ
ﻚﻟﺫ
ﻪﺘﺤﺻﻭ
ﻋﺒﻴﺪﺓ ﻟﺪﻯ
ﺮﺴﻔﻳ
ﻓﻴﻪ ﻏﺮ
ﺤﻮﻳﺔ ﺍﻟﱵ
ﳛ
ﺑﻌﺪ ﺗﺄﻟﻴﻔﻪ ﻟﻜ
ﻭﺑﻴﺎﻥ
ﻪﺌﻄﺧ
ﺀ ﻭﺍﻷﺻﻤﻌﻲ
ﻳﻔﺴﺮ ﺍﻟﻘﺮﺁﻥ
ﻲ.)٤( ﺪﻗﻭ
ﻈﻳ
١٧٥-١٧٦
. ﺎﻤﻴﺳ
ﺫﺍﻮﺸﻟﺍ
ﺍﻟﺼﺤﻴﺢ ﻻ
ﺐ ﺇﱃ
ﻡﺪﻋ
ﺻ
ﺫ ﻋﻤﻼ ﺟﻮﺍﺯ
ﺀﺎﻤﻠﻌﻟﺍ
ﰊﻷ
ﺮﻳﺐ ﺍﻟﻘﺮﺁﻥ،
ﺾ ﺍﳌﺴﺎﺋﻞ ﺍﻟﻨﺤ
ﻤﺎﺀ ﻋﺼﺮﻩ
ﺑ
ﻪﺒﻳﺬ ﺪﻌﺑ
،
ﺃﻣﺜﺎﻝ ﺍﻟﻔﺮﺍﺀ
ﻳ ﻭﺻﻔﻮﻩ ﺑﺄﻧﻪ
ﻦﻋ ﻲﻌﻤﺻﻷﺍ
٢٥٠ . ﻃﺒﻘﺎﺕ ﺍﻟﻨﺤﻮﻳﲔ:
- . ٧١
ﺣﻮﻟﻴﺔ
ﺳ ﻰ ﺑﻌﺾ، ﻻ
ﺕ ﻫﻮ ﺍﻟﻨﻘﻞ
. ﲪﺪ ﱂ ﻳﺬﻫﺐ
ﻋﻨﻪ ﻗﻮﻻ ﻭﻋ
ﺍ ﻧﻘﺪ ﺑﻌﺾ
ﰲ ﺗﻔﺴﲑ ﻏﺮ
ﺭﺓ ﺇﱃ ﺑﻌﺾ
ﲔ ﺑﻌﺾ ﻋﻠﻤ
ﺘﺎﺑﻪ ﻫﺬﺍ ﺇﻻ
،ﻭﻣﻨﻬﺠﻪ ﻓﻴﻪ
ﻦﻳ،
)٣( ﱴﺣ ﻭ
ﻭﺍﻩ ﺍﻟﺘﻮﺯﻱ
ﻋ
:٢٤٣
-٢٤٤ ﻭ
٠
ﲔﻛﺰﺳ :١٧٦
ﻃﻭ
ﺯ ﺍﻟﻘﺮﺁﻥ :٦١/١
•ﺣ
ﺑﻌﻀﻬﺎ ﻋﻠﻰ
ﻮﺕ ﺍﻟﻘﺮﺍﺀﺍﺕ
ﻦ ﳏﻞ ﺍﻟﱰﺍﻉ
ﺃﻥ ﺍﻹﻣﺎﻡ ﺃﲪ
ﻋ ﺑﻞ ﺍﻟﺜﺎﺑﺖ
ﻣﺎ ﺭﻭﻱ ﻣﻦ
ﺃﻭﻝ ﻣﺼﻨﻒ
ﺏ، ﻣﻊ ﺇﺷﺎﺭ
ﺼﻮﻣﺔ ﺑﻴﻨﻪ ﻭﺑﲔ
ﻬﻢ ﻗﺮﺍﺀﺓ ﻛﺘﺎ
ﰲ ﻣﺴﻠﻜ
ﻭ ﻪ
ﻭﺍﳌﱪﺩ ﻭﺁﺧﺮﻳﻦ
ﻦ ﺍﳌﱪﺩ ﻭﺭﻭ
ﻟﺪﺭﺍﺳﺎﺕ ﺍﻟﻨﺤﻮﻳﺔ
ﲔﻳ :١٦٧
. ﺯ ﺍﻟﻘﺮﺁﻥ :(ﺳ ﻓﺆﺍﺩ
ﺪﻘﻣﻭ
ﻣﺔ ﳏﻘﻖ ﳎﺎﺯ
ﺕ ﺃﻭ ﻟﺘﺮﺟﻴﺢ
ﻷﻥ ﻃﺮﻳﻖ ﺛﺒﻮ
ﻀﺎ ﺧﺎﺭﺝ ﻋﻦ
ﻫﺬﺍ ﻳﺘﺒﲔ ﻟﻨﺎ
ﰲ ﺍﳉﻤ
،ﺔﻠ
ﺔﻣﺯﻼﻟﺍ
. ﺔﻬ ﺔﻴﻧﺎﺜﻟﺍ :
ﻘﺮﺁﻥ(، ﻭﻫﻮ
ﻭﻛﻼﻡ ﺍﻟﻌﺮﺏ
(
ﺣﺪﺛﺖ ﺧﺼ
ﻓﺤﺮﻡ ﺑﻌﻀﻬ
ﺍﻟﻨﻜﲑ ﻋﻠﻴﻪ
ﺝ ﺎﺤﻨﻟﺍﻭ
ﺱ ﻭ
ﺍﻟﺒﻐﺪﺍﺩﻱ ﻋﻦ
ﱘﺮﻜﻟﺍ
ﻩﺮﺛﺃﻭ
ﰲ ﺍﻟ
ﺕ ﺍﻟﻨﺤﻮﻳﲔ ﻭﺍﻟﻠﻐﻮﻳﲔ
ﻖﻘﳏ
ﻛﺘﺎﺏ
) ﺯﺎﳎ
ﺑﻐﺪﺍﺩ
:١٣ / ٢٥٥
ﺍﺮﻘﻟﺍ
ﺕﺍﺀ
ﻷ ،ﻚﻟﺫ
ﻭﻫﺬﺍ ﺃﻳﻀ
ﺪﻌﺑ ﻫ
ﲑﺴﻔﺘﻟﺍ
ﻪﻃﻭﺮﺷ
ﻬﺒﺸﻟﺍ
)ﳎﺎﺯ ﺍﻟﻘ
ﺮﻌﺸﻟﺎﺑ
ﲎﻌﳌﺍ
.)١(
ﺪﻗﻭ
،ﺮﻛﺬﻟﺍ
ﻥﻭﺮﺧﺁ
ﺝﺎﺟﺰﻟﺍﻭ
ﻣﺎ ﺭﻭﺍﻩ
)١( ﻥﺁﺮﻘﻟﺍ
)٢(
ﻃﺒﻘﺎﺕ
)٣( ﺔﻣﺪﻘﻣ
)٤( ﺦﻳﺭﺎﺗ ﺎﻣ
ﻭﺃ
ﺏ
ﺔﻣﺯﻼﻟﺍ
. ﺔﻬ ﺔﻴﻧﺎﺜﻟﺍ :
ﻘﺮﺁﻥ(، ﻭﻫﻮ
ﻭﻛﻼﻡ ﺍﻟﻌﺮﺏ
(
ﻪﻃﻭﺮﺷ
ﻬﺒﺸﻟﺍ
)ﳎﺎﺯ ﺍﻟﻘ
ﺮﻌﺸﻟﺎﺑ
ﲎﻌﳌﺍ
.)١( ﻪﺠﻬﻨﻣﻭ
ﻦ،
)٣(
ﻭ
ﻦ ﻱ)ﻱ . (٢ ﺩ ﺍﳋﺎﻣﺲ
ﺍﻟﻌﺪﺩ
٦٢٣ /٢: ﻱ
. ﻦﻳﺮﺴﻔﳌﺍ
:
ﺩﻭﺍﺪﻟﺍ
ﺣﻮﻟﻴﺔ
ﻦﻳ :٢/ ٣٢٧
. ١/ ٢٥٥
ﺒﻃﻭ
ﻘﺎﺕ
•ﺣ
١ ﻭﻃﺒﻘﺎﺕ ﺍﳌﻔﺴﺮﻳ
، ﺗﺎﺭﻳﺦ ﺑﻐﺪﺍﺩ
٣:
ﺕ
ﲔﻳﻮﺤﻨﻟﺍ
٥٧١ :
ﺕ
ﲔﻳﻮﺤﻨﻟﺍ
: ١٧٦
)١(
ﻃﺒﻘﺎﺕ
)٢(
ﻃﺒﻘﺎﺕ ﺩ ﺍﳋﺎﻣﺲ
ﺍﻟﻌﺪﺩ
٦٢٣ /٢: ﻱ
. ﻦﻳﺮﺴﻔﳌﺍ
:
ﺩﻭﺍﺪﻟﺍ
ﺣﻮﻟﻴﺔ
ﻦﻳ :٢/ ٣٢٧
. (٢)
ﻪﺑ . ١/ ٢٥٥
ﺒﻃﻭ
ﻘﺎﺕ
•ﺣ
١ ﻭﻃﺒﻘﺎﺕ ﺍﳌﻔﺴﺮﻳ
، ﺗﺎﺭﻳﺦ ﺑﻐﺪﺍﺩ
٣:
ﺕ
ﲔﻳﻮﺤﻨﻟﺍ
٥٧١ :
ﺕ
ﲔﻳﻮﺤﻨﻟﺍ
: ١٧٦
)١(
ﻃﺒﻘﺎﺕ
)٢(
ﻃﺒﻘﺎﺕ ﺎﻬ.)١( ﺤﻪ.) (٢ (٣).ﺪﻩﻓ ﺩ ﺍﳋﺎﻣﺲ
ﻮﺤﻨ ﰊﺮﻌﻟﺍ
٣٨ :
ﺍﻟﻌﺪﺩ
١٧ . ﻭﺍﻟﻘﺮﺍﺀﺍﺕ ﰲ
ﻨﻟﺍ
ﺎﺯ ﳏﻘﻖ ﺍ
٧/١:
٢٥٥ . ٧٤٢ ﻭﺃﺛﺮ ﺍﻟﻘﺮﺁﻥ
ﺣﻮﻟﻴﺔ
١٣/٢٥٥
ﺔﻣﺪﻘﻣﻭ
ﻢ ﺍﻷﺩﺑﺎﺀ :٥/ ٦١
ﺔﻳ :١٢٤ ﻭ١٨٣ ﻭ
٧
•ﺣ
، ﺗﺎﺭﻳﺦ ﺑﻐﺪﺍﺩ
٣:
-٨٤ ﺮﻈﻨﻳﻭ
:
ﻢﺠﻌﻣ
ﺍﻟﺪﺭﺍﺳﺎﺕ ﺍﻟﻨﺤﻮﻳ
/ . ٩١ﻭ٥١
ﺕ ﲔﻳﻮﺤﻨﻟﺍ
: ١٧٦
ﺎﺯﳏﻘﻖ ﺍ
٣٨ : -
ﺍﻟﻜﺮﱘ ﻭﺃﺛﺮﻩ ﰲ
ﳏﺔ
ﺎﺯ ﻘﻖ ﺍ
١: /
)١(
ﻃﺒﻘﺎﺕ
)٢( ﺔﻣﺪﻘﻣ
)٣( ﻥﺁﺮﻘﻟﺍ
ﺔﻣﺪﻘﻣﻭ ﺩ ﺍﳋﺎﻣﺲ
ﻮﺤﻨ ﰊﺮﻌﻟﺍ
٣٨ :
ﺍﻟﻌﺪﺩ
١٧ . ﻭﺍﻟﻘﺮﺍﺀﺍﺕ ﰲ
ﻨﻟﺍ
ﺎﺯ ﳏﻘﻖ ﺍ
٧/١:
٢٥٥ . ٧٤٢ ﻭﺃﺛﺮ ﺍﻟﻘﺮﺁﻥ
ﺣﻮﻟﻴﺔ
١٣/٢٥٥
ﺔﻣﺪﻘﻣﻭ
ﻢ ﺍﻷﺩﺑﺎﺀ :٥/ ٦١
ﺔﻳ :١٢٤ ﻭ١٨٣ ﻭ
٧
•ﺣ
، ﺗﺎﺭﻳﺦ ﺑﻐﺪﺍﺩ
٣:
-٨٤ ﺮﻈﻨﻳﻭ
:
ﻢﺠﻌﻣ
ﺍﻟﺪﺭﺍﺳﺎﺕ ﺍﻟﻨﺤﻮﻳ
/ . ٩١ﻭ٥١
ﺕ ﲔﻳﻮﺤﻨﻟﺍ
: ١٧٦
ﺎﺯﳏﻘﻖ ﺍ
٣٨ : -
ﺍﻟﻜﺮﱘ ﻭﺃﺛﺮﻩ ﰲ
ﳏﺔ
ﺎﺯ ﻘﻖ ﺍ
١: /
)١(
ﻃﺒﻘﺎﺕ
)٢( ﺔﻣﺪﻘﻣ
)٣( ﻥﺁﺮﻘﻟﺍ
ﺔﻣﺪﻘﻣﻭ ﻮﺤﻨ ﰊﺮﻌﻟﺍ
٣:
١٧ . ﻭﺍﻟﻘﺮﺍﺀﺍﺕ ﰲ
ﻨﻟﺍ
ﺎﺯ ﳏﻘﻖ ﺍ
٧/١:
٢٥٥ . ٧٤٢ ﻭﺃﺛﺮ ﺍﻟﻘﺮﺁﻥ
١٣/٢٥٥
ﺔﻣﺪﻘﻣﻭ
ﻢ ﺍﻷﺩﺑﺎﺀ :٥/ ٦١
ﺔﻳ :١٢٤ ﻭ١٨٣ ﻭ
٧
، ﺗﺎﺭﻳﺦ ﺑﻐﺪﺍﺩ
٣:
-٨٤ ﺮﻈﻨﻳﻭ
:
ﻢﺠﻌﻣ
ﺍﻟﺪﺭﺍﺳﺎﺕ ﺍﻟﻨﺤﻮﻳ
/ . ٩١ﻭ٥١
ﺕ ﲔﻳﻮﺤﻨﻟﺍ
: ١٧٦
ﺎﺯﳏﻘﻖ ﺍ
٣٨ : -
ﺍﻟﻜﺮﱘ ﻭﺃﺛﺮﻩ ﰲ
ﳏﺔ
ﺎﺯ ﻘﻖ ﺍ
١: /
)١(
ﻃﺒﻘﺎﺕ
)٢( ﺔﻣﺪﻘﻣ
)٣( ﻥﺁﺮﻘﻟﺍ
ﺔﻣﺪﻘﻣﻭ ﰊﺮﻌ
:
òÌÜÛa
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bèj’ ﺩ ﺍﳋﺎﻣﺲ
ﰲ ،ﺻﺤﻴﺤﻪ
ﻓﺘﺢ ﺍﻟﺒﺎﺭﻱ،
ﺇﻻ ﺍﻟﺸﻲﺀ
ﻦ ﺃﻥ ﺍﻹﻣﺎﻡ
ﻪﺑﺎ)ﲑﺴﻔﺘﻟﺍ
- ﻰﻠﻋ ﺔﻐﻠﻟﺍ
ﻋﻬﺪ ﺍﻟﺘﺎﺑﻌﲔ
ﻐﺔ، ﻓﺄﻧﻜﺮﻭﺍ
«.)٤( ﺭﺎﺷﺃﻭ
ﺪﻣﺔ ﺗﻔﺴﲑﻩ:
. ٣
ﺍﻟﻌﺪﺩ
ﰲ ﻱﺭﺎﺨﺒﻟﺍ
ﻓ ﻦ ﺣﺠﺮ ﰲ
ﻲ ﻋﻦ ﺍﻟﻌﺮﺏ
ﲔ ﺍﻟﺬﻫﱯ ﻣﻦ
ﺟﺎﺀﺪ
ﰲ ﻛﺘﺎ
ﺎ ﺍﺑﻦ ﻋﺒﺎﺱ-
ﻋ ﻄﺮﻳﻘﺔ ﺇﱃ
ﺎﺀ ﻭﺃﻫﻞ ﺍﻟﻠﻐ
ﺻﻼ ﻟﻠﻘﺮﺁﻥ«
ﻮﺭﻱ ﰲ ﻣﻘﺪ
. ﻱﺩﻭﺍﺪﻟﺍ
٨٢/٢:
ﻣﻨﻪ، ﻣﻨﻬﻢ:
ﺍﻟﻐﺮﻳﺐ، ﻭﺍﺑﻦ
ﻪﻧﺇ ﻻ ﻲﻜﳛ
ﻕ«.) (٣
ﳏﻤﺪ
ﲔﺴﺣ
ﺍﻟﺘﻔﺴﲑ، ﻓﻘﺪ
ﺑﺔ-ﻻ ﺳﻴﻤﺎ
ﻤﺮﺕ ﻫﺬﻩ ﺍﻟﻄ
ﻮﺭﻋﻲ ﺍﻟﻔﻘﻬﺎ
ﻌﻠﺘﻢ ﺍﻟﺸﻌﺮ ﺃﺻ
ﻹﻣﺎﻡ ﺍﻟﻨﻴﺴﺎﺑﻮ
ﺸﺮ ﺇﱃ ﻏﲑﻩ. ﻃﺒﻘﺎﺕ ﺍﳌﻔﺴﺮﻳﻦ:
ﺣﻮﻟﻴﺔ
ﻣ ﻪ ﻭﺃﺧﺬﻭﺍ
ﰲ ﺍﳌﺸﻜﻞ ﻭ
ﲏﻳﺪﳌﺍ
» :
ﺩ :
»ﻮﻫ ﻕﺪﺻ
ﺷﺎﺭ ﺇﻟﻴﻬﺎ ﺩ. (٢)
ﻪﺑ . ﺍ ﺝ ﺑﺎﻟﻠﻐﺔ ﰲ
ﻋﺘﻤﺎﺩ ﺍﻟﺼﺤﺎ
:»ﺪﻗﻭ ﻤﺘﺳﺍ
ﺼﻮﻣﺔ ﺑﲔ ﻣﺘﻮ
ﻌﻠﺘﻢ ﺫﻟﻚ ﺟﻌ
ﻗﺎﻝ ﺑﺬﻟﻚ ﺍ
:( ٦/١
ﻭﱂ ﻳﺸ
ﺩﺍﺪﻐ:١٣ / ٢٥٢
ﻃﻭ
•ﺣ
ﻋﺘﻤﺪﻭﺍ ﻛﺘﺎﺑﻪ
ﰲ ﻭﺍﺑﻦ ﻗﺘﻴﺒﺔ
ﱴ ﻗﺎﻝ ﻋﻨﻪ
ﻋﻨﻪ ﺃﺑﻮ ﺩﺍﻭﺩ
ﻭﻫﻲ ﺍﻟﱵ ﺃﺷ
ﻮﻥ ﺍﻻﺣﺘﺠﺎﺝ
ﻛﻼﻣﻪ ﻋﻦ ﺍﻋ
ﻘﺮﺁﻥ، ﻗﻮﻟﻪ:
ﺣﺪﺛﺖ
ﺼﺧ
ﻭﻗﺎﻟﻮﺍ ﺇﻥ
ﻌﻓ
ﺺ ﺇﱃ ﺃﻥ ﳑﻦ
ﺎﺋﺐ ﺍﻟﻔﺮﻗﺎﻥ:
١ ﺮﻈﻨﻳﻭ
:
ﺗﺎﺭﻳﺦ ﺑﻐ
٢ . . ٢٣
/ . ٠٨
ﻌﻠﻤﺎﺀ ﻗﺪ ﺍﻋ
،ﻱ ﰲ ﺗﻔﺴﲑﻩ
ﲑﺜﻛ
ﺣﱴ . (١)
ﺢ«.)٢( ﻝﺎﻗﻭ
ﺔﻬ ﺔﺜﻟﺎﺜﻟﺍ
: ﻭ
ﺭﻱ ﳑﻦ ﳝﻨﻌﻮ
ﻭﻥ (ﻛ ﻋﻘﺐ
ﲑ ﻏﺮﻳﺐ ﺍﻟﻘ
ﻬﻢ، ﺇﱃ ﺃﻥ
،ﻫﺬﻩ ﺍﻟﻄﺮﻳﻘﺔ
ﺶ ﻫﺬﺍ ﺍﻟﻨﺺ
ﺍﻟﻘﺮﺁﻥ ﻭﺭﻏﺎ
ﺎﺯﳏﻘﻖ ﺍ
:١/١٧
ﺑﻐﺪﺍﺩ
٧٥٢ / ٣١ :
ﺕ
ﻦﻳﺮﺴﻔﳌﺍ
٨/٢:
ﲑ
ﻥﻭﺮﺴﻔﳌﺍﻭ
١: /
ﻛﺒﺎﺭ ﺍﻟﻌ
ﻱﱪﻄﻟﺍﻭ
ﻢﻫﲑﻏﻭ
ﺢﻴﺤﺼﻟﺍ
ﻬﺒﺸﻟﺍ
ﺭﻮﺑﺎﺴﻴﻨﻟﺍ
ﻭﺮﺴﻔﳌﺍﻭ
ﰲ ﺗﻔﺴﲑ
ﻭﻣﻦ ﻳﻠﻴﻬ
ﻫ ﻢﻬﻴﻠﻋ
ﰲ ﻫﺎﻣﺶ
)
ﻏﺮﺍﺋﺐ
)١( ﺔﻣﺪﻘﻣ
)٢( ﺦﻳﺭﺎﺗ
)٣(
ﻃﺒﻘﺎﺕ
)٤(
ﲑﺴﻔﺘﻟﺍ ﺩ ﺍﳋﺎﻣﺲ
ﰲ ،ﺻﺤﻴﺤﻪ
ﻓﺘﺢ ﺍﻟﺒﺎﺭﻱ،
ﺇﻻ ﺍﻟﺸﻲﺀ
ﻦ ﺃﻥ ﺍﻹﻣﺎﻡ
ﻪﺑﺎ)ﲑﺴﻔﺘﻟﺍ
- ﻰﻠﻋ ﺔﻐﻠﻟﺍ
ﻋﻬﺪ ﺍﻟﺘﺎﺑﻌﲔ
ﻐﺔ، ﻓﺄﻧﻜﺮﻭﺍ
«.)٤( ﺭﺎﺷﺃﻭ
ﺪﻣﺔ ﺗﻔﺴﲑﻩ:
. ٣
ﺍﻟﻌﺪﺩ
ﰲ ﻱﺭﺎﺨﺒﻟﺍ
ﻓ ﻦ ﺣﺠﺮ ﰲ
ﻲ ﻋﻦ ﺍﻟﻌﺮﺏ
ﲔ ﺍﻟﺬﻫﱯ ﻣﻦ
ﺟﺎﺀﺪ
ﰲ ﻛﺘﺎ
ﺎ ﺍﺑﻦ ﻋﺒﺎﺱ-
ﻋ ﻄﺮﻳﻘﺔ ﺇﱃ
ﺎﺀ ﻭﺃﻫﻞ ﺍﻟﻠﻐ
ﺻﻼ ﻟﻠﻘﺮﺁﻥ«
ﻮﺭﻱ ﰲ ﻣﻘﺪ
. ﻱﺩﻭﺍﺪﻟﺍ
٨٢/٢:
ﻣﻨﻪ، ﻣﻨﻬﻢ:
ﺍﻟﻐﺮﻳﺐ، ﻭﺍﺑﻦ
ﻪﻧﺇ ﻻ ﻲﻜﳛ
ﻕ«.) (٣
ﳏﻤﺪ
ﲔﺴﺣ
ﺍﻟﺘﻔﺴﲑ، ﻓﻘﺪ
ﺑﺔ-ﻻ ﺳﻴﻤﺎ
ﻤﺮﺕ ﻫﺬﻩ ﺍﻟﻄ
ﻮﺭﻋﻲ ﺍﻟﻔﻘﻬﺎ
ﻌﻠﺘﻢ ﺍﻟﺸﻌﺮ ﺃﺻ
ﻹﻣﺎﻡ ﺍﻟﻨﻴﺴﺎﺑﻮ
ﺸﺮ ﺇﱃ ﻏﲑﻩ. ﻃﺒﻘﺎﺕ ﺍﳌﻔﺴﺮﻳﻦ:
ﺣﻮﻟﻴﺔ
ﻣ ﻪ ﻭﺃﺧﺬﻭﺍ
ﰲ ﺍﳌﺸﻜﻞ ﻭ
ﲏﻳﺪﳌﺍ
» :
ﺩ :
»ﻮﻫ ﻕﺪﺻ
ﺷﺎﺭ ﺇﻟﻴﻬﺎ ﺩ. ﺍ ﺝ ﺑﺎﻟﻠﻐﺔ ﰲ
ﻋﺘﻤﺎﺩ ﺍﻟﺼﺤﺎ
:»ﺪﻗﻭ ﻤﺘﺳﺍ
ﺼﻮﻣﺔ ﺑﲔ ﻣﺘﻮ
ﻌﻠﺘﻢ ﺫﻟﻚ ﺟﻌ
ﻗﺎﻝ ﺑﺬﻟﻚ ﺍ
:( ٦/١
ﻭﱂ ﻳﺸ
ﺩﺍﺪﻐ:١٣ / ٢٥٢
ﻃﻭ
•ﺣ
ﻋﺘﻤﺪﻭﺍ ﻛﺘﺎﺑﻪ
ﰲ ﻭﺍﺑﻦ ﻗﺘﻴﺒﺔ
ﱴ ﻗﺎﻝ ﻋﻨﻪ
ﻋﻨﻪ ﺃﺑﻮ ﺩﺍﻭﺩ
ﻭﻫﻲ ﺍﻟﱵ ﺃﺷ
ﻮﻥ ﺍﻻﺣﺘﺠﺎﺝ
ﻛﻼﻣﻪ ﻋﻦ ﺍﻋ
ﻘﺮﺁﻥ، ﻗﻮﻟﻪ:
ﺣﺪﺛﺖ
ﺼﺧ
ﻭﻗﺎﻟﻮﺍ ﺇﻥ
ﻌﻓ
ﺺ ﺇﱃ ﺃﻥ ﳑﻦ
ﺎﺋﺐ ﺍﻟﻔﺮﻗﺎﻥ:
١ ﺮﻈﻨﻳﻭ
:
ﺗﺎﺭﻳﺦ ﺑﻐ
٢ . . ٢٣
/ . ٠٨
ﻌﻠﻤﺎﺀ ﻗﺪ ﺍﻋ
،ﻱ ﰲ ﺗﻔﺴﲑﻩ
ﲑﺜﻛ
ﺣﱴ . (٢)
ﻪﺑ . (١)
ﺢ«.)٢( ﻝﺎﻗﻭ
ﺔﻬ ﺔﺜﻟﺎﺜﻟﺍ
: ﻭ
ﺭﻱ ﳑﻦ ﳝﻨﻌﻮ
ﻭﻥ (ﻛ ﻋﻘﺐ
ﲑ ﻏﺮﻳﺐ ﺍﻟﻘ
ﻬﻢ، ﺇﱃ ﺃﻥ
،ﻫﺬﻩ ﺍﻟﻄﺮﻳﻘﺔ
ﺶ ﻫﺬﺍ ﺍﻟﻨﺺ
ﺍﻟﻘﺮﺁﻥ ﻭﺭﻏﺎ
ﺎﺯﳏﻘﻖ ﺍ
:١/١٧
ﺑﻐﺪﺍﺩ
٧٥٢ / ٣١ :
ﺕ
ﻦﻳﺮﺴﻔﳌﺍ
٨/٢:
ﲑ
ﻥﻭﺮﺴﻔﳌﺍﻭ
١: /
ﻛﺒﺎﺭ ﺍﻟﻌ
ﻱﱪﻄﻟﺍﻭ
ﻢﻫﲑﻏﻭ
ﺢﻴﺤﺼﻟﺍ
ﻬﺒﺸﻟﺍ
ﺭﻮﺑﺎﺴﻴﻨﻟﺍ
ﻭﺮﺴﻔﳌﺍﻭ
ﰲ ﺗﻔﺴﲑ
ﻭﻣﻦ ﻳﻠﻴﻬ
ﻫ ﻢﻬﻴﻠﻋ
ﰲ ﻫﺎﻣﺶ
)
ﻏﺮﺍﺋﺐ
)١( ﺔﻣﺪﻘﻣ
)٢( ﺦﻳﺭﺎﺗ
)٣(
ﻃﺒﻘﺎﺕ
)٤(
ﲑﺴﻔﺘﻟﺍ ١٦٧
ﺩ ﺍﳋﺎﻣﺲ
ﺟﻌﺔ ﻣﻘﺪﻣﺔ
ﻩ، ﺗﺒﲔ ﺃﻥ
ﻓﻴﻪ ﺗﺼﺮﻳﺢ
ﺮﺁﻥ ﻭﺇﻋﺮﺍﺑﻪ
ﻗﻮﻟﻪ ﺗﻌﺎﱃ
:
h
i
n
ﻧﻪ ﰲ ﺣﻜﻢ
ﹺ
ﻞﺴﹾ
ﻠﺴ
(ﻣﻔﻌﻮﻝ ﻟﻪ
ﺲ ﺑﻌﺮ ،ﰊ ﺻﺤﻴﺢ
ﺔﻐﻠﻟ :
،ﺍﺑﻦ ﺩﺭﻳﺪ
ﻢﻜﶈﺍ
:
ﹺﻳﺺ
ﺮﺒﻟﺍﻭ
ﻝ ﰲ ﻳﺎﻗﻮﺕ ﺑﻌﺪ
ﺎﻬﻌﲨﺄﺑ،
ﺃﻻ ﺗﺮﺍﻩ
ﺭﺩ ﺍﻟﱪﻳﺺ(
ﻥﺎﺴﻟ
،
ﺍﻟﻘﺪﺡﹾﺖ
ﻘﹶ
ﻔﺻ
َ
ﺍﳌﺎﺀﹶﺖ ﺍﻟﺮﻳﺢ
ﻘﹶ
ﻔﺻ
ﹰ
ﹰ ﻭﴰﺎﻻ
ﻪ ﳝﻴﻨﺎﺘﹶﺒ
ﻠﹶ
ﻗ
ﻦ ﺛﺎﺑﺖ. ﺍﻟﻌﺪﺩ
،
ﻓﻠﺪﻯ ﻣﺮﺍ
ﰲ
ﻩﲑﺴﻔﺗ
ﻰ ﺍﻟﻌﻜﺲ،
ﺴﲑ ﻟﻐﺔ ﺍﻟﻘﺮ
ﻚ ﻳﻘﻮﻝ ﰲ
e
f
g
h
ﺏ ﺍﻟﺼﻴﺐ ﻷ
ﹺ ﺍﻟﺴ
ﻖﻴﺣﺮﻟﺎﺑ ﻖ
ﺕﻮﻤﹾ
ﻟﺍ ﺭﹶ
ﺬﺣ(
ﻮﺍ، ﺑﺪﻣﺸﻖ؛ ﻭﻟﻴﺲ
ﺑﺪﻣﺸﻖ. ﲨﻬﺮﺓ ﺍﻟ
ﺸﻖﻣ ﰲ ﺩﺮ،
ﰲﻭ
ﺮ ﺑﺪﻣﺸﻖ
«ﻝﺎﻗ
ﺑ ﱪﻳﺺ ﺍﱘ ﺍﻟﻐﻮﻃﺔ
ﺮ ﺩﻣﺸﻖ ﻣﻦ ﻭﺭ
ﻮﹸ
ﻔﺼﻴﹺﻧﺎﺀ ﻟ
ﹺﱃ ﺇ
ﺀ ﺇ،
ﻲﻌﻤﺻَ
ﻝ ﺍﻷ
، ﻭﺻ
ﺍﺫﹺ
ﺍﻟﺸﻲﺀ ﺇﻳﺢ ﺍﻟﺮ
ﻬﺎ ﺍﻟﺒﻴﺖ ﳊﺴﺎﻥ ﺑﻦ
،ﻱﺭﻮﺑﺎﺴﻴﻨﻟﺍ
ﻈﺎﻥ ﺍﳌﺴﺄﻟﺔ
ﻴ
ﻪ، ﺑﻞ ﻋﻠﻰ
ﺎ ﰲ ﺗﻔﺴ ﺝ
، ﻓﻤﻦ ﺫﻟﻚ
c
d
e
ﺇﱃ ﺃﺻﺤﺎﺏ
ﻖﱠ
ﻔﺼﻯ ﻳﺩﺮﺑ
ﻖﻔﺼ.)١( ﻭ)ﺣ
ﻳﺺ
:
ﻣﻮﺿﻊ، ﻗﺎﻟﻮ
ﺑ ﺮ ﹶﻰ، ﻭﻫﻮ
ﻠﻌﹶ
ﻓ
ﺮﺏ :(
ﺮﻬ ﻧﹺﻳﺺ
ﺮﺑﻭ
)
ﻗﻮﻟﻪ
»
ﻭﺍﻟﱪﻳﺺ
ﺪﻻﻥ ﻋﻠﻰ ﺃﻥ ﺍﻟﱪ
ﻥ ﻣﺎﺀ ﺑﺮﺩﻯ ﻭﻫﻮ
ﺀﺎﻧﹺ
ﻟﻪ ﻣﻦ ﺇﹶﻪ ﺣﻮ
ﻘﹶ
ﻔ
ﱟ ﰲ ﻗﻮ
ﻥﹺﱃ ﺩ
ﱟ ﺇ
ﻥﺩ
ﻄﺮﺏ،
ﹶﺖ
ﻘﱠ
ﻔﺻﻭ
ﻫﺬﻩ ﺍﳌﺼﺎﺩﺭ ﲨﻴﻌﻬ
ﺣﻮﻟﻴﺔ
ﳌﻨﺴﻮﺏ ﺇﱃ
ﺳﺮﻳﻊ ﰲ ﻣﻈ
ﱂ ﻳﺬﻫﺐ ﺇﻟﻴ
ﻪ
ﰲ ﺍﻻﺣﺘﺠﺎﺝ
ﺑﻌﺾ ﻣﻨﻬﺎ
`
a
b
ﰲ)ﻥﻮﻠﻌﳚ
(
ﻢﻬﻴﻠ
... ﺀ ﺑﺮﺩﻯ ﻳﺼ
ﻱﺮﻫﺯﻷﺍﻭ
: ﹺ
ﺮﺒﻟﺍﻭ
ﻞﺻﻷﺍ
. ﻯﺩﺮﺑ
ﺟﺎﺀ ﰲ)ﻟﺴﺎﻥ ﺍﻟﻌﺮ
ﰲ ﺍﳍﺎﻣﺶ
ﻪﻟﻮﻘﺑ
:
ﻭﻫﺬﺍﻥ ﺍﻟﺸﻌﺮﺍﻥ ﻳﺪ
ﻥ ﻓﺎﻧﻪ ﻳﻘﻮﻝ ﻳﺴﻘﻮﻥ
ﹶ
ﻔﺻﹶ
ﹶﻪ ﻭﺃ
ﻘﱠ
ﻔﹶﻪ ﻭﺻ
ﻘﹶ
ﻔ
ﺩ ﹸ ﺍﻟﺸﺮﺍﺏ ﻣﻦ
ﻞﻳﻮ
ﹶ
ﺃ ﻖﻔﹶ
ﻄﺼﺘ
ﻱ: ﻄﻀﺗ
)ﻖﻔﺻ
(. ﻫ ﻭﻧﺴﺒﺖ
•ﺣ
òÌÜÛa
@
À
@
ÐnÛa
ﻫﻮ ﲢﻘﻴﻖ ﺍﳌ
ﺳ ﻊ ﺍﺳﺘﻄﺮﺍﺩ
ﺑﺬﻟﻚ ﻭﱂ
ﺴﲑﻩ ﻃﺎﻓﺢ
ﺔ، ﻧﺸﲑ ﺇﱃ
^
_
`
ﰲ ﻮﻉ ﺍﻟﻀﻤﲑ
ﺮﻌﺷ
:
ﻋﻠﺮﻳﺺ ﺍﻟﺒﺭﺩ
ﻥ ﺍﳌﻌﲎ :ﻣﺎ
١. ﻗﺎﻝ ﺍﺑﻦ ﺩﺭﻳﺪ
، ﻭﺃﺣﺴﺒﻪ ﺭﻭﻣﻲ
:
ﻣﺎﺩﺓ ﺑﺮﺹ
ﻭﺟ . (٢)
ﻪﺑ . ﺣﺐ ﺍﳊﺎﺷﻴﺔ ﻋﻠﻴﻪ
ﻛﻮﺭﻳﻦ ﻣﺎ ﻧﺼﻪ: ﻭ
ﺺ؟ ﻭﻛﺬﻟﻚ ﺣﺴﺎﻥ
ﺼﻔﻖ، ﻓﻴﻘﺎﻝ :ﹶ
ﻔﺻ
ﲢﻮﻴﻖﻔﺼْﺗﻪ ﻭﺍﻟﺘ
ﻸ
ﺘﻓ ﺭﺎﺠﺷَ
ﻷﺍ ﻖﻔﺼﺗ
١٠ / ٢٠٠
، ﻣﺎﺩﺓ )
pb
@
Þìy
@
òîvy
@a
ﻢ ﻟﺪﻳﻨﺎ ﻫﻨﺎ
ﻭﺧﺎﲤﺘﻪ ﻣﻊ
ﺭﻱ ﱂ ﻳﻘﻞ
ﺠﺎﺝ ﺎ، ﻭﺗﻔﺴ
ﱐﺎﻌﳌ ﺔﻠﻤﺘﶈﺍ
[
\
]
ﺯﺎﺟﻭ
ﻮﺟﺭ
ﻗﺎﻝ ﺣﺴﺎﻥ،
ﻭﺭﻦﹶ ﻣ
ﻥﻮﹸ
ﻘﺴ
)ﻖﻔﺼﻳ
ﻷﻥ (
ﺐ
ﻥﺁﺮﻘﻟﺍ
٧٦/١:
ﹼﻤﺖ ﺑﻪ ﺍﻟﻌﺮﺏ
ﻠﻜ
ﺐ ﺍﻟﻠﻐﺔ :ﺍﻷﺯﻫﺮﻱ
ﻣﺸﻖ .ﻭﻋﻠﻖ ﺻﺎﺣ
ﺫﻟﻚ ﻭﺍﻟﺒﻴﺘﲔ ﺍﳌﺬﻛ
ﺎﺭ ﺇﱃ ﺍﻟﱪﻳﺺ ﺍﻷ
ﻭ .٥/٧:
ﺃﻣﺎ ﻳﺼ
ﻸﹺﺫﺍ ﻣ
ﻪ ﺇﹾﺘ
ﻘﹶ
ﻔﺻﹶ
ﻪ ﻭﺃﺘ
ﻪﺘﱠ
ﻔﺼﻓ
،
ﺢﻳﺮﻟﺍﻭﺗ
ﻪ .ﻟﺴﺎﻥ ﺍﻟﻌﺮﺏ
:
@@@@@@@
bèj’
ﻢﻬﳌﺍﻭ
ﻩﲑﺴﻔﺗ
ﺭﻮﺑﺎﺴﻴﻨﻟﺍ
ﺎﺠﺘﺣﻻﺎﺑ
ﻭﺑﻴﺎﻥ
ﳌﺍ
o
Z
[
ﺓﺮﻘﺒﻟﺍ
٩١ :
،ﺭﻮﻛﺬﳌﺍ
ﺴﻳ
ﹶ
ﺫ
ﱠ
ﻛﺮ
ﻪﻟﻮﻘﻛ
:
)١(
ﻏﺮﺍﺋﺐ
ﻭﻗﺪ ﺗﻜ
ﺬﻳﺐﻭ
ﻣﺪﺑ ﺮ
ﺫ ﺮﻛﺫ
ﻧﺴﺐ
ﺏﺮﻌﻟﺍ
ﺘﹾ
ﻘﱠ
ﻔﺻﻭ
ﻪﺘﺑﺮﺿ
ﻪﺗﺩﺩﺭﻭ ﺲ ﺑﻌﺮ ،ﰊ ﺻﺤﻴﺢ
ﺔﻐﻠﻟ :
،ﺍﺑﻦ ﺩﺭﻳﺪ
ﻢﻜﶈﺍ
:
ﹺﻳﺺ
ﺮﺒﻟﺍﻭ
ﻝ ﰲ ﻳﺎﻗﻮﺕ ﺑﻌﺪ
ﺎﻬﻌﲨﺄﺑ،
ﺃﻻ ﺗﺮﺍﻩ
ﺭﺩ ﺍﻟﱪﻳﺺ(
ﻥﺎﺴﻟ
،
ﺍﻟﻘﺪﺡﹾﺖ
ﻘﹶ
ﻔﺻ
َ
ﺍﳌﺎﺀﹶﺖ ﺍﻟﺮﻳﺢ
ﻘﹶ
ﻔﺻ
ﹰ
ﹰ ﻭﴰﺎﻻ
ﻪ ﳝﻴﻨﺎﺘﹶﺒ
ﻠﹶ
ﻗ
ﻦ ﺛﺎﺑﺖ. ﻮﺍ، ﺑﺪﻣﺸﻖ؛ ﻭﻟﻴﺲ
ﺑﺪﻣﺸﻖ. ﲨﻬﺮﺓ ﺍﻟ
ﺸﻖﻣ ﰲ ﺩﺮ،
ﰲﻭ
ﺮ ﺑﺪﻣﺸﻖ
«ﻝﺎﻗ
ﺑ ﱪﻳﺺ ﺍﱘ ﺍﻟﻐﻮﻃﺔ
ﺮ ﺩﻣﺸﻖ ﻣﻦ ﻭﺭ
ﻮﹸ
ﻔﺼﻴﹺﻧﺎﺀ ﻟ
ﹺﱃ ﺇ
ﺀ ﺇ،
ﻲﻌﻤﺻَ
ﻝ ﺍﻷ
، ﻭﺻ
ﺍﺫﹺ
ﺍﻟﺸﻲﺀ ﺇﻳﺢ ﺍﻟﺮ
ﻬﺎ ﺍﻟﺒﻴﺖ ﳊﺴﺎﻥ ﺑﻦ
ﻳﺺ
:
ﻣﻮﺿﻊ، ﻗﺎﻟﻮ
ﺑ ﺮ ﹶﻰ، ﻭﻫﻮ
ﻠﻌﹶ
ﻓ
ﺮﺏ :(
ﺮﻬ ﻧﹺﻳﺺ
ﺮﺑﻭ
)
ﻗﻮﻟﻪ
»
ﻭﺍﻟﱪﻳﺺ
ﺪﻻﻥ ﻋﻠﻰ ﺃﻥ ﺍﻟﱪ
ﻥ ﻣﺎﺀ ﺑﺮﺩﻯ ﻭﻫﻮ
ﺀﺎﻧﹺ
ﻟﻪ ﻣﻦ ﺇﹶﻪ ﺣﻮ
ﻘﹶ
ﻔ
ﱟ ﰲ ﻗﻮ
ﻥﹺﱃ ﺩ
ﱟ ﺇ
ﻥﺩ
ﻄﺮﺏ،
ﹶﺖ
ﻘﱠ
ﻔﺻﻭ
ﻫﺬﻩ ﺍﳌﺼﺎﺩﺭ ﲨﻴﻌﻬ
ﻱﺮﻫﺯﻷﺍﻭ
: ﹺ
ﺮﺒﻟﺍﻭ
ﻞﺻﻷﺍ
. ﻯﺩﺮﺑ
ﺟﺎﺀ ﰲ)ﻟﺴﺎﻥ ﺍﻟﻌﺮ
ﰲ ﺍﳍﺎﻣﺶ
ﻪﻟﻮﻘﺑ
:
ﻭﻫﺬﺍﻥ ﺍﻟﺸﻌﺮﺍﻥ ﻳﺪ
ﻥ ﻓﺎﻧﻪ ﻳﻘﻮﻝ ﻳﺴﻘﻮﻥ
ﹶ
ﻔﺻﹶ
ﹶﻪ ﻭﺃ
ﻘﱠ
ﻔﹶﻪ ﻭﺻ
ﻘﹶ
ﻔ
ﺩ ﹸ ﺍﻟﺸﺮﺍﺏ ﻣﻦ
ﻞﻳﻮ
ﹶ
ﺃ ﻖﻔﹶ
ﻄﺼﺘ
ﻱ: ﻄﻀﺗ
)ﻖﻔﺻ
(. ﻫ ﻭﻧﺴﺒﺖ
١. ﻗﺎﻝ ﺍﺑﻦ ﺩﺭﻳﺪ
، ﻭﺃﺣﺴﺒﻪ ﺭﻭﻣﻲ
:
ﻣﺎﺩﺓ ﺑﺮﺹ
ﻭﺟ . (٢)
ﻪﺑ . ﺣﺐ ﺍﳊﺎﺷﻴﺔ ﻋﻠﻴﻪ
ﻛﻮﺭﻳﻦ ﻣﺎ ﻧﺼﻪ: ﻭ
ﺺ؟ ﻭﻛﺬﻟﻚ ﺣﺴﺎﻥ
ﺼﻔﻖ، ﻓﻴﻘﺎﻝ :ﹶ
ﻔﺻ
ﲢﻮﻴﻖﻔﺼْﺗﻪ ﻭﺍﻟﺘ
ﻸ
ﺘﻓ ﺭﺎﺠﺷَ
ﻷﺍ ﻖﻔﺼﺗ
١٠ / ٢٠٠
، ﻣﺎﺩﺓ )
ﺐ
ﻥﺁﺮﻘﻟﺍ
٧٦/١:
ﹼﻤﺖ ﺑﻪ ﺍﻟﻌﺮﺏ
ﻠﻜ
ﺐ ﺍﻟﻠﻐﺔ :ﺍﻷﺯﻫﺮﻱ
ﻣﺸﻖ .ﻭﻋﻠﻖ ﺻﺎﺣ
ﺫﻟﻚ ﻭﺍﻟﺒﻴﺘﲔ ﺍﳌﺬﻛ
ﺎﺭ ﺇﱃ ﺍﻟﱪﻳﺺ ﺍﻷ
ﻭ .٥/٧:
ﺃﻣﺎ ﻳﺼ
ﻸﹺﺫﺍ ﻣ
ﻪ ﺇﹾﺘ
ﻘﹶ
ﻔﺻﹶ
ﻪ ﻭﺃﺘ
ﻪﺘﱠ
ﻔﺼﻓ
،
ﺢﻳﺮﻟﺍﻭﺗ
ﻪ .ﻟﺴﺎﻥ ﺍﻟﻌﺮﺏ
:
)١(
ﻏﺮﺍﺋﺐ
ﻭﻗﺪ ﺗﻜ
ﺬﻳﺐﻭ
ﻣﺪﺑ ﺮ
ﺫ ﺮﻛﺫ
ﻧﺴﺐ
ﺏﺮﻌﻟﺍ
ﺘﹾ
ﻘﱠ
ﻔﺻﻭ
ﻪﺘﺑﺮﺿ
ﻪﺗﺩﺩﺭﻭ
òÌÜÛa
@
À
@
ÐnÛa
pb
@
Þìy
@
òîvy
@a
@@@@@
bèj’ ﻣﺎﺮ.) (١
ﻘﺎﻕ ﺍﻟﺼﻼ
«.) (٢
nﺓﺮﻘﺒﻟﺍ
٦٢ :
ﻮﻥ ﺍﳌﺬﻛﻮ
ﹺ ﺗﻜﺮ
ﹺ ﺍﻟﻠﺌﻴﻢ
ﻢﺘﺷ
ﻝﺎﻗ
ﻭﺍﺷﺘﻘ » :
ﺎ ﻭﺍﺭﺗﺴﻢ
. ﺮﻌﺷ
:
ﹺ
ﱘﺪﺘﺴﻤﻛ ﻙ«
r
s
t
n
ﺠﺐ ﺃﻥ ﻳﻜﻮ
ﺷ ﻋﻦﹺﺽ
ﺮﻋﺃ
ﻣﻮﺿﻊ ﺁﺧﺮ
ﺻﻠﻰ ﺎﻋﻠﻰ ﺩ
ﻬﺎ ﻭﻗﻮﻣﺘﻬﺎ،
ﺼﺎﻙﱠﻰ ﻋ
ﻠﺻ
o
p
q
r
ﻲﺀ ﺍﳊﻘﲑ، ﻓﻴﺠ
... ﹸ
ﺃﻭ
)ﺓﻼﺼﻟﺍ
ﻣ ( ﰲ
ﺎ
...ﺻﻭ
ﺑﺎﻟﻨﺎﺭ ﺇﺫﺍ ﻟﻴﻨﺘﻬ
ﻪ
... ﺎﻤﻓ
m
n
o
ﻞﻴﺜﻤﺘﻟ ﻲﺸﻟﺎﺑ
ﻩﺧﺎﺭﹺ ﺍﺩ
ﱘﺮﻜ
ﻔﺴﲑ ﻟﻔﻈﺔ)
ﻰﺸﻋ :
ﺎﺍﻟﺮﻳﺢ ﰲ ﺩ
ﺻﻠﻴﺖ ﺍﻟﻌﺼﺎ
ﻣ
ﹺ
ﺮﻙ ﻪﻣﺪﺘﺳﺍﻭ
o
j
k
l
m
ﱃ ﳝﻨﻊ ﻣﻦ
ﺘﻟﺍ
ﻋ ﺮﻔﹾ
ﻏ
َ ﺍﻟﻜ
ﺀﺍﺭﻮ
ﻤﺎ ﺗﻌﺮﺽ
ﻔﺘﻟ
ﺪﻋﺎﺀ ﻗﺎﻝ
ﻋﻷﺍ
ﺎﻬﻠﺑﺎﻘﻓ
ﻦﻣ ﻢﳍﻮﻗ
ﺻ:
ﹾ ﺑﺄﻣ
ﻞﺠﻌﻓﻼ ﺗ
ﻪﻟﻮﻗ ﱃﺎﻌﺗ
: o
»ﻥﺇ
ﺍﷲ ﺗﻌﺎﱃ
ﹾ
ﻏﹶ
ﺃﻭ
ﻤﻨﻴﺣﻭ
ﲟﻌﲎ ﺍﻟﺪ
ﺎﻣﺇﻭ ﻣ
ﻓ
ﰲﻭ ﻗ
ﻝﻮﻘﻳ
: » »: ﻮﻝﺇ» ﺩ ﺍﳋﺎﻣﺲ
٤
/٦١٢
، ﻣﺎﺩﺓ
ﻴﻪﻠﺮﻩ ﻭﻳﹶﻣ
ﺃ ﺮﺑﺪﻳ
. ﺍﻟﻨﺎﺭ ﻟﺘﺴﺘﻘﻴﻢ
،
١/ ٣١٢
، ﻭ
ﻟﺴﺎﻥ
ﲑﺜ ﺍﺪﺟ. ﻭﺍﻟﺒﻴﺖ
ﺍﻟﻜﺘﺎﺏ ﻟﺴﻴﺒﻮﻳﻪ :
ﺓﺃﺮﻣ،
ُﺀﺎﻴﹾ
ﻠﻌﹾ
ﻟﺍﻭ
: =
ﺍﻟﻌﺪﺩ
،ﻟﺴﺎﻥ ﺍﻟﻌﺮﺏ
: ٤
ﻱﹶ
ﺃﺼﺎ ﻓﻼﻥﻠﻲ ﻋ
ﺎ ﻋﻠﻰﻌﺼﺎ ﺇﺩﺍﺭ
ﻱﺮﺸ :١/ ١٤٣
ﻭ
٨ ﻭ٩٦
ﻭﻫﻮ
ﲑﺜﻛ
ﺍ ﻭﻭﺭﺩ ﺍﻟﺒﻴﺖ ﰲ
ﺪﻣ .ﻭﹸ
ﺔﻴﻣ:
ﺍﺳﻢ ﺍﻣ
( ﺏ،ﳊﺎﰎ ﺍﻟﻄﺎﺋﻲ
ﻝﺎﻘﻳﻭ
:
ﻲﱢ
ﻠﺼﹲ ﻳ
ﻥﻼﻓ
ﹰ
ﺪﺍﻳﻭ .ﹸ ﺍﻟﻌ
ﺔﻴﻠﺼﺗﻭ
ﺱ ﺍﻟﺒﻼﻏﺔ
:
ﺸﳐﺰﻟﺍ
ـﺟﻭ
٣٠ /٨ ﻭ
٩
ﻟﻨﻌﻤﺎﻥ ﺑﻦ ﺍﳌﻨﺬﺭ
. ﺍﻷﺑﺪ ﺑﺪﻻ ﻣﻦ ﺍﻷﻣ
ﺣﻮﻟﻴﺔ
ﺖ ﰲ)ﻟﺴﺎﻥ ﺍﻟﻌﺮ
ﺑﻦ ﺯﻫﲑ ﺍﻟﻌﺒﺴﻲ،
ﻭﹰ ﺭ
ﻭﻳﺪﺍﻬﺎ ﺭﺘﺤﺴ
ﻲﻧﹶ
ﺄﻫﺎ ﻛﺎﻟﺘﺮﻣ .ﺃﺳﺎ
ﻪﻴﻓ ﺎﻀﻳﺃ
:
١٩٩
ﻭ
ﺎﻥ ﺎ ﺇﱃ ﺍ ﻳﻌﺘﺬﺭ
) ﺪﺼﻗ
(. ﻭﺭﻭﻱ
•ﺣ
١٦
.ﻭﻧﺴﺐ ﺍﻟﺒﻴﺖ
. ١
ﺑ ﺍﻟﺒﻴﺖ ﻟﻘﻴﺲ
ﺴﻤﹶ
ﹶﻬﺎ ﻓ
ﻔﻴﻘﹾ
ﺜﺗ ﺕﺩﺭ
ﻱﺃ: ﻣﹶ
ﺃ ﻢﹶ
ﻜﺣﹶ
ﻣﺎ ﺃ
)
ﺩﻭﻡ، ﻭﻋﺼﺎ
(. (٢)
ﻪﺑ . ١-١٩٧
ﺮﻈﻨﻳﻭ
ﻓ
ﻝ ﻗﺼﻴﺪﺗﻪ
ﱵﻟﺍ
ﺎﻛ
ﺏ :/ ٣
٣٥٣
ﺓﺩﺎﻣ
ﺐ
ﻥﺁﺮﻘﻟﺍ
٨٦/١:
. ﺐ
ﻥﺁﺮﻘﻟﺍ
٤٣/١:
ﺎﺼﻌﻟﺍ ﺖ :
ﺇﺫﺍ ﺃﺭ
ﺍﻣﺘﻬﺎ: ﻲ ﻓﻴﻬﺎﹶﻧ
ﺄﺘﻟﺍ
،
: ١٢ / ٢١٢
ﺓﺩﺎﻣ
ﺐ ﻥﺁﺮﻘﻟﺍ
٦٩/١:
ﺍ
ﻟﺬﺑﻴﺎﱐ، ﻭﻫﻮ ﺃﻭﻝ
١
، ﻭﻟﺴﺎﻥ ﺍﻟﻌﺮﺏ
)١(
ﻏﺮﺍﺋﺐ
) ﺭﻮﻋ(
)٢(
ﻏﺮﺍﺋﺐ
ﻭ
ﺖﻴﻠﺻ
ﺍﺪﺘﺳﺍﻭ
ﺏﺮﻌﻟﺍ
)٣(
ﻏﺮﺍﺋﺐ
ﺔﻐﺑﺎﻨﻠﻟ
١/٥٦ ٤
/٦١٢
، ﻣﺎﺩﺓ
ﻴﻪﻠﺮﻩ ﻭﻳﹶﻣ
ﺃ ﺮﺑﺪﻳ
. ﺍﻟﻨﺎﺭ ﻟﺘﺴﺘﻘﻴﻢ
،
١/ ٣١٢
، ﻭ
ﻟﺴﺎﻥ
ﲑﺜ ﺍﺪﺟ. ﻭﺍﻟﺒﻴﺖ
ﺍﻟﻜﺘﺎﺏ ﻟﺴﻴﺒﻮﻳﻪ :
ﺓﺃﺮﻣ،
ُﺀﺎﻴﹾ
ﻠﻌﹾ
ﻟﺍﻭ
: =
،ﻟﺴﺎﻥ ﺍﻟﻌﺮﺏ
: ٤
ﻱﹶ
ﺃﺼﺎ ﻓﻼﻥﻠﻲ ﻋ
ﺎ ﻋﻠﻰﻌﺼﺎ ﺇﺩﺍﺭ
ﻱﺮﺸ :١/ ١٤٣
ﻭ
٨ ﻭ٩٦
ﻭﻫﻮ
ﲑﺜﻛ
ﺍ ﻭﻭﺭﺩ ﺍﻟﺒﻴﺖ ﰲ
ﺪﻣ .ﻭﹸ
ﺔﻴﻣ:
ﺍﺳﻢ ﺍﻣ
( ﺏ،ﳊﺎﰎ ﺍﻟﻄﺎﺋﻲ
ﻝﺎﻘﻳﻭ
:
ﻲﱢ
ﻠﺼﹲ ﻳ
ﻥﻼﻓ
ﹰ
ﺪﺍﻳﻭ .ﹸ ﺍﻟﻌ
ﺔﻴﻠﺼﺗﻭ
ﺱ ﺍﻟﺒﻼﻏﺔ
:
ﺸﳐﺰﻟﺍ
ـﺟﻭ
٣٠ /٨ ﻭ
٩
ﻟﻨﻌﻤﺎﻥ ﺑﻦ ﺍﳌﻨﺬﺭ
. ﺍﻷﺑﺪ ﺑﺪﻻ ﻣﻦ ﺍﻷﻣ
ﺖ ﰲ)ﻟﺴﺎﻥ ﺍﻟﻌﺮ
ﺑﻦ ﺯﻫﲑ ﺍﻟﻌﺒﺴﻲ،
ﻭﹰ ﺭ
ﻭﻳﺪﺍﻬﺎ ﺭﺘﺤﺴ
ﻲﻧﹶ
ﺄﻫﺎ ﻛﺎﻟﺘﺮﻣ .ﺃﺳﺎ
ﻪﻴﻓ ﺎﻀﻳﺃ
:
١٩٩
ﻭ
ﺎﻥ ﺎ ﺇﱃ ﺍ ﻳﻌﺘﺬﺭ
) ﺪﺼﻗ
(. ﻭﺭﻭﻱ
١٦
.ﻭﻧﺴﺐ ﺍﻟﺒﻴﺖ
. ١
ﺑ ﺍﻟﺒﻴﺖ ﻟﻘﻴﺲ
ﺴﻤﹶ
ﹶﻬﺎ ﻓ
ﻔﻴﻘﹾ
ﺜﺗ ﺕﺩﺭ
ﻱﺃ: ﻣﹶ
ﺃ ﻢﹶ
ﻜﺣﹶ
ﻣﺎ ﺃ
)
ﺩﻭﻡ، ﻭﻋﺼﺎ
(. ١-١٩٧
ﺮﻈﻨﻳﻭ
ﻓ
ﻝ ﻗﺼﻴﺪﺗﻪ
ﱵﻟﺍ
ﺎﻛ
ﺏ :/ ٣
٣٥٣
ﺓﺩﺎﻣ
ﺐ
ﻥﺁﺮﻘﻟﺍ
٨٦/١:
. ﺐ
ﻥﺁﺮﻘﻟﺍ
٤٣/١:
ﺎﺼﻌﻟﺍ ﺖ :
ﺇﺫﺍ ﺃﺭ
ﺍﻣﺘﻬﺎ: ﻲ ﻓﻴﻬﺎﹶﻧ
ﺄﺘﻟﺍ
،
: ١٢ / ٢١٢
ﺓﺩﺎﻣ
ﺐ ﻥﺁﺮﻘﻟﺍ
٦٩/١:
ﺍ
ﻟﺬﺑﻴﺎﱐ، ﻭﻫﻮ ﺃﻭﻝ
١
، ﻭﻟﺴﺎﻥ ﺍﻟﻌﺮﺏ
)١(
ﻏﺮﺍﺋﺐ
) ﺭﻮﻋ(
)٢(
ﻏﺮﺍﺋﺐ
ﻭ
ﺖﻴﻠﺻ
ﺍﺪﺘﺳﺍﻭ
ﺏﺮﻌﻟﺍ
)٣(
ﻏﺮﺍﺋﺐ
ﺔﻐﺑﺎﻨﻠﻟ
١/٥٦
òÌÜÛa
@
À
@
ÐnÛa
pb
@
Þìy
@
òîvy
@a
@@@@@@@
bèj’
òÌÜÛa
@
À
@
ÐnÛa
pb
@
Þìy
@
òîvy
@a
@@@@@@
bèj’ ﰒ ﺍﻟﺘﻔﺴﲑ
ﻼﺣﻪ، ﻭﺇﲤﺎﻡ
ﻭﻣﻊ ﻛﻞ ﻣﺎ
، ﻓﺈﻥ ﺫﻟﻚ
،ﻑ ﺍﳌﻌﻠﻼﺕ
ﻣﺎ ﳚﺐ ﺇﺻﻼ
ﻻ،ﺕﺎﺿﺍﺮﺘﻋ
ﺕ ﺍﳌﻌﻘﺪﺍﺕ
ﺕ، ﻭﺍﻟﻮﻗﻮﻑ
ﻣﻊ ﺇﺻﻼﺡ
ﲑ ﺍﻟﻜﺒﲑ (ﻭﺍﻻ
ﻯﻮﺳ
ﺎﻴﺑﻷﺍ
ﺕﺍﺀ ﺕﺍﱪﺘﻌﳌﺍ
،ﺕﺎﻳﻮﻨﻌﳌﺍﻭ
ﺓﰲ )
ﲑﺴﻔﺘﻟﺍ
،ﺕﻼﻀﻌﳌﺍ
ﻭﺃﺛﺒﺖ
ﺀﺍﺮﻘﻟﺍ
ﺚ ﺍﻟﻠﻔﻈﻴﺎﺕ
ﳌﺴﺎﺋﻞ ﺍﳌﻮﺭﺩﺓ
ﻣﻦ ﺍﳌﻮﺍﺿﻊ
ﺮﻱ،
ﻝﺎﻗ:»
ﻋﻠﻰ ﺍﳌﺒﺎﺣﺚ
ﺇﲤﺎﻣﻪ ﻣﻦ ﺍﳌ
ﰲ)ﺍﻟﻜﺸﺎﻑ
(
ﺮﺸﳐﺰﻟﺍﻭ
ﻞﻤﺘﺸﳌ
ﺎ ﻳﻨﺒﻐﻲ
ﰲ ﺪﺟﻮ ﻤ
ﺎ.)٢(
ﻤ ﺩ ﺍﳋﺎﻣﺲ
ﹺ
ﻞﺒﺠﹾ
ﹺ ﺍﻟ
ﻞ،
ﺕﻮﹾ
ﻗﹶ
ﺃ
:
ﺍﻟﻌﺪﺩ
: ﻞﺻﹶ
ﻲ ﺃ ﻓﹶﻊ
ﻔﺗﺮﻤﹾ
ﻟﺍ
ﺪﻣﻷﺍﻭ
: ﺮﻫﺪﻟﺍ
. (٢)
ﻪﺑ . ﹸ
ﺔﻴﻟﺎﻌﹾ
ﺽ ﺍﻟ،
ﺪﻨﺴﻟﺍﻭ:
ﺪﺑَ
ﺍﻷﹶ ﻭ
ﻞﺧﺩ ﺪﺣ ﻦ
ﺣﻮﻟﻴﺔ
ﹺ ﺍﻷﺭﺽ
ﻲ ﺍﻷﺻﻞ ﻓ
ﺎﻟﻒ:
ﻦﻲ ﻣﺎﺿﹾﻤ
ﻟﺍ
•ﺣ
ﻚﻟﹶ
ﺬﹶ
ﻛ،
ﺀﺎﻴﹾ
ﻠﻌﹾ
ﻟﺍﻭ
ﹸ
ﺔﻴﻟﺎﺨﹾ
ﺽ ﺍﻟ،
َﻭﺎﺴﻟﺍ
ﻮﺭﻱ : . ٦/١
٢ . ﻢ ﻣﻮﺿﻊ،
ﺪﻨﺴﻟﺍﻭ
:
ﹶﺖ
ﻠﺧ،
ُ ﺍﻷﺭﺽ
ﺀﺍﻮﹶ
ﻘﹾ
ﻟﺍﻭ
ﺐ
ﻥﺁﺮﻘﻟﺍ
:
ﻮﺑﺎﺴﻴﻨﻟﺍ
ﺐ
ﻥﺁﺮﻘﻟﺍ
٤٢٢ /٣:
=ﻢﺳﺍ
ﻱﹶ
ﺃ: ﺧ
)١(
ﻏﺮﺍﺋﺐ
)٢(
ﻏﺮﺍﺋﺐ ﹺ
ﻞﺒﺠﹾ
ﹺ ﺍﻟ
ﻞ،
ﺕﻮﹾ
ﻗﹶ
ﺃ
:
: ﻞﺻﹶ
ﻲ ﺃ ﻓﹶﻊ
ﻔﺗﺮﻤﹾ
ﻟﺍ
ﺪﻣﻷﺍﻭ
: ﺮﻫﺪﻟﺍ
. ﹸ
ﺔﻴﻟﺎﻌﹾ
ﺽ ﺍﻟ،
ﺪﻨﺴﻟﺍﻭ:
ﺪﺑَ
ﺍﻷﹶ ﻭ
ﻞﺧﺩ ﺪﺣ ﻦ
ﹺ ﺍﻷﺭﺽ
ﻲ ﺍﻷﺻﻞ ﻓ
ﺎﻟﻒ:
ﻦﻲ ﻣﺎﺿﹾﻤ
ﻟﺍ
ﻚﻟﹶ
ﺬﹶ
ﻛ،
ﺀﺎﻴﹾ
ﻠﻌﹾ
ﻟﺍﻭ
ﹸ
ﺔﻴﻟﺎﺨﹾ
ﺽ ﺍﻟ،
َﻭﺎﺴﻟﺍ
ﻮﺭﻱ : . ٦/١
٢ . ﻢ ﻣﻮﺿﻊ،
ﺪﻨﺴﻟﺍﻭ
:
ﹶﺖ
ﻠﺧ،
ُ ﺍﻷﺭﺽ
ﺀﺍﻮﹶ
ﻘﹾ
ﻟﺍﻭ
ﺐ
ﻥﺁﺮﻘﻟﺍ
:
ﻮﺑﺎﺴﻴﻨﻟﺍ
ﺐ
ﻥﺁﺮﻘﻟﺍ
٤٢٢ /٣:
=ﻢﺳﺍ
ﻱﹶ
ﺃ: ﺧ
)١(
ﻏﺮﺍﺋﺐ
)٢(
ﻏﺮﺍﺋﺐ ﹺ
ﻞﺒﺠﹾ
ﹺ ﺍﻟ
ﻞ،
ﺕﻮﹾ
ﻗﹶ
ﺃ
:
: ﻞﺻﹶ
ﻲ ﺃ ﻓﹶﻊ
ﻔﺗﺮﻤﹾ
ﻟﺍ
ﺪﻣﻷﺍﻭ
: ﺮﻫﺪﻟﺍ
. ﹸ
ﺔﻴﻟﺎﻌﹾ
ﺽ ﺍﻟ،
ﺪﻨﺴﻟﺍﻭ:
ﺪﺑَ
ﺍﻷﹶ ﻭ
ﻞﺧﺩ ﺪﺣ ﻦ
ﹺ ﺍﻷﺭﺽ
ﻲ ﺍﻷﺻﻞ ﻓ
ﺎﻟﻒ:
ﻦﻲ ﻣﺎﺿﹾﻤ
ﻟﺍ
ﻚﻟﹶ
ﺬﹶ
ﻛ،
ﺀﺎﻴﹾ
ﻠﻌﹾ
ﻟﺍﻭ
ﹸ
ﺔﻴﻟﺎﺨﹾ
ﺽ ﺍﻟ،
َﻭﺎﺴﻟﺍ
ﻮﺭﻱ : . ٦/١
٢ . ﻢ ﻣﻮﺿﻊ،
ﺪﻨﺴﻟﺍﻭ
:
ﹶﺖ
ﻠﺧ،
ُ ﺍﻷﺭﺽ
ﺀﺍﻮﹶ
ﻘﹾ
ﻟﺍﻭ
ﺐ
ﻥﺁﺮﻘﻟﺍ
:
ﻮﺑﺎﺴﻴﻨﻟﺍ
ﺐ
ﻥﺁﺮﻘﻟﺍ
٤٢٢ /٣:
=ﻢﺳﺍ
ﻱﹶ
ﺃ: ﺧ
)١(
ﻏﺮﺍﺋﺐ
)٢(
ﻏﺮﺍﺋﺐ
òÌÜÛa
@
À
@
ÐnÛa
pb
@
Þìy
@
òîvy
@a
@@@@@@@
bèj’ » ﺔﻴﺑ«
). (١ ﻪ) :ﻮﻟﻪﺳ ﺩ ﺍﳋﺎﻣﺲ
ﺍﻟﻌﺪﺩ
ﺣﻮﻟﻴﺔ
•ﺣ
- .٧
ﺭ
ﻖﺑﺎﺴﻟﺍ
٦/ ١: -
)١(
ﺭﺪﺼﳌﺍ ﺩ ﺍﳋﺎﻣﺲ
ﻭﻫﺬﺍ ﺃﻣﺮ ﻻ
ﱯ-
)١( ﻰﻠﻋ
ﻚ، ﻓﻬﻮ ﻗﺪ
ﻠﻰ ﻣﻌﺎﻥ ﻗﺪ
ﻋﻠﻰ ﻣﺎ ﳚﺪﻩ
ﺮﺏ، ﻣﻊ ﺃﻥ
ﻘﺎﻡ ﰲ ﳎﺎﻝ
ﺔﻐﻠﻟﺍ
ﺎﻬﻠﻟﺫﻭ
ﺮ ﺍﻟﻨﺤﻮ ﰲ
ﻟﻴﺎ ﻓﺈﻧﺎ ﻧﺮﻯ
ﻝﻮﻘ :
»ﺪﻗﻭ
ﺍﳌﻌﺘﺰﻟﺔ ﻋﻦ
ﺍﻟﻌﺪﺩ
،ﺔﺣﺎﺼﻔﻟﺍﻭ
ﺍﻟﺸﻴﺦ ﺍﻟﺬﻫﱯ
ﺿﻮﻋﻴﺘﻪ ﻛﺬﻟﻚ
ﻞ ﺍﻷﻟﻔﺎﻅ ﻋﻠ
ﻋ ﻳﻞ، ﺍﻋﺘﻤﺎﺩﺍ
ﰲ ﻛﻼﻡ ﺍﻟﻌﺮ
ﺫﺇ
ﺍ ﻛﺎﻥ ﺍﳌﻘ
ﻡ ﻱﺮﺸﳐﺰﻟﺍ
ﻱﺮﺸﳐ ﺮﺨﺴﻳ
ﻬﺎ ﻣﺒﺪﺃ ﺍﻋﺘﺰﺍﻟ
ﱄ«.)٤( ﻮﻘﻳﻭ
ﺪﻣﺔ ﻓﻜﺮﺓ
ﺤﺎﻧﻪ ﻳﻮﻡ
ﺔﻣﺎﻴﻘﻟﺍ
. ﻣﻦ ﺍﻟﺼﺤﺔ
ﲔ- ﻢﻬﻨﻣﻭ
ﻭﻋﻠﻰ ﻣﻮﺿ
ﺼﻮﺹ، ﻭﳛﻤﻞ
ﻇﺎﻫﺮ ﰲ ﺍﻟﺘﺄﻭﻳﻞ
ﺎﺩﺭﺓ ﺍﻟﻮﺭﻭﺩ
، ﻭﻻ ﺳﻴﻤﺎ
:)٣(»
ﻡﺪﺨﺘﺳﺍ
ﺔﻳﻮ :
»ﺸﳐﺰﻟﺍﻭ
ﻫﺎ ﺃﻭ ﺗﺄﻭﻳﻠﻬ
ﻌﺘﻘﺪ ﺍﻻﻋﺘﺰﺍﱄ
ﺨﻴﻴﻞ ﰲ ﺧﺪ
ﺅﻳﺔ ﺍﳌﺆﻣﻨﲔ ﷲ ﺳﺒﺤ
ﺣﻮﻟﻴﺔ
ﻟﺒﻴﺎﻥ ﺩ
ﺎﻬﺘﺟﺭ
ﺪﻦﻣ
ﲔﺜﺣﺎﺒﻟﺍ
ﻋﻠﻰ ﺍﳌﻌﺎﱐ،
ﻷﻟﻔﺎﻅ ﻭﺍﻟﻨﺼ
ﻇﺎ ﻭ ﺑﺘﻌﺴﻒ
ﰲ ﻧﻔﺴﻬﺎ، ﻧﺎ
ﺽ ﺍﳌﺮﺍﺩ ﻣﻨﻪ
ﻱﻭﺎﺼﻟﺍ ﻪﻨﻋ:
ﺮﳚﺎﺗﻪ ﺍﻟﻨﺤﻮ
ﺔ ﳝﺲ ﻇﺎﻫﺮ
ﻼ ﻟﻴﻨﺼﺮ ﺍﳌﻌ
ﺘﻤﺜﻴﻞ ﻭﺍﻟﺘﺨ
ﰲ ﺗﺄﻭﻳﻠﻪ ﻟﻠﻤﺮﺍﺩ ﺑﺮﺅ
. •ﺣ
ﻰﻠﻋ
ﻪﻤﻈﻧ
ﺒﻟ
ﻻﺣﻈﻪ ﺍﻟﻌﺪﻳﺪ
ﻝﻻﺪﺘﺳﻻﺍ
ﺴﲑﻩ ﻟﺒﻌﺾ ﺍﻷ
ﺑﺒﻌﺪ ﻛﺒﲑ، ﺃ
ﻛﺎﻧﺖ ﻏﺮﻳﺒﺔ
،ﻨﺎﻩ ﻭﺍﺳﺘﻔﺎﺽ
ﱄ)٢(. ﺍﻳﻘﻮﻝ
ﰲ ﳎﺎﻝ ﲣﺮ
ﺍ ﻛﺎﻧﺖ ﺍﻵﻳﺔ
ﻌﺴﻔﺎ ﻣﺘﻤﺤﻼ
ﺃﺳﻠﻮﺏ ﺍﻟﺘ
/٤٤٥
-٤٤٧
. ٤/ ١٩٢ ﻭ٢٣٢
ﰲ
ﲑ ﺍﻟﻘﺮﺁﻥ :٢٤١
. . . ﻋﻠﻴﻬﺎ، ﻓﺘﻘﺎﺱ
ﻴﻪ، ﻭﻫﻮ ﻣﺎ
ﺰﳐﺸﺮﻱ ﰲ
ﺣﻴﺎﻧﺎ ﰲ ﺗﻔﺴ
ﻬﺎ
ﻆﻔﻠﻟﺍ
ﻻﺇ
ﻫﺪ ﳍﺎ
ﻥﺇﻭ ﻛ
ﺪ ﺍﺷﺘﻬﺮ
ﻨﻌﻣ
ﺒﻫﺬ ﻪ
ﱄﺍﺰﺘﻋﻻﺍ
ﻝ«
.ﻝﻮﻘﻳﻭ
ﻻﻋﺘﺰﺍﻝ، ﻓﺈﺫﺍ
ﻱ ﳓﻮﻳﺎ ﻣﺘﻌ
ﳐﺰﻟﺍ
ﻱﺮﺸ
«.) (٥
ﲑ
ﻥﻭﺮﺴﻔﳌﺍﻭ
١: /
ﺗﻔﺴﲑ ﺍﻟﻜﺸﺎﻑ :
ﺍﻟﺰﳐﺸﺮﻱ ﰲ ﺗﻔﺴﲑ
ﻱﺮﺸﳐﺰﻟﺍ :١٤٧
ﺭ
ﻖﺑﺎﺴﻟﺍ
٦٤١ :
. ﻋ ﺔﺠﳊﺍ
ﺟﺪﺍﻝ ﻓﻴ
ﻣﻨﻬﺞ ﺍﻟﺰ
ﻳﻐﺮﺏ
ﺣﺃ
ﻻ ﳛﺘﻤﻠﻬ
ﻣﻦ ﺷﻮﺍﻫ
ﺍﻟﻠﻔﻆ ﻗﺪ
ﻧﺼﺮﺓ ﻣﺬ
ﻝﺍﺰﺘﻋﻼﻟ
ﺔﻣﺪﺧ
ﻻﺍ
ﻱﺮﺸﳐﺰﻟﺍ
ﻡﺪﺨﺘﺳﺍ
ﺪﻴﺣﻮﺘﻟﺍ«
)١(
ﲑﺴﻔﺘﻟﺍ
)٢( ﺮﻈﻨﻳ
ﺗ:
)٣( ﺞﻬﻨﻣ
ﺍ
)٤( ﺞﻬﻨﻣ
ﺍ
)٥(
ﺭﺪﺼﳌﺍ ﱯ-
)١(
ﱯ ﻘﺎﻡ ﰲ ﳎﺎ
ﺔﻐﻠﻟﺍ
ﻬﻠﻟﺫﻭ
ﺮ ﺍﻟﻨﺤﻮ
ﻟﻴﺎ ﻓﺈﻧﺎ ﻧﺮ
ﻝﻮﻘ :
»ﻗﻭ
ﺫﺇ
ﺍ ﻛﺎﻥ ﺍﳌﻘ
ﻡ ﻱﺮﺸﳐﺰﻟﺍ
ﻱﺮﺸﳐ ﺮﺨﺴﻳ
ﻬﺎ ﻣﺒﺪﺃ ﺍﻋﺘﺰﺍﻟ
ﱄ«.)٤( ﻮﻘﻳﻭ
، ﻭﻻ ﺳﻴﻤﺎ
:)٣(»
ﻡﺪﺨﺘﺳﺍ
ﺔﻳﻮ :
»ﺸﳐﺰﻟﺍﻭ
ﻫﺎ ﺃﻭ ﺗﺄﻭﻳﻠﻬ
ﻌﺘﻘﺪ ﺍﻻﻋﺘﺰﺍﱄ
ﺽ ﺍﳌﺮﺍﺩ ﻣﻨﻪ
ﻱﻭﺎﺼﻟﺍ ﻪﻨﻋ:
ﺮﳚﺎﺗﻪ ﺍﻟﻨﺤﻮ
ﺔ ﳝﺲ ﻇﺎﻫﺮ
ﻼ ﻟﻴﻨﺼﺮ ﺍﳌﻌ
،ﻨﺎﻩ ﻭﺍﺳﺘﻔﺎﺽ
ﱄ)٢(. ﺍﻳﻘﻮﻝ
ﰲ ﳎﺎﻝ ﲣﺮ
ﺍ ﻛﺎﻧﺖ ﺍﻵﻳﺔ
ﻌﺴﻔﺎ ﻣﺘﻤﺤﻼ
ﺪ ﺍﺷﺘﻬﺮ
ﻨﻌﻣ
ﺒﻫﺬ ﻪ
ﱄﺍﺰﺘﻋﻻﺍ
ﻝ«
.ﻝﻮﻘﻳﻭ
ﻻﻋﺘﺰﺍﻝ، ﻓﺈﺫﺍ
ﻱ ﳓﻮﻳﺎ ﻣﺘﻌ
ﻆ ﻗﺪ
ﺮﺓ ﻣﺬ
ﻝﺍﺰﺘ
ﺔﻣ
ﻻﺍ
ﻱﺮﺸﳐ ،)١(
ﻙ ﻫﺒﻪ .« ﺩ ﺍﳋﺎﻣﺲ
ﺍﻟﻌﺪﺩ
ﺣﻮﻟﻴﺔ
:٢٣٣ ﻭ٢٣٥
. ١٧٤-١٧٦
. ﻪ) :ﻮﻟﻪﺳ •ﺣ
ﻟﺪﺭﺍﺳﺎﺕ ﺍﻟﻨﺤﻮﻳﺔ
ﻭﻳﻨﻈﺮ ﻣﻨﻪ ﺃﻳﻀﺎ :٤
ﺍﻟﻜﺮﱘ ﻭﺃﺛﺮﻩ ﰲ ﺍﻟ
ﻱﺮﺸﳐﺰﻟﺍ :٤١ ﻭ
)١( ﻥﺁﺮﻘﻟﺍ
)٢( ﺞﻬﻨﻣ
ﺍ ﺩ ﺍﳋﺎﻣﺲ
ﺍﻟﻌﺪﺩ
ﺣﻮﻟﻴﺔ
:٢٣٣ ﻭ٢٣٥
. ١٧٤-١٧٦
. •ﺣ
ﻟﺪﺭﺍﺳﺎﺕ ﺍﻟﻨﺤﻮﻳﺔ
ﻭﻳﻨﻈﺮ ﻣﻨﻪ ﺃﻳﻀﺎ :٤
ﺍﻟﻜﺮﱘ ﻭﺃﺛﺮﻩ ﰲ ﺍﻟ
ﻱﺮﺸﳐﺰﻟﺍ :٤١ ﻭ
)١( ﻥﺁﺮﻘﻟﺍ
)٢( ﺞﻬﻨﻣ
ﺍ ﺩ ﺍﳋﺎﻣﺲ
ﺍﻟﻌﺪﺩ
ﺣﻮﻟﻴﺔ
:٢٣٣ ﻭ٢٣٥
. ١٧٤-١٧٦
. •ﺣ
ﻟﺪﺭﺍﺳﺎﺕ ﺍﻟﻨﺤﻮﻳﺔ
ﻭﻳﻨﻈﺮ ﻣﻨﻪ ﺃﻳﻀﺎ :٤
ﺍﻟﻜﺮﱘ ﻭﺃﺛﺮﻩ ﰲ ﺍﻟ
ﻱﺮﺸﳐﺰﻟﺍ :٤١ ﻭ
)١( ﻥﺁﺮﻘﻟﺍ
)٢( ﺞﻬﻨﻣ
ﺍ ١٧٣
òÌÜÛa
@
À
@
ÐnÛa
pb
@
Þìy
@
òîvy
@a
@@@@@@@
bèj’
òÌÜÛa
@
À
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ÐnÛa
pb
@
Þìy
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òîvy
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bèj’ (. ﲔ)١ ،ﻪ)٢( )٢( ﺩ ﺍﳋﺎﻣﺲ
ﺍﺗﺮ ﻭﺍﻵﺣﺎﺩ
ﺎﻣﻌﺔ ﺑﻐﺪﺍﺩ، ﺳﻨﺔ
ﺍﻟﻌﺪﺩ
ﺮﻱ ﻓﻴﻪ ﺍﻟﺘﻮ
ﻡﻮﻠ،ﺔﻴﻣﻼﺳﻹﺍ
ﺎﺟ
.ﻭ
،ﻞﻘﻨﻟﺍ
ﺮﳚﻭ
/ ١١٧
. ﰲ ﳎﻠﺔ ﻛﻠﻴﺔ ﺍﻟﻌﻠ
ﺞ ﺍﳌﻌﻘﻞ:٧٧١ /١
ﺣﻮﻟﻴﺔ
ﻪ ﺍﻟﻌﻘﻞ ﻋﻠﻰ
١/٢٧٦
ﻭﺍﳌﺰﻫﺮ
١
ﻊ ﻭﺍﻟﻈﻦ، ﻣﻨﺸﻮﺭ
ﻷﺍ
ﺳﻨﻮﻱ، ﻭﻣﻨﺎﻫﺞ
•ﺣ
ﻣﺎ ﺭﻛﺐ ﻓﻴﻪ
ﻝﻮﺼﶈﺍ :ﻕ
١
ﺝ
١
ﺍﻟﻘﺮﺁﻥ ﺑﲔ ﺍﻟﻘﻄﻊ
٩٢ ﺎﻳﺔ ﺍﻟﺴﻮﻝﻭ
:
ﻣ ﻭﺇﻥ ﻣﻨﻬﺎ
(
ﰲ ﺗﻔﺼﻴﻞ ﺭﺃﻳﻬﻢ
ﺍ:
ﲝﺜﻨﺎ :ﺩﻻﻟﺔ ﺃﻟﻔﺎﻅ
١
.ﻡ
ﻝ :٤٩٢ /١-٦٩
،ﺔﻴﻠﻘﻌﻟﺍ
ﺎﻀﻳﺃ
.)٣(
)١(
ﻳﻨﻈﺮ
ﰲ
)٢(
ﲝ ﺮﻈﻨﻳ
١٩٩٩
)٣(
ﻝﻮﺼﶈﺍ ﺩ ﺍﳋﺎﻣﺲ
ﺍﺗﺮ ﻭﺍﻵﺣﺎﺩ
ﺎﻣﻌﺔ ﺑﻐﺪﺍﺩ، ﺳﻨﺔ
ﺍﻟﻌﺪﺩ
ﺮﻱ ﻓﻴﻪ ﺍﻟﺘﻮ
ﻡﻮﻠ،ﺔﻴﻣﻼﺳﻹﺍ
ﺎﺟ
.ﻭ
،ﻞﻘﻨﻟﺍ
ﺮﳚﻭ
/ ١١٧
. ﰲ ﳎﻠﺔ ﻛﻠﻴﺔ ﺍﻟﻌﻠ
ﺞ ﺍﳌﻌﻘﻞ:٧٧١ /١
ﺣﻮﻟﻴﺔ
ﻪ ﺍﻟﻌﻘﻞ ﻋﻠﻰ
١/٢٧٦
ﻭﺍﳌﺰﻫﺮ
١
ﻊ ﻭﺍﻟﻈﻦ، ﻣﻨﺸﻮﺭ
ﻷﺍ
ﺳﻨﻮﻱ، ﻭﻣﻨﺎﻫﺞ
•ﺣ
ﻣﺎ ﺭﻛﺐ ﻓﻴﻪ
ﻝﻮﺼﶈﺍ :ﻕ
١
ﺝ
١
ﺍﻟﻘﺮﺁﻥ ﺑﲔ ﺍﻟﻘﻄﻊ
٩٢ ﺎﻳﺔ ﺍﻟﺴﻮﻝﻭ
:
ﻣ ﻭﺇﻥ ﻣﻨﻬﺎ
(
ﰲ ﺗﻔﺼﻴﻞ ﺭﺃﻳﻬﻢ
ﺍ:
ﲝﺜﻨﺎ :ﺩﻻﻟﺔ ﺃﻟﻔﺎﻅ
١
.ﻡ
ﻝ :٤٩٢ /١-٦٩
،ﺔﻴﻠﻘﻌﻟﺍ
ﺎﻀﻳﺃ
.)٣(
)١(
ﻳﻨﻈﺮ
ﰲ
)٢(
ﲝ ﺮﻈﻨﻳ
١٩٩٩
)٣(
ﻝﻮﺼﶈﺍ ( ﺎﻀ.)٣( ﺓﱪ.)١( ﻪ
òÌÜÛa
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bèj’
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bèj’ ﺩ ﺍﳋﺎﻣﺲ
ﺔ، ، ١
١٩٧٨
. ، ١
١٩٨٠
. ١٩٧٤
.ﻡ
ﺭﻣﻀﺎﻥ، ﺩﻣﺸﻖ،
ﺭﺍ
،ﺭﺎﺼﻧﻷﺍ
، ٢
ﻜﺘﺐ ،ﺍﻟﻌﺮﺑﻴﺔ
، ١
١٩٧٩
. ١٩ . ﺩﺭﺍﻮﳌﺍ
ﻟﻌﺒﺪ ﺍﷲ
ﺍﻟﻌﺪﺩ
١
. ﻡ
ﺩﺍﺭ ﺍﻟﻜﺘﺐ
ﺔﻴﻓﺎﻘﺜﻟﺍ
ﺮﰊ،
١٩٦٨
. ﺍﻵﻓﺎﻕ ﺍﳉﺪﻳﺪﺓ،
ﺎﻣﻌﺔ ﻋﲔ ﴰﺲ،
٤
ﻳﻦ ﻋﺒﺪ
ﻦﲪﺮﻟﺍ
ﺭ
ﺬﻳﺐ، ﺍﻟﻘﺎﻫﺮﺓ، ﺩ
ﺮﺓ، ﺩﺍﺭ ﺇﺣﻴﺎﺀ ﺍﻟﻜ
،ﺪ، ﺩﺍﺭ ﺍﻟﻔﻜﺮ
٣
،
ﻣﻌﺔ ﺍﳌﻮﺻﻞ،
٩٨٩
ﺏ،
١٩٨٧
. ـ . ،، ١ﺪﱐ
١٩٨٥
. ﻥ، ﻭﺑﺬﻳﻠﻪ ﺃﻋﺬﺏ
ﻊﺟﺍﺮﳌﺍﻭ
،ﱯﻠﳊﺍ
١٥٩ ، ٣
ﻟﻠﺒﺪﻱ، ﺍﻟﻜﻮﻳﺖ،
، ﺩﺍﺭﺓ ﺍﻻﲢﺎﺩ ﺍﻟﻌﺮ
ﺎﻛﺮ، ﺑﲑﻭﺕ، ﺩﺍﺭ
. ﺕ . ﻪ) :ﻮﻟﻪﺳ ﺩ
، ٢
١٩٥٩
. ،ﺓﺮﻫﺎﻘﻟﺍ ،
ﺮﺸﻧ
ﺎﺟ
ﻱ، ﲢﻘﻴﻖ ﳏﻲ ﺍﻟﺪ
ﺩ . ﻋﺒﺪ ﺍﻟﻌﻈﻴﻢ ﺍﻟﺬ
ﻀﻞ ﺇﺑﺮﺍﻫﻴﻢ، ﺍﻟﻘﺎﻫﺮ
ﺴﻠﻔﻴﺔ، ﺩ. . ﺕ
ﻂ ﻋﺒﺪ ﺍﻟﺮﲪﻦ ﳏﻤﺪ
ﻳﺔ ﺩﺍﺭ ﺍﻟﻜﺘﺐ، ﺟﺎ
. ١
ﺎﻣﺔ ﺍﳌﺼﺮﻳﺔ ﻟﻠﻜﺘﺎﺏ
ﺍﻟﻘﻠﻢ، ﺩ
. ﺕ . ، ١ﲑﻳﺔ،
١٣٠٦
ـﻫ
ﻤﺸﺔ، ﺟﺪﺓ، ﺩﺍﺭ ﺍﳌﺪ
ﻦ ﳏﻤﺪ ﺑﻦ ﺳﻠﻴﻤﺎﻥ
ﺣﻮﻟﻴﺔ
ﺔ ﺍﳌﺼﺎﺩﺭ
ﻭ
ﺓ، ﻣﺼﻄﻔﻰ ﺍﻟﺒﺎﰊ
ﳏﻤﺪ ﲰﲑ ﳒﻴﺐ ﺍﻟ
ﺑﻦ ﳏﻤﺪ، ﺍﻟﻘﺎﻫﺮﺓ
ﻘﻴﻖ ﺃﲪﺪ ﳏﻤﺪ ﺷﺎ
،ﺮﺓ، ﺩﺍﺭ ﺍﳊﺪﻳﺚ
ﻔﻜﺮ، ﺏ . . ﺕ
ﻱ، ﺑﻐﺪﺍﺩ، ﺍﻟﺮﺍﺑﻄﺔ،
ﺪﺒ
،ﺍﶈﺴﻦ ﺍﻟﺘﺮﻛﻲ
ﺃﺑﻮ ﺑﻜﺮ ﺍﻷﻧﺒﺎﺭﻱ
،ﲏﻳﻮﳉﺍ
ﻖﻴﻘﲢ
ﻘﻴﻖ ﳏﻤﺪ ﺃﺑﻮ ﺍﻟﻔﻀ
ﳌﻨﻮﺭﺓ، ﺍﳌﻜﺘﺒﺔ ﺍﻟﺴﻠ
ﻟﻘﻠﻢ، ﺩ
. . ﺕ
ﺒﺎﺭﻛﻔﻮﺭﻱ، ﺿﺒﻂ
ﺪ، ﺍﳌﻮﺻﻞ، ﻣﺪﻳﺮﻳ
، ﺩﺍﺭ ﺍﻟﻔﻜﺮ
٩٨٨
ﺍﻟﻘﺎﻫﺮﺓ، ﺍﳍﻴﺌﺔ ﺍﻟﻌﺎ
ﻫﱯ، ﺑﲑﻭﺕ، ﺩﺍﺭ
ﺯﺍﱐ، ﺍ
ﻟﻘﺎﻫﺮﺓ، ﺍﳋﲑ
ﻣﻔﻴﺪ ﳏﻤﺪ ﺃﺑﻮ ﻋﻤﺸ
ﺪﺋﺍﻭﺰﻟﺍ
:
ﳏﻤﺪ ﺑﻦ
•ﺣ
ﺔﻤﺋﺎﻗ
ﺍﻟﺴﻴﻮﻃﻲ، ﺍﻟﻘﺎﻫﺮ
ﺍﻟﻨﺤﻮ ﺍﻟﻌﺮﰊ
:
ﺩ.ﳏ
ﻜﺎﻡ :ﺍﻵﻣﺪﻱ ﻋﻠﻲ
ﻜﺎﻡ :ﺍﺑﻦ ﺣﺰ
ﻡ، ﲢﻘ
ﺣﺎﻣﺪ ﺍﻟﻐﺰﺍﱄ، ﺍﻟﻘﺎﻫﺮ
ﱐ، ﺍﻟﻘﺎﻫﺮﺓ، ﺩﺍﺭ ﺍﻟﻔ
ﻋﻠﻲ ﻧﻘﻲ ﺍﳊﻴﺪﺭﻱ
ﺪ :ﺩ. ﻋﺒﺪ ﺍﷲ
ﺒﻋ
ﰲ ﻛﺘﺎﺏ ﺍﷲ
:
ﻘﻪ :ﺇﻣﺎﻡ ﺍﳊﺮﻣﲔ
ﻥ :ﺍﻟﺰﺭﻛﺸﻲ، ﲢﻘ
ﻟﺒﻐﺪﺍﺩﻱ، ﺍﳌﺪﻳﻨﺔ ﺍﳌ
ﺒﻮﺭ ﺷﺎﻫﲔ، ﺩﺍﺭ ﺍ
ﺟﺎﻣﻊ ﺍﻟﺘﺮﻣﺬﻱ :ﺍﳌﺒ
.ﳏﺴﻦ ﻋﺒﺪ ﺍﳊﻤﻴﺪ
ﻥﺎﻴﺒﻟ :(ﺩ ، ﺕﻭﲑﺑ
ﻷﺣﻜﺎﻡ ﺍﻟﻘﺮﺁﻥ
(:
. ﳏﻤﺪ ﺣﺴﲔ ﺍﻟﺬ
ﺳﻌﺪ ﺍﻟﺪﻳﻦ ﺍﻟﺘﻔﺘﺎﺯ
ﳊﻨﺒﻠﻲ، ﲢﻘﻴﻖ ﺩ .ﻣ
ﻊ ﺍﻷﺻﻮﻝ ﻭﳎﻤﻊ
،ﺓﺭﻮﻨﳌ
١٩٦١
. ﻥﰲ
ﻋﻠﻮﻡ ﺍﻟﻘﺮﺁﻥ،
ﻥﺁﺮ
ﻭﺍﻟﻘﺮﺍﺀﺍﺕ ﰲ
ﰲﻡ
ﺃﺻﻮﻝ ﺍﻷﺣﻜ
ﻡﰲ
ﺃﺻﻮﻝ ﺍﻷﺣﻜ
ﻡﻮﻠﻋ ﻦﻳﺪﻟﺍ
:
ﺣ ﻮﺑﺃ
ﻝﻮﺤﻔﻟﺍ :ﱐﺎﻛﻮﺸﻟﺍ
ﻁﺎﺒﻨﺘﺳﻻﺍ :ﺪﻴﺴﻟﺍ
ﻣﺬﻫﺐ ﺍﻹﻣﺎﻡ ﺃﲪﺪ
ﺍﻟﻮﻗﻒ ﻭﺍﻻﺑﺘﺪﺍﺀ
ﻭﻧﻴﺔ،
١٩٧١
. ﰲﺎﻥ
ﺃﺻﻮﻝ ﺍﻟﻔﻘ
١٤ ـﻫ
. ﰲﻥ
ﻋﻠﻮﻡ ﺍﻟﻘﺮﺁﻥ
١٩ . ﺦﺩﺍﺪﻐﺑ
:
ﺍﳋﻄﻴﺐ ﺍ
ﺦ ﻥﺁﺮﻘﻟﺍ
:
ﻋﺒﺪ ﺍﻟﺼﺒ
ﻱﺫﻮﺣﻷﺍ
ﺑﺮﺸﺡ ﺟ
ﺗﻔﺴﲑ ﺍﻟﻘﺮﺁﻥ
:
ﺩ. ﲑ
ﻱﱪﻄﻟﺍ
)
ﺟﺎﻣﻊ ﺍﻟ
ﲑ
ﱯﻃﺮﻘﻟﺍ
) ﻊﻣﺎﳉﺍ
ﲑ
ﻥﻭﺮﺴﻔﳌﺍﻭ
:
ﺩ
ﻋﻠﻰﺢ
ﺢﻴﺿﻮﺘﻟﺍ
:
ﻴﺪ :ﺃﺑﻮ ﺍﳋﻄﺎﺏ ﺍﳊ
ﺪﺋﺍﻮﻔﻟﺍ
ﻣﻦ ﺟﺎﻣﻊ
ﻢ ﺍﳌﺪﱐ، ﺍﳌﺪﻳﻨﺔ ﺍﳌﻨ
١- ﻥﺎﻘﺗﻹﺍ
٢ -
ﺮﺛﺃ
ﺮﻘﻟﺍ
٣ - ﻡﺎﻜﺣﻹﺍ
٤ - ﻡﺎﻜﺣﻹﺍ
٥ -
ﺀﺎﻴﺣﺇ
ﻋ
٦ -
ﺩﺎﺷﺭﺇ
ﺍ
٧ -
ﻝﻮﺻﺃ
ﺍ
٨ - ﻝﻮﺻﺃ
ﻣ
٩ -
ﺡﺎﻀﻳﺇ
ﻭﺎﻌﺘﻟﺍ
١٠-
ﺎﻫﱪﻟﺍ
٤٠٠
١١- ﺎﻫﱪﻟﺍ
٩٥٧
١٢- ﺦﻳﺭﺎﺗ
١٣- ﻳﺭﺎﺗﺦ
١٤- ﺔﻔﲢ ﺍ
١٥- ﺭﻮﻄﺗ
١٦- ﲑﺴﻔﺗ
١٧- ﲑﺴﻔﺗ
١٨- ﲑﺴﻔﺘﻟﺍ
١٩- ﺢﻳﻮﻠﺘﻟﺍ
٢٠- ﻴﻬﻤﺘﻟﺍ
٢١- ﻊﲨ
ﻢﺷﺎﻫ
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bèj’ ﺩ ﺍﳋﺎﻣﺲ
، ١ﻼﻝ ،
١٩٧٢
. ﻝﻼﻘﺘﺳ
،ﻯﱪﻜﻟﺍ
ـ )
ﺔﻴﺷﺎﲝ
ﲑﺴﻔﺗ
١٩٧٨ . ﺀ
،ﺍﻟﻜﺘﺐ ﺍﻟﻌﺮﺑﻴﺔ
، ١
١٩٧٩
. ﺸﻖ، ﺩﺍﺭ ،ﺍﻟﻔﻜﺮ
ﻬﻮﻝ(، ﻧﺸﺮ ﺁﺭﺛﺮ
ﻤﺪ ﻋﻠﻲ ،ﺻﺒﻴﺢ
ﺍﻟﻌﺪﺩ
١٩٨ . ﻪ) :ﻮﻟﻪﺳ ،ﺔﻴﻧﺎ
١٩٨٨
. ﺮ، ﻣﺼﺮ، ﺍﻻﺳﺘﻘﻼ
،ﻋﻤﺮ، ﺍﻟﻘﺎﻫﺮﺓ
ﺳﻻﺍ
ﻴﻤﻨﻴﺔ،
١٣٢١
ـﻫ
ﺲ،
١٩٦٦
. ﺖ، ﺍﻟﻌﺼﺮﻳﺔ،
٨، ٢
ﺍﻟﻘﺎﻫﺮﺓ، ﺩﺍﺭ
ﺀﺎﻴﺣﺇ
، ٢ﻟﺔ،
١٩٨١
. ﻳﺎﺽ، ﺍﻟﻔﺮﺩﻭﺱ،
ﻈﻬﺮ ﺍ
ﻟﺴﻘﺎﺀ، ﺩﻣﺸ
ﺐ ﺍﻟﻌﺮﺑﻴﺔ، ﺩ..ﺕ
١٩ . ﻢ ﺍﳌﻌﺎﱐ
ﳌﺆﻟﻒ
ﻬﳎ
ﺩ، ﺩ. . ﺕ
ﺸﻲ، ﺍﻟﻘﺎﻫﺮﺓ، ﳏﻤ
،ﻄﻔﻰ ﺍﻟﺒﺎﰊ
٨٨ ، ١
ﺍﻟﺪﺍﺭ ﺍﳌﺼﺮﻳﺔ ﺍﻟﻠﺒﻨﺎ
ﺖ ﻣﻨﲑ،
١٩٨٦
. ﻌﺮﺑﻴﺔ، ﺩ ، ﺕ . ﻴﻖ ﻋﻠﻰ ﳏﻤﺪ ﻋﻤﺮ
ﻋ ﲢﻘﻴﻖ ﻋﻠﻲ ﳏﻤﺪ
ﺑﻮﺭﻱ، ﻣﺼﺮ، ﺍﳌﻴ
ﲑﻭﺕ، ﺩﺍﺭ ﺍﻷﻧﺪﻟﺲ
ﺳﺎﱂ ﻣﻜﺮﻡ، ﺍﻟﻜﻮﻳﺖ
، ﻓﺆﺍﺩ ﻋﺒﺪ ﺍﻟﺒﺎﻗﻲ
ﺕ، ﻣﺆﺳﺴﺔ ﺍﻟﺮﺳﺎﻟ
ﺟﺎﺑﺮ ﺍﻟﻌﻠﻮﺍﱐ، ﺍﻟﺮﻳ
ﻡ، ﲢﻘﻴﻖ ﳏﻤﺪ ﻣﻈ
ﺓ، ﺩﺍﺭ ﺃﺣﻴﺎﺀ ﺍﻟﻜﺘﺐ
ﻫﺮﺓ، ﺍﳌﺪﱐ،
٩٦٤
. ﺕ. ﺎﺏ ﺍﳌﺒﺎﱐ ﰲ ﻧﻈﻢ
ﳏﻤﻮﺩ ﻧﺼﺎﺭ، ﺑﻐﺪﺍ
ﺩ
ﺑﻦ ﺍﳊﺴﻦ ﺍﻟﺒﺪﺧﺸ
ﺣﻮﻟﻴﺔ
ﻛﺮ، ﺍﻟﻘﺎﻫﺮﺓ، ﻣﺼﻄ
،ﺠﺴﺘﺎﱐ، ﺍﻟﻘﺎﻫﺮﺓ
ﺩ، ﻣﻄﺒﻌﺔ ﻭﺃﻭﻓﺴﺖ
ﺭ ﺇﺣﻴﺎﺀ ﺍﻟﻜﺘﺐ ﺍﻟﻌ
ﻋﻠﻲ ﺍﻟﺪﺍﻭﺩﻱ، ﲢﻘﻴ
ﲢ ،ﳊﺴﻦ ﺍﻟﺰﺑﻴﺪﻱ
ﻦ ﺑﻦ ﳏﻤﺪ ﺍﻟﻨﻴﺴﺎ
ﻔﺪﺍﺀ ﺍﺑﻦ ﻛﺜﲑ، ﺑﲑ
ﺔ، ﺩ . . ﺕ
ﺔ :ﺩ. ﺳﺎ ﻋﺒﺪ ﺍﻟﻌﺎﻝ
ﻜﺮ ،
١٩٨٣
. ﺒﻠﻲ، ﲢﻘﻴﻖ ﳏﻤﺪ
ﺆﺍﺩ ﺳﺰﻛﲔ، ﺑﲑﻭﺕ
ﺯﻱ، ﲢﻘﻴﻖ ﺩ. ﻪﻃ
ﺪﲪﺃ
:
ﺍﺑﻦ ﺍﻟﻠﺤﺎﻡ
ﻭﺁﺧﺮﻭﻥ، ﺍﻟﻘﺎﻫﺮ
ﻦ ﻋﺒﺪ ﺍﳊﻤﻴﺪ، ﺍﻟﻘﺎﻫ
ﺩﺍﺭ ﺍﳌﺴﺘﺸﺮﻕ، ﺩ
ﻋﻄﻴﺔ، ﻭﻣﻘﺪﻣﺔ ﻛﺘﺎ
١ . ﳏ ﻴﻖ، ﳏﻤﻮﺩ ﳏﻤﺪ
ﻱﻭﺎﻀﻴﺒﻠ :ﺪﻤﳏ
•ﺣ
ﻖ ﺃﲪﺪ ﳏﻤﺪ ﺷﺎﻛ
ﺑﻦ ﺍﻷﺷﻌﺚ ﺍﻟﺴﺠ
ﺷﻴﺔ ﺍﻟﺴﻨﺪﻱ، ﺑﻐﺪﺍﺩ
ﺭﺍﺩ ،
ﺍﳊﺪﻳﺚ ﻭﺩﺍﺭ
ﻋ ﺲ ﺍﻟﺪﻳﻦ ﳏﻤﺪ ﺑﻦ
ﻮﻳﲔ :ﳏﻤﺪ ﺑﻦ ﺍﳊ
ﺐ ﺍﻟﻔﺮﻗﺎﻥ :ﺍﳊﺴﻦ
ﻋﻤﺎﺩ ﺍﻟﺪﻳﻦ ﺃﰊ ﺍﻟﻔ
ﺍﻟﻘﺎﻫﺮﺓ، ﺍﻻﺳﺘﻘﺎﻣﺔ
ﺍﻟﺪﺭﺍﺳﺎﺕ ﺍﻟﻨﺤﻮﻳﺔ
ﺍﻟﻘﺎﻫﺮﺓ ، ﺩﺍﺭ ﺍﻟﻔﻜ
ﺠﺎﺭ ﺍﻟﻔﺘﻮﺣﻲ ﺍﳊﻨﺒ
ﲢﻘﻴﻖ ﺩ،
.ﳏﻤﺪ ﻓﺆ
ﻪﻘﻔﻟﺍ
:
ﺍﻟﻔﺨﺮ ﺍﻟﺮﺍﺯ
ﻔﻘﻪ ﻋﻠﻰ ﻣﺬﻫﺐ
ﻖ ﳏﻤﺪ ﺟﺎﺩ ﺍﳌﻮﱄ
ﻖ ﳏﻤﺪ ﳏﻲ ﺍﻟﺪﻳﻦ
،ﺍﳊﻤﻮﻱ، ﺑﲑﻭﺕ
ﺁﻥ) :ﻋ ﻣﻘﺪﻣﺔ ﺍﺑﻦ
ﺒﺔ ﺍﳋﺎﳒﻲ،
١٩٥٤
ﲑ :ﺍﺑﻦ ﺗﻴﻤﻴﺔ، ﲢﻘﻴﻖ
ﻣﻨﻬﺎﺝ ﺍﻟﻮﺻﻮﻝ ﻟﻠ
ﺔﻟ :
،ﻲﻌﻓﺎﺸﻟﺍ
ﻖﻴﻘﲢ
ﰊﺃ ﺩﻭﺍﺩ
:
ﻥﺎﻤﻴﻠﺳ
ﺢ
ﻱﺭﺎﺨﺒﻟﺍ
ﲝﺎﺷﻴ :
ﺢ
ﻢﻠﺴﻣ
: ،ﺍﻟﻘﺎﻫﺮﺓ
ﺕ
ﻦﻳﺮﺴﻔﳌﺍ
ﴰﺲ :
ﺕ ﺍﻟﻨﺤﻮﻳﲔ ﻭﺍﻟﻠﻐﻮ
١٩٧٢ . ﺐ
ﻥﺁﺮﻘﻟﺍ
ﻭ
ﻏﺎﺋﺐﺭ
ﻱﱪ . (
ﻞ ﻥﺁﺮﻘﻟﺍ
:
ﻆﻓﺎﳊﺍ
ﺳﺖ : ﺍﺑﻦ
ﺍﻟﻨﺪﱘ،
ﻥ
ﱘﺮﻜﻟﺍ
ﻭﺃﺛﺮﻩ ﰲ
ﺎﻑ :
،ﻱﺮﺸﳐﺰﻟﺍ
ﺍ
ﻛﺐ
ﲑﻨﳌﺍ
:
ﺍﺑﻦ ﺍﻟﻨﺠ
١٩٥٧
. . ﻥﺁﺮﻘﻟ
: ،ﺃﺑﻮ ﻋﺒﻴﺪﺓ
ﻝﻮﰲ
ﻋﻠﻢ ﺃﺻﻮﻝ
ﰲﺼﺮ
ﺃﺻﻮﻝ ﺍﻟﻔ
،ﻭﻓﺴﺖ
١٩٨٠
. ﺮ :
،ﻲﻃﻮﻴﺴﻟﺍ
ﻖﻴﻘﲢ
ﺓﺩ :ﻝﺁ
ﺗﻴﻤﻴﺔ، ﲢﻴﻖ
ﻢ
ﺀﺎﺑﺩﻷﺍ
:
ﺍ ﺕﻮﻗﺎﻳ
ﺘﺎﻥ ﰲ ﻋﻠﻮﻡ ﺍﻟﻘﺮﺁ
ﻱ، ﺍﻟﻘﺎﻫﺮﺓ، ﻣﻜﺘﺒﺔ
ﰲﺔ
ﺃﺻﻮﻝ ﺍﻟﺘﻔﺴﲑ
ﺞ
ﻝﻮﻘﻌﻟﺍ
ﻣ ﺡﺮﺷ
ﺕ . ﻪ) :ﻮﻟﻪﺳ ٢٢- ﻟﺎﺳﺮﻟﺍ
٢٣-
ﻦﻨﺳﺃ
٢٤- ﺢﻴﺤﺻ
٢٥- ﺢﻴﺤﺻ
٦٢- ﻃﺒﻘﺎﺕ
٧٢- ﻃﺒﻘﺎﺕ
٢، ١
٨٢-ﻏﺮﺍﺋﺐ
ﱪﻄﻟﺍ
٢٩- ﻞﺋﺎﻀﻓ
٣٠- ﺳﺮﻬﻔﻟﺍ
٣١- ﻥﺁﺮﻘﻟﺍ
٣٢- ﺎﺸﻜﻟﺍ
٣٣- ﻛﻮﻜﻟﺍ
٧، ١
٣٤-
ﺯﺎﳎﺍ
٣٥- ﻮﺼﶈﺍ
٣٦-
ﺼﺘﺨﳌﺍ
ﻭﻷﺎﺑ
٣٧- ﺮﻫﺰﳌﺍ
٣٨- ﺩﻮﺴﳌﺍ
٣٩- ﻢﺠﻌﻣ
٤٠- ﺘﻣﺪﻘﻣ
ﺮﻔﺟ
٤١- ﺔﻣﺪﻘﻣ
٤٢-
ﺞﻫﺎﻨﻣ
ﺩ.ﺕ ﺩ ﺍﳋﺎﻣﺲ
، ١ﻼﻝ ،
١٩٧٢
. ﻝﻼﻘﺘﺳ ﻯﱪﻜﻟﺍ
ـ )
ﺔﻴﺷﺎﲝ
ﺴﻔﺗ
١٩٧٨ . ﺀ ﺍﻟﻜﺘﺐ ﺍﻟﻌﺮﺑﻴﺔ
، ١
١٩٧٩
. ﺸﻖ، ﺩﺍﺭ
ﺮﻜﻔﻟﺍ
ﻬﻮﻝ(، ﻧﺸﺮ ﺁﺭﺛ
ﻤﺪ ﻋﻠﻲ
ﺢﻴﺒﺻ
ﺍﻟﻌﺪﺩ
١٩٨ . ،ﺔﻴﻧﺎ
١٩٨٨
. ﺮ، ﻣﺼﺮ، ﺍﻻﺳﺘﻘﻼ
،ﻋﻤﺮ، ﺍﻟﻘﺎﻫﺮﺓ
ﺳﻻﺍ
ﻴﻤﻨﻴﺔ،
١٣٢١
ـﻫ
ﺲ،
١٩٦٦
. ﺖ، ﺍﻟﻌﺼﺮﻳﺔ،
٨، ٢
ﺍﻟﻘﺎﻫﺮﺓ، ﺩﺍﺭ
ﺀﺎﻴﺣﺇ
، ٢ﻟﺔ،
١٩٨١
. ﻳﺎﺽ، ﺍﻟﻔﺮﺩﻭﺱ،
ﻈﻬﺮ ﺍ
ﻟﺴﻘﺎﺀ، ﺩﻣﺸ
ﺐ ﺍﻟﻌﺮﺑﻴﺔ، ﺩ..ﺕ
١٩ . ﻢ ﺍﳌﻌﺎﱐ
ﳌﺆﻟﻒ
ﻬﳎ
ﺩ، ﺩ. . ﺕ
ﺸﻲ، ﺍﻟﻘﺎﻫﺮﺓ، ﳏﻤ
،ﻄﻔﻰ ﺍﻟﺒﺎﰊ
٨٨ ، ١
ﺍﻟﺪﺍﺭ ﺍﳌﺼﺮﻳﺔ ﺍﻟﻠﺒﻨﺎ
ﺖ ﻣﻨﲑ،
١٩٨٦
. ﻌﺮﺑﻴﺔ، ﺩ ، ﺕ . ﻴﻖ ﻋﻠﻰ ﳏﻤﺪ ﻋﻤﺮ
ﻋ ﲢﻘﻴﻖ ﻋﻠﻲ ﳏﻤﺪ
ﺑﻮﺭﻱ، ﻣﺼﺮ، ﺍﳌﻴ
ﲑﻭﺕ، ﺩﺍﺭ ﺍﻷﻧﺪﻟﺲ
ﺳﺎﱂ ﻣﻜﺮﻡ، ﺍﻟﻜﻮﻳﺖ
، ﻓﺆﺍﺩ ﻋﺒﺪ ﺍﻟﺒﺎﻗﻲ
ﺕ، ﻣﺆﺳﺴﺔ ﺍﻟﺮﺳﺎﻟ
ﺟﺎﺑﺮ ﺍﻟﻌﻠﻮﺍﱐ، ﺍﻟﺮﻳ
ﻡ، ﲢﻘﻴﻖ ﳏﻤﺪ ﻣﻈ
ﺓ، ﺩﺍﺭ ﺃﺣﻴﺎﺀ ﺍﻟﻜﺘﺐ
ﻫﺮﺓ، ﺍﳌﺪﱐ،
٩٦٤
. ﺕ. ﺎﺏ ﺍﳌﺒﺎﱐ ﰲ ﻧﻈﻢ
ﳏﻤﻮﺩ ﻧﺼﺎﺭ، ﺑﻐﺪﺍ
ﺩ
ﺑﻦ ﺍﳊﺴﻦ ﺍﻟﺒﺪﺧﺸ
ﺣﻮﻟﻴﺔ
ﻛﺮ، ﺍﻟﻘﺎﻫﺮﺓ، ﻣﺼﻄ
،ﺠﺴﺘﺎﱐ، ﺍﻟﻘﺎﻫﺮﺓ
ﺩ، ﻣﻄﺒﻌﺔ ﻭﺃﻭﻓﺴﺖ
ﺭ ﺇﺣﻴﺎﺀ ﺍﻟﻜﺘﺐ ﺍﻟﻌ
ﻋﻠﻲ ﺍﻟﺪﺍﻭﺩﻱ، ﲢﻘﻴ
ﲢ ،ﳊﺴﻦ ﺍﻟﺰﺑﻴﺪﻱ
ﻦ ﺑﻦ ﳏﻤﺪ ﺍﻟﻨﻴﺴﺎ
ﻔﺪﺍﺀ ﺍﺑﻦ ﻛﺜﲑ، ﺑﲑ
ﺔ، ﺩ . . ﺕ
ﺔ :ﺩ. ﺳﺎ ﻋﺒﺪ ﺍﻟﻌﺎﻝ
ﻜﺮ ،
١٩٨٣
. ﺒﻠﻲ، ﲢﻘﻴﻖ ﳏﻤﺪ
ﺆﺍﺩ ﺳﺰﻛﲔ، ﺑﲑﻭﺕ
ﺯﻱ، ﲢﻘﻴﻖ ﺩ. ﻪﻃ
ﺪﲪﺃ
:
ﺍﺑﻦ ﺍﻟﻠﺤﺎﻡ
ﻭﺁﺧﺮﻭﻥ، ﺍﻟﻘﺎﻫﺮ
ﻦ ﻋﺒﺪ ﺍﳊﻤﻴﺪ، ﺍﻟﻘﺎﻫ
ﺩﺍﺭ ﺍﳌﺴﺘﺸﺮﻕ، ﺩ
ﻋﻄﻴﺔ، ﻭﻣﻘﺪﻣﺔ ﻛﺘﺎ
١ . ﳏ ﻴﻖ، ﳏﻤﻮﺩ ﳏﻤﺪ
ﻱﻭﺎﻀﻴﺒﻠ :ﺪﻤﳏ
•ﺣ
ﻖ ﺃﲪﺪ ﳏﻤﺪ ﺷﺎﻛ
ﺑﻦ ﺍﻷﺷﻌﺚ ﺍﻟﺴﺠ
ﺷﻴﺔ ﺍﻟﺴﻨﺪﻱ، ﺑﻐﺪﺍﺩ
ﺭﺍﺩ ،
ﺍﳊﺪﻳﺚ ﻭﺩﺍﺭ
ﻋ ﺲ ﺍﻟﺪﻳﻦ ﳏﻤﺪ ﺑﻦ
ﻮﻳﲔ :ﳏﻤﺪ ﺑﻦ ﺍﳊ
ﺐ ﺍﻟﻔﺮﻗﺎﻥ :ﺍﳊﺴﻦ
ﻋﻤﺎﺩ ﺍﻟﺪﻳﻦ ﺃﰊ ﺍﻟﻔ
ﺍﻟﻘﺎﻫﺮﺓ، ﺍﻻﺳﺘﻘﺎﻣﺔ
ﺍﻟﺪﺭﺍﺳﺎﺕ ﺍﻟﻨﺤﻮﻳﺔ
ﺍﻟﻘﺎﻫﺮﺓ ، ﺩﺍﺭ ﺍﻟﻔﻜ
ﺠﺎﺭ ﺍﻟﻔﺘﻮﺣﻲ ﺍﳊﻨﺒ
ﲢﻘﻴﻖ ﺩ،
.ﳏﻤﺪ ﻓﺆ
ﻪﻘﻔﻟﺍ
:
ﺍﻟﻔﺨﺮ ﺍﻟﺮﺍﺯ
ﻔﻘﻪ ﻋﻠﻰ ﻣﺬﻫﺐ
ﻖ ﳏﻤﺪ ﺟﺎﺩ ﺍﳌﻮﱄ
ﻖ ﳏﻤﺪ ﳏﻲ ﺍﻟﺪﻳﻦ
،ﺍﳊﻤﻮﻱ، ﺑﲑﻭﺕ
ﺁﻥ) :ﻋ ﻣﻘﺪﻣﺔ ﺍﺑﻦ
ﺒﺔ ﺍﳋﺎﳒﻲ،
١٩٥٤
ﲑ :ﺍﺑﻦ ﺗﻴﻤﻴﺔ، ﲢﻘﻴﻖ
ﻣﻨﻬﺎﺝ ﺍﻟﻮﺻﻮﻝ ﻟﻠ
ﺔﻟ :
،ﻲﻌﻓﺎﺸﻟﺍ
ﻖﻴﻘﲢ
ﰊﺃ ﺩﻭﺍﺩ
:
ﻥﺎﻤﻴﻠﺳ
ﺢ
ﻱﺭﺎﺨﺒﻟﺍ
ﲝﺎﺷﻴ :
ﺢ
ﻢﻠﺴﻣ
: ،ﺍﻟﻘﺎﻫﺮﺓ
ﺕ
ﻦﻳﺮﺴﻔﳌﺍ
ﴰﺲ :
ﺕ ﺍﻟﻨﺤﻮﻳﲔ ﻭﺍﻟﻠﻐﻮ
١٩٧٢ . ﺐ
ﻥﺁﺮﻘﻟﺍ
ﻭ
ﻏﺎﺋﺐﺭ
ﻱﱪ . ﻪ) :ﻮﻟﻪﺳ (
ﻞ ﻥﺁﺮﻘﻟﺍ
:
ﻆﻓﺎﳊﺍ
ﺳﺖ : ﺍﺑﻦ
ﺍﻟﻨﺪﱘ،
ﻥ
ﱘﺮﻜﻟﺍ
ﻭﺃﺛﺮﻩ ﰲ
ﺎﻑ :
،ﻱﺮﺸﳐﺰﻟﺍ
ﺍ
ﻛﺐ
ﲑﻨﳌﺍ
:
ﺍﺑﻦ ﺍﻟﻨﺠ
١٩٥٧
. . ﻥﺁﺮﻘﻟ
: ،ﺃﺑﻮ ﻋﺒﻴﺪﺓ
ﻝﻮﰲ
ﻋﻠﻢ ﺃﺻﻮﻝ
ﰲﺼﺮ
ﺃﺻﻮﻝ ﺍﻟﻔ
،ﻭﻓﺴﺖ
١٩٨٠
. ﺮ :
،ﻲﻃﻮﻴﺴﻟﺍ
ﻖﻴﻘﲢ
ﺓﺩ :ﻝﺁ
ﺗﻴﻤﻴﺔ، ﲢﻴﻖ
ﻢ
ﺀﺎﺑﺩﻷﺍ
:
ﺍ ﺕﻮﻗﺎﻳ
ﺘﺎﻥ ﰲ ﻋﻠﻮﻡ ﺍﻟﻘﺮﺁ
ﻱ، ﺍﻟﻘﺎﻫﺮﺓ، ﻣﻜﺘﺒﺔ
ﰲﺔ
ﺃﺻﻮﻝ ﺍﻟﺘﻔﺴﲑ
ﺞ
ﻝﻮﻘﻌﻟﺍ
ﻣ ﺡﺮﺷ
ﺕ . ٢٢- ﻟﺎﺳﺮﻟﺍ
٢٣-
ﻦﻨﺳﺃ
٢٤- ﺢﻴﺤﺻ
٢٥- ﺢﻴﺤﺻ
٦٢- ﻃﺒﻘﺎﺕ
٧٢- ﻃﺒﻘﺎﺕ
٢، ١
٨٢-ﻏﺮﺍﺋﺐ
ﱪﻄﻟﺍ
٢٩- ﻞﺋﺎﻀﻓ
٣٠- ﺳﺮﻬﻔﻟﺍ
٣١- ﻥﺁﺮﻘﻟﺍ
٣٢- ﺎﺸﻜﻟﺍ
٣٣- ﻛﻮﻜﻟﺍ
٧، ١
٣٤-
ﺯﺎﳎﺍ
٣٥- ﻮﺼﶈﺍ
٣٦-
ﺼﺘﺨﳌﺍ
ﻭﻷﺎﺑ
٣٧- ﺮﻫﺰﳌﺍ
٣٨- ﺩﻮﺴﳌﺍ
٣٩- ﻢﺠﻌﻣ
٤٠- ﺘﻣﺪﻘﻣ
ﺮﻔﺟ
٤١- ﺔﻣﺪﻘﻣ
٤٢-
ﺞﻫﺎﻨﻣ
ﺩ.ﺕ
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https://openalex.org/W639438969
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https://europepmc.org/articles/pmc4474600?pdf=render
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English
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Changes in Extremely Hot Summers over the Global Land Area under Various Warming Targets
|
PloS one
| 2,015
|
cc-by
| 7,003
|
Changes in Extremely Hot Summers over the
Global Land Area under Various Warming
Targets Lei Wang, Jianbin Huang*, Yong Luo, Yao Yao, Zongci Zhao Lei Wang, Jianbin Huang*, Yong Luo, Yao Yao, Zongci Zhao
Ministry of Education Key Laboratory for Earth System Modeling, Center for Earth System Science, and Joint
Center for Global Change Studies (JCGCS), Tsinghua University, Beijing, China Ministry of Education Key Laboratory for Earth System Modeling, Center for Earth System Science, and Joint
Center for Global Change Studies (JCGCS), Tsinghua University, Beijing, China * jbh@mail.tsinghua.edu.cn * jbh@mail.tsinghua.edu.cn OPEN ACCESS Citation: Wang L, Huang J, Luo Y, Yao Y, Zhao Z
(2015) Changes in Extremely Hot Summers over the
Global Land Area under Various Warming Targets. PLoS ONE 10(6): e0130660. doi:10.1371/journal. pone.0130660 Editor: Shang-Ping Xie, University of California San
Diego, UNITED STATES
Received: February 26, 2015
Accepted: May 22, 2015
Published: June 19, 2015
Copyright: © 2015 Wang et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited. Editor: Shang-Ping Xie, University of California San
Diego, UNITED STATES Editor: Shang-Ping Xie, University of California San
Diego, UNITED STATES
Received: February 26, 2015
Accepted: May 22, 2015
Published: June 19, 2015
Copyright: © 2015 Wang et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited. Data Availability Statement: The model data used
in this study are available from Earth System Grid
Federation at http://cmip-pcmdi.llnl.gov/cmip5/. The
GISS data are available from Goddard Institute for
Space Studies at http://data.giss.nasa.gov/gistemp/
and CRU data are available from Climate Research
Unit at http://www.cru.uea.ac.uk/cru/data/hrg/. Funding: This study was supported by the China
Meteorological Administration Special Public Welfare
Research Fund (GYHY201306019, http://guoqing. china.com.cn/2012-03/23/content_24968247.htm—
received by JH), the Tsinghua University Initiative
Scientific Research Program (Grant No. Abstract Summer temperature extremes over the global land area were investigated by comparing
26 models of the fifth phase of the Coupled Model Intercomparison Project (CMIP5)
with observations from the Goddard Institute for Space Studies (GISS) and the Climate
Research Unit (CRU). Monthly data of the observations and models were averaged for
each season, and statistics were calculated for individual models before averaging them to
obtain ensemble means. The summers with temperature anomalies (relative to 1951–1980)
exceeding 3σ (σ is based on the local internal variability) are defined as “extremely hot”. The models well reproduced the statistical characteristics evolution, and partly captured the
spatial distributions of historical summer temperature extremes. If the global mean tempera-
ture increases 2°C relative to the pre-industrial level, “extremely hot” summers are projected
to occur over nearly 40% of the land area (multi-model ensemble mean projection). Sum-
mers that exceed 5σ warming are projected to occur over approximately 10% of the global
land area, which were rarely observed during the reference period. Scenarios reaching
warming levels of 3°C to 5°C were also analyzed. After exceeding the 5°C warming target,
“extremely hot” summers are projected to occur throughout the entire global land area, and
summers that exceed 5σ warming would become common over 70% of the land area. In
addition, the areas affected by “extremely hot” summers are expected to rapidly expand by
more than 25%/°C as the global mean temperature increases by up to 3°C before slowing
to less than 16%/°C as the temperature continues to increase by more than 3°C. The area
that experiences summers with warming of 5σ or more above the warming target of 2°C is
likely to maintain rapid expansion of greater than 17%/°C. To reduce the impacts and dam-
age from severely hot summers, the global mean temperature increase should remain low. RESEARCH ARTICLE Hot Summers under Various Warming Targets human health and property, have garnered much attention [5]. For example, the European
heat waves in 2003 and 2006 [6–8], the great California heat wave in 2006 [9], the rare high
temperatures in Russia in 2010 [10, 11] and the extremely hot Australian summer in 2013, are
all well-known for their severity and damage. Additionally, studies have indicated that heat
waves will occur with higher intensity and frequency if global warming continues [12–17]. 20131089356, http://kyxxxt.cic.tsinghua.edu.cn/—
received by JH), and the National Natural Science
Foundation of China (Grant No.41175066, http://www. nsfc.gov.cn/—received by YL). The funders had no
role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript. Heat waves also occur on monthly and seasonal scales as temperature extremes [18–22]. Because monthly data are relatively easy to access, monthly temperatures are widely used to
examine changes in past and future climate extremes [23]. The number of warm monthly tem-
perature extremes has significantly increased in the past decade [24, 25]. Hansen et al. [18]
indicated that the occurrence of extremely hot summers, which rarely occurred in 1951–1980
(less than 1% of the land area), became extensive in the last decade (approximately 10% of the
land area). In addition, the latest results from the fifth phase of the Coupled Model Intercom-
parison Project (CMIP5) models suggest that extremely hot monthly temperatures will affect
approximately 85% of the land area in 2100 under the Representative Concentration Pathway
8.5 (RCP8.5) [19]. Competing Interests: The authors have declared
that no competing interests exist. A global mean temperature increase of 2°C relative to pre-industrial level has been accepted
by most countries and organizations as a critical target because it seems achievable and would
limit climate and ecological impacts [26–30]. Controlling the increase in the global mean tem-
perature is as an effective method for avoiding or reducing losses caused by global warming. Consequently, quantifying the responses of climate extremes to a 2°C warming and other
warming rates relative to the pre-industrial level will help policymakers, researchers and the
public understand the possible consequences of inaction. The seasonal temperatures of the 26
CMIP5 models, which include historical simulations and three RCP scenarios, were analyzed
in this study. Compared with the ocean, land areas are more closely associated with human
livelihood. Introduction With rapid increases in global mean temperature over the past decade, climate extremes have
become stronger and more frequent [1–4]. Particularly, heat waves, which severely threaten PLOS ONE | DOI:10.1371/journal.pone.0130660
June 19, 2015 1 / 11 PLOS ONE | DOI:10.1371/journal.pone.0130660
June 19, 2015 Data and Methods Monthly surface air temperatures from 26 CMIP5 models, which include historical data and
three simulated scenarios (RCP2.6, RCP4.5 and RCP8.5) (S1 Table), were used in our research. Historical observations were obtained from the Goddard Institute for Space Studies (GISS)
[31] and the Climate Research Unit (CRU) [32]. The monthly GISS data (1880 to the present),
which are smoothed to 250 km to include as much regional variability as possible, have a spa-
tial resolution of 2°x 2° [31], and the monthly CRU data (1901 to the present) have a high spa-
tial resolution of 0.5° x 0.5° [32]. The durations of the 26 CMIP5 historical runs are shown in
S1 Table, most of which extend from 1850 to 2005. Most of the 21st century projections, which
follow the three emission scenarios, extend from 2006 to 2100. Monthly data are averaged for each season at the beginning, and the summer means of JJA
data from the North Hemisphere and DJF data from the South Hemisphere are concurrently
analyzed in the following. In our research, the period of 1951–1980 was chosen as a reference period because of its
advantages. As mentioned by Hansen et al. [18], the temperature during this period was rela-
tively stable and almost without rapid increase, which quite a number of older people have
experienced. The definition of temperature extremes used in this study is similar to that proposed by
Hansen et al. [18], except that a value of +5σ is included in our research. In a normal distribu-
tion, the values between ±0.43σ are defined as “typical” summers; the values greater (less) than
+0.43σ (-0.43σ) are defined as “hot” (“cold”) summers; the values greater (less) than +2σ (-2σ)
and +3σ (-3σ) are defined as “very hot” (“very cold”) and “extremely hot” (“extremely cold”)
summers, respectively. As global warming continues, “extremely hot” events will become more
common. Additionally, changes in the occurrence of more severe summers have been quanti-
fied: the summers with temperature anomalies greater than +5σ in a normal distribution are
referred to as “exceeding 5σ hot” summers. To calculate the local σ, we first fit the summer data from 1901 (the first year of CRU data)
to 2005 using a least squares polynomial method and determined the long-term non-linear
trend. Next, the summer time series were detrended by deducting the long-term trend. Accordingly, we focused on changes in temperature extremes over global land areas
in response to various warming targets. Summer is the focus of our research because humans
are more sensitive to hot extremes during the summer than during other seasons. Hansen et al. [18] and Coumou and Robinson [19] also research on the extreme tempera-
tures. However, Hansen et al. [18] only investigated the observations by using GISS data, while
Coumou and Robinson [19] mainly predicted the area percentage with monthly heat extremes
during boreal summer in 21st century and their spatial patterns at the end 20 years of 21st cen-
tury under RCP2.6 and RCP8.5 scenarios. In contrast, based on 26 CMIP5 models, a systemic
evaluation of the long-term global extreme summer temperatures was performed to quantify
the capability of models in simulating extreme summer temperatures in our study, including
not only the spatial distributions and total area percentages but also the probability of occur-
rence. The global extreme summer temperatures include June-July-August (JJA) in the North-
ern Hemisphere and December-January-February (DJF) in the Southern Hemisphere, while
boreal summer of JJA was analyzed in both hemispheres in Coumou and Robinson [19]. In
addition, we mainly focus on the response of extremely hot summers to the different warming
magnitudes relative to the pre-industrial level. Therefore, all simulations from RCP2.6, RCP4.5
and RCP8.5 scenarios (actually 26 simulations in each RCP scenario) are together used in our
research to quantify the various warming magnitudes regardless of different emission scenar-
ios. So our analysis emphasizes the response of summer climate extremes to different warming
targets rather than different emission scenarios as Coumou and Robinson [19]. Furthermore,
another big difference between this work and Coumou and Robinson [19] is that the seasonal
data are used in this study instead of monthly data in the research of Coumou and Robinson
[19]. Since the standard deviation of monthly data is larger than that of seasonal data, the pro-
jected future increase of the hot extreme temperatures analyzed by using seasonal data is gener-
ally larger than those analyzed by using monthly data. 2 / 11 PLOS ONE | DOI:10.1371/journal.pone.0130660
June 19, 2015 Hot Summers under Various Warming Targets This paper is structured as follows. Section 1 introduces the data and methods used in this
research. Next, 26 CMIP5 models are evaluated for simulating historical temperature extremes
in section 2. In section 3, the responses of the temperature extremes to various warming targets
are analyzed. Finally, a summary and discussion of our finding is presented in section 4. PLOS ONE | DOI:10.1371/journal.pone.0130660
June 19, 2015 Evolution of historical summer temperature extremes Comparing the results from the multi-model ensemble mean and two observations of GISS
and CRU reveals that the CMIP5 models perform extremely well when reproducing the evolu-
tion of the statistical distribution of summer temperature extremes between 1946 and 2005
(Fig 1). Because the historical CMIP5 runs end in 2005, the temperature extremes are counted
every ten years from 1946 to 2005 for the model results and two observational datasets. The
curves of the summer temperatures are smoother in the CRU dataset than in the GISS dataset
because of the additional observational grids in the CRU data. The distributions of the multi-
model ensemble mean are highly consistent with those of the observations. The curves shift to
the right and become broader over time, particularly following the 1980s. The probability of an
“extremely hot” summer is indicated by a gradually increasing area of the right tail. The curve
shifts rightward as the mean temperature increases, and the broader trend may result from the
heterogeneous warming rates in different regions rather than the increase of variability [33]. Fei Ji et al. [34] indicated that the most dramatic and rapid warming has occurred in arid or
semiarid regions in the Northern Hemisphere at mid-latitudes, and that warming since 1900
has varied spatially and unevenly. However, the global temperature variability has remained
stable [35]. As shown in Fig 1, rapid warming has occurred since the 1970s, which resulted in
significant rightward curve shifts. In addition, the spatial combined statistics from regions with
different warming rates contributes to the broadening of the curves. The occurrence probability
of an “extremely hot” summer during each period is shown in Table 1, and the occurrence
probability is somewhat underestimated by the multi-model ensemble mean. The multi-model ensemble mean performs well in terms of the total area affected by tem-
perature extremes (Fig 2). The “extremely hot” area has rapidly increased since the late 1970s,
and the “cold” area has generally decreased, with the exception of a relatively stable period
from the early 1940s to the mid-1970s. The trends in the hot and cold areas are highly consis-
tent with those of the observations. The averaging process results in smoother multi-model
ensemble mean curves than observed for the other two observations. The spatial distributions
of the “extremely hot” summers during 1986–2005 are shown in Fig 3. Data and Methods The first, second and third graphs show the GISS, CRU and multi-
model ensemble mean results, respectively. To better compare the results from observations and multi-
model ensemble mean, the frequencies during the periods of 1951–1980 (reference period), 1976–1985,
1986–1995 and 1996–2005 are shown in the fourth graph. doi:10.1371/journal.pone.0130660.g001 Data and Methods Then,
we estimated σ by using this detrended 105 data, which include a sufficient number of data
points to estimate the local year-to-year variability. Considering the starting time of the historical simulations of the 26 models (S1 Table), the
average temperature from 1861 to 1880 is used to represent the pre-industrial level. When the
11-year running mean global mean temperature exceeds 2°C, the global mean temperature
reaches the 2°C target. Additionally, the 20-year climate centered on the year reaching 2°C tar-
get is determined. The same methods are used to examine the climate when the global mean
temperature reaches various warming targets relative to the pre-industrial level, such as 3°C,
4°C and 5°C. In this analysis, the statistical characteristics of the ensemble mean of the 26 mod-
els are obtained after calculating each model independently by considering the weight of each
grid area. That is to say, the statistics were first calculated for individual models, and then the
results from all models were averaged to present the multi-model ensemble mean. Addition-
ally, JJA in the Northern Hemisphere and DJF in the Southern Hemisphere are considered
jointly when presenting the summer characteristics. 3 / 11 PLOS ONE | DOI:10.1371/journal.pone.0130660
June 19, 2015 Hot Summers under Various Warming Targets Fig 1. Frequency of summer local temperature anomalies. The temperature anomalies (relative to the
1951–1980 mean) are divided by the local standard deviation of 1901–2005 and binned into intervals of 0.05. The local standard deviation is calculated from the long-term non-linearly detrended time series (details in
data and method section). For the multi-model ensemble mean, this process was conducted for each model,
and the final frequencies were averaged. The first, second and third graphs show the GISS, CRU and multi-
model ensemble mean results, respectively. To better compare the results from observations and multi-
model ensemble mean, the frequencies during the periods of 1951–1980 (reference period), 1976–1985,
1986–1995 and 1996–2005 are shown in the fourth graph. Fig 1. Frequency of summer local temperature anomalies. The temperature anomalies (relative to the
1951–1980 mean) are divided by the local standard deviation of 1901–2005 and binned into intervals of 0.05. The local standard deviation is calculated from the long-term non-linearly detrended time series (details in
data and method section). For the multi-model ensemble mean, this process was conducted for each model,
and the final frequencies were averaged. PLOS ONE | DOI:10.1371/journal.pone.0130660
June 19, 2015 Evolution of historical summer temperature extremes When disregarding the
missing data in GISS, the locations with “extremely hot” summers are similar between the two
observations, except for the west of South America and the middle of Africa. The present spa-
tial distribution and areal coverage of “extremely hot” summer that are simulated using the
multi-model ensemble mean is more similar to the GISS results than the CRU results, in which PLOS ONE | DOI:10.1371/journal.pone.0130660
June 19, 2015 4 / 11 Hot Summers under Various Warming Targets Table 1. Occurrence probability of an “extremely hot” summer over the global land area. Period
GISS (%)
CRU (%)
Multi-model ensemble mean
Reference period
0.1
0.1
0.1
1946–1955
0.3
0.5
0.3
1956–1965
0.1
0.1
0.1
1966–1975
0.1
0.2
0.2
1976–1985
0.6
0.7
0.4
1986–1995
1.7
2.0
1.4
1996–2005
4.8
5.1
4.3
The second, third and fourth columns in the table present results from the two observational datasets and
multi-model ensemble mean. doi:10 1371/journal pone 0130660 t001 Table 1. Occurrence probability of an “extremely hot” summer over the global land area. The second, third and fourth columns in the table present results from the two observational datasets and
multi-model ensemble mean. doi:10.1371/journal.pone.0130660.t001 important locations, such as West Europe, South America and North Africa, are all significant,
but their exact frequency of “extremely hot” summers is underestimated. important locations, such as West Europe, South America and North Africa, are all significant,
but their exact frequency of “extremely hot” summers is underestimated. Response of hot summer extremes to various warming targets doi:10.1371/journal.pone.0130660.g003 Fig 3. Spatial distribution of the “extremely hot” summers frequency from 1986 to 2005. The left,
middle and right maps represent the GISS, CRU and averages of the individual models results, respectively. doi:10.1371/journal.pone.0130660.g003 doi:10.1371/journal.pone.0130660.g003 different regions [33, 35]. The details regarding changes in probability are shown in Table 2. Notably the occurrence probability of an “extremely hot” summer would increase to 40.3% as
the global temperature reaches the 2°C warming target and 93.3% as the global temperature
reaches the 5°C warming target. The probability was only 0.1% during the reference period. When the increase of the global mean temperature exceeds 2°C or 5°C relative to the pre-
industrial level, the occurrence probabilities of having an “exceeding 5σ hot” summer may
increase to 12.9% and 79.6%, respectively. This probability was zero during the reference
period. different regions [33, 35]. The details regarding changes in probability are shown in Table 2. Notably the occurrence probability of an “extremely hot” summer would increase to 40.3% as
the global temperature reaches the 2°C warming target and 93.3% as the global temperature
reaches the 5°C warming target. The probability was only 0.1% during the reference period. When the increase of the global mean temperature exceeds 2°C or 5°C relative to the pre-
industrial level, the occurrence probabilities of having an “exceeding 5σ hot” summer may
increase to 12.9% and 79.6%, respectively. This probability was zero during the reference
period. Which regions will be vulnerable to hot summer extremes in the future when warming tar-
gets are reached? The frequencies of “extremely hot” and “exceeding 5σ hot” summers in
response to different warming rates (2°C to 5°C) relative to the pre-industrial level are shown
in Fig 5. When the 2°C target is reached, an “extremely hot” summer is projected to occur
twice every ten years at most low-mid latitudes. Central and western Asia, central and northern
Africa and northern South America are relatively more vulnerable than other areas. Further-
more, many areas seem to experience “extremely hot” summers 7 out of 10 years and may even
suffer an “exceeding 5σ hot” summer. When the 3°C warming target is reached, this situation Fig 4. Frequency of local summer temperature anomalies. These results are based on different
magnitudes of warming relative to the pre-industrial level. Response of hot summer extremes to various warming targets The multi-model ensemble mean well reproduces the statistical characteristics of the probabil-
ity density distribution and covering area percentage of the present temperature extremes over
the global land area, and partially captures the locations of “extremely hot” summers. This
systematic evaluation of the performance of the models on hot summer extremes increases the
confidence in the projected future changes in temperature extremes. Under the RCP2.6 sce-
nario, the global mean temperatures of 8 models (8/26) reach the 2°C target, even though this
scenario is designed to limit the global mean temperature increase to within 2°C relative to the
pre-industrial level. A 3°C warming target was reached by 8 models in the RCP4.5 scenario and
by all 26 models in the RCP8.5 scenario. Regarding the 4°C and 5°C warming targets, 19 and 9
models, respectively, from the RCP8.5 scenario reached these targets. In this research, we only
focused on the response of the temperature extremes to different magnitudes of warming rela-
tive to the pre-industrial level. Thus, the emission scenarios (RCP2.6, RCP4.5 and RCP8.5) are
not distinguished in the analysis. As a result, 78 simulations are used, from 26 CMIP5 models,
each simulating the three scenarios. As shown in Fig 4, the curves of the probability density widen and shift right as the global
mean temperature increases. Thus, “extremely hot” summers will occur more readily in the
future. The rightward shift is more severe than that during the 1990s due to the gradual aggra-
vation of global warming, and significant broadening may result from various warming rates in Fig 2. Percentages of global land areas hit by summer temperature extremes. The time series spans
from 1901 to 2005. The thick lines are the results of 26 individual models averages. The shading in the plots
denotes the 1σ uncertainty range of the 26 CMIP5 models. Fig 2. Percentages of global land areas hit by summer temperature extremes. The time series spans
from 1901 to 2005. The thick lines are the results of 26 individual models averages. The shading in the plots
denotes the 1σ uncertainty range of the 26 CMIP5 models. doi:10.1371/journal.pone.0130660.g002 doi:10.1371/journal.pone.0130660.g002 5 / 11 PLOS ONE | DOI:10.1371/journal.pone.0130660
June 19, 2015 Hot Summers under Various Warming Targets Fig 3. Spatial distribution of the “extremely hot” summers frequency from 1986 to 2005. The left,
middle and right maps represent the GISS, CRU and averages of the individual models results, respectively. doi:10.1371/journal.pone.0130660.g004 Response of hot summer extremes to various warming targets The temperature anomalies (relative to 1951–1980)
are divided by the local standard deviation of 1901–2005 and binned into intervals of 0.05 for individual
models before averaging the statistical results. The local standard deviation is calculated from the long-term
non-linearly detrended time series (details in data and method section). The total number of simulations that
reach the warming targets is shown in brackets. The shading represents the 1σ range of uncertainty. doi:10.1371/journal.pone.0130660.g004 Fig 4. Frequency of local summer temperature anomalies. These results are based on different
magnitudes of warming relative to the pre-industrial level. The temperature anomalies (relative to 1951–1980)
are divided by the local standard deviation of 1901–2005 and binned into intervals of 0.05 for individual
models before averaging the statistical results. The local standard deviation is calculated from the long-term
non-linearly detrended time series (details in data and method section). The total number of simulations that
reach the warming targets is shown in brackets. The shading represents the 1σ range of uncertainty. doi:10.1371/journal.pone.0130660.g004 6 / 11 PLOS ONE | DOI:10.1371/journal.pone.0130660
June 19, 2015 Hot Summers under Various Warming Targets Table 2. Probability of “extremely hot” (>3σ) and “exceeding 5σ hot” (>5σ) summers in response to
different magnitudes of warming relative to the pre-industrial level. Warming targets
>3σ (%)
>5σ (%)
Reference period
0.1
0
1°C
11.0
1.3
2°C
40.3
12.9
3°C
70.4
39.1
4°C
87.3
64.0
5°C
93.3
79.6
doi:10.1371/journal.pone.0130660.t002 Table 2. Probability of “extremely hot” (>3σ) and “exceeding 5σ hot” (>5σ) summers in response to
different magnitudes of warming relative to the pre-industrial level. becomes worse. Both “extremely hot” and “exceeding 5σ hot” summers are projected to occur
more frequently at a 3°C than at a 2°C warming. Nearly all land areas would experience
“exceeding 5σ hot” summers during a 10-year period, excluding Australia. When the global
mean temperature reaches the 4°C and 5°C warming targets, essentially no land areas would be
excluded from “extremely hot” summer every year. Warming by 5°C relative to pre-industrial
level would also make “exceeding 5σ hot” summers extremely common in most land areas. Notably, the impacts on Australia, India and Antarctica are relatively small compared with
other locations. Fig 5. Spatial distribution of the frequency of “extremely hot” (>3σ) and “exceeding 5σ hot” summers. The maps show the frequency of “extremely hot” and “exceeding 5σ hot” when the temperature increases by
from 2°C to 5°C relative to pre-industrial level. Response of hot summer extremes to various warming targets The σ is calculated from the long-term non-linearly detrended
time series (details in data and method section). The left and right columns present the “extremely hot” and
“exceeding 5σ hot” summers at different warming targets (2°C, 3°C, 4°C and 5°C in rows 1, 2, 3, and 4,
respectively). The upper and lower halves of each plot describe the boreal summer (JJA) and austral summer
(DJF), respectively. The color bar is different from that shown in Fig 3. Units: times/10 yrs. doi:10 1371/journal pone 0130660 g005 Fig 5. Spatial distribution of the frequency of “extremely hot” (>3σ) and “exceeding 5σ hot” summers. The maps show the frequency of “extremely hot” and “exceeding 5σ hot” when the temperature increases by
from 2°C to 5°C relative to pre-industrial level. The σ is calculated from the long-term non-linearly detrended
time series (details in data and method section). The left and right columns present the “extremely hot” and
“exceeding 5σ hot” summers at different warming targets (2°C, 3°C, 4°C and 5°C in rows 1, 2, 3, and 4,
respectively). The upper and lower halves of each plot describe the boreal summer (JJA) and austral summer
(DJF), respectively. The color bar is different from that shown in Fig 3. Units: times/10 yrs. doi:10.1371/journal.pone.0130660.g005 doi:10.1371/journal.pone.0130660.g005 7 / 11 PLOS ONE | DOI:10.1371/journal.pone.0130660
June 19, 2015 Hot Summers under Various Warming Targets The responses of the areas affected by “extremely hot” and “exceeding 5σ hot” summers to
different warming targets are specifically examined based on 78 CMIP5 simulations (Fig 6). All
78 simulations reached the 1°C warming target, and 55, 34, 19 and 9 of the simulations reached
the 2°C, 3°C, 4°C, 5°C warming targets, respectively. When the 1°C warming target is reached,
9.7% of the global land is projected to experience an “extremely hot” summer, and an “exceed-
ing 5σ hot” summer would rarely occur over land. After exceeding the 3°C warming target,
more than half of the global land area (67.0%) could experience an “extremely hot” summer. In
addition, almost all of the land areas (91.4%) would experience “extremely hot” summers when
the 5°C warming target is reached. “Exceeding 5σ hot” summers are projected to occur over
more than half of the land area when the 4°C warming target is exceeded. Response of hot summer extremes to various warming targets Notably, the area
affected by “extremely hot” summers expands rapidly between warming targets of a 1°C and
3°C, with an area increase of more than 25% per degree. Above the 3°C warming target, the
area increases by less than 16% per degree. However, the areas that experience “exceeding 5σ
hot” summers continues to rapidly increase by more than 17% per degree after the 2°C warm-
ing target is reached. Summary and Discussions Global warming may result in various impacts on the ecology and environment. Our research
focused on the changes in summer temperature extremes over land areas around the world in
response to different magnitude of warming, showing one aspect of impacts from global warm-
ing. To use the models’ future prediction for quantifying the temperature extremes, their simu-
lating ability need to be evaluated first. A systematic evaluation of the performance of the
models on predicting hot summer extremes showed the multi-model ensemble mean of the 26
CMIP5 models well reproduced the statistical characteristics of historical summer tempera-
tures and captured the main locations of “extremely hot” summers. The performance of the
multi-model ensemble mean is important for projecting future changes in extreme summer
temperatures. The frequency, areal extent and intensity of summer temperature extremes are
all projected to increase as global warming continues and higher warming results in more
severe hot summers. Mid-low latitude areas are projected to experience more summer temper-
ature extremes than high latitudes. Exploring the response of summer temperature extremes to
various warming targets will improve our understanding in the importance of controlling the
magnitude of warming. To reduce the impacts and damage from severely hot summers, global
warming should be limited as much as possible. The observations indicate that the global mean Fig 6. Percentages of the areas with “extremely hot” (left) and “exceeding 5σ hot” (right) summers. The maps show the responses to different magnitudes of warming relative to pre-industrial level. The σ is
calculated from the long-term non-linearly detrended time series (details in data and method section). The
colors indicate the number of simulations. The line in the middle of each box represents the ensemble mean
of the individual model results, and the upper and bottom edges of the box show the range of the 1σ
uncertainty. The two bars outside the box represent the maximum and minimum simulation values. doi:10.1371/journal.pone.0130660.g006 Fig 6. Percentages of the areas with “extremely hot” (left) and “exceeding 5σ hot” (right) summers Fig 6. Percentages of the areas with “extremely hot” (left) and “exceeding 5σ hot” (right) summers. The maps show the responses to different magnitudes of warming relative to pre-industrial level. The σ is
calculated from the long-term non-linearly detrended time series (details in data and method section). The
colors indicate the number of simulations. Supporting Information S1 Table. Affiliations and atmospheric model resolutions of the 26 CMIP5 models. The
durations of the historical and RCP runs are also provided. (DOCX) Acknowledgments We acknowledge the World Climate Research Programme’s Working Group on Coupled
Modeling, which is responsible for CMIP. We also thank the climate groups (S1 Table) for pro-
viding the model output. Our work is completed on the “Explorer 100” cluster system of Tsin-
ghua National Laboratory for Information Science and Technology. We also would like to
thank the anonymous reviewers for their comments and suggestions that much improved the
original manuscript. Author Contributions Conceived and designed the experiments: LW JH YL. Performed the experiments: LW JH YL
ZZ. Analyzed the data: LW YY. Contributed reagents/materials/analysis tools: LW JH YL ZZ. Wrote the paper: LW JH. Wrote the paper: LW JH. Hot Summers under Various Warming Targets temperature has currently increased by 0.8°C since the industrial revolution [36]. Thus, stricter
greenhouse gas emission policies should be enacted to mitigate global warming as soon as
possible. The quantification of temperature extremes relative to local climate mean and variability
offers of useful way to analyze the extreme temperatures. Furthermore, distinct from heat
waves defined by daily temperatures, this kind of definition emphasizes the overall climate
change. The heat waves are of concern mainly because of their significant impacts on the
human health, while hot extremes on seasonal scales may have a long-term impact on the eco-
logical aspects, such as crop growth and animal breeding. However, one limitation of this defi-
nition method is to neglect the absolute temperature. For example, the standard deviations for
most high latitudes in the Northern Hemisphere are relatively greater than those in the tropics,
and the mean temperature is lower at the high latitude. When the 2°C target is reached, the
temperatures exceeding 3σ in most of the high latitude areas remain less than 30°C, while the
temperatures less than 3σ in some areas of tropics are higher than 30°C. Thus, this definition of
temperature extremes cannot reflect the local absolute temperatures. In addition, only the changes in the extremely hot summer temperatures are analyzed in
this study, while the reasons for changes are not discussed. Some researchers have indicated
that temperature extremes may be linked to weakening summer circulations, anomalous pat-
terns of mid-latitudinal planetary waves, soil and atmospheric moisture, the interactions with
precipitation extremes and other factors [7, 37–41]. The temperatures in different regions are
affected by various circulation systems and other local characteristics. The mechanisms that
cause the changes in extremely hot summers under various warming targets at different loca-
tions should be studied in the future. 2.
Pezza AB, van Rensch P, Cai W (2012) Severe Heat Waves in Southern Australia: Synoptic Climatol-
ogy and Large Scale Connections. Climate Dynamics 38: 209–224. 1.
Peterson TC, Heim RR Jr, Hirsch R, Kaiser DP, Brooks H, Diffenbaugh NS, et al. (2013) Monitoring and
Understanding Changes in Heat Waves, Cold Waves, Floods, and Droughts in the United States: State
of Knowledge. Bulletin of the American Meteorological Society 94: 821–834. Summary and Discussions The line in the middle of each box represents the ensemble mean
of the individual model results, and the upper and bottom edges of the box show the range of the 1σ
uncertainty. The two bars outside the box represent the maximum and minimum simulation values. Fig 6. Percentages of the areas with “extremely hot” (left) and “exceeding 5σ hot” (right) summers. The maps show the responses to different magnitudes of warming relative to pre-industrial level. The σ is
calculated from the long-term non-linearly detrended time series (details in data and method section). The
colors indicate the number of simulations. The line in the middle of each box represents the ensemble mean
of the individual model results, and the upper and bottom edges of the box show the range of the 1σ
uncertainty. The two bars outside the box represent the maximum and minimum simulation values. doi:10.1371/journal.pone.0130660.g006 doi:10.1371/journal.pone.0130660.g006 8 / 11 PLOS ONE | DOI:10.1371/journal.pone.0130660
June 19, 2015 PLOS ONE | DOI:10.1371/journal.pone.0130660
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23457264 11 / 11
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Comparison of Clinical Features Between Patients with Positive and Negative Appendectomy
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Comparison of Clinical Features Between Patients
with Positive and Negative Appendectomy Guner Cakmak
(
guner_cakmak@yahoo.com
)
Sakarya Üniversitesi Eğitim Ve Araştırma Hastanesi: Sakarya Universitesi Egitim ve Arastirma Hastanesi
Baris Mantoglu
Sakarya Üniversitesi Eğitim Ve Araştırma Hastanesi: Sakarya Universitesi Egitim ve Arastirma Hastanesi
https://orcid.org/0000-0002-2161-3629
Emre Gonullu
Sakarya Üniversitesi Eğitim Ve Araştırma Hastanesi: Sakarya Universitesi Egitim ve Arastirma Hastanesi
Kayhan Ozdemir
Sakarya Üniversitesi Eğitim Ve Araştırma Hastanesi: Sakarya Universitesi Egitim ve Arastirma Hastanesi
Burak Kamburoglu
Sakarya Üniversitesi Eğitim Ve Araştırma Hastanesi: Sakarya Universitesi Egitim ve Arastirma Hastanesi
Research
Keywords: acute appendicitis, mean platelet volume, total bilirubin, negative appendectomy
Posted Date: December 3rd, 2020
DOI: https://doi.org/10.21203/rs.3.rs-117309/v1 Guner Cakmak
(
guner_cakmak@yahoo.com
) Comparison of Clinical Features Between Patients
with Positive and Negative Appendectomy
Guner Cakmak
(
guner_cakmak@yahoo.com
)
Sakarya Üniversitesi Eğitim Ve Araştırma Hastanesi: Sakarya Universitesi Egitim ve Arastirma Hastanesi
Baris Mantoglu
Sakarya Üniversitesi Eğitim Ve Araştırma Hastanesi: Sakarya Universitesi Egitim ve Arastirma Hastanesi
https://orcid.org/0000-0002-2161-3629
Emre Gonullu
Sakarya Üniversitesi Eğitim Ve Araştırma Hastanesi: Sakarya Universitesi Egitim ve Arastirma Hastanesi
Kayhan Ozdemir
Sakarya Üniversitesi Eğitim Ve Araştırma Hastanesi: Sakarya Universitesi Egitim ve Arastirma Hastanesi
Burak Kamburoglu
Sakarya Üniversitesi Eğitim Ve Araştırma Hastanesi: Sakarya Universitesi Egitim ve Arastirma Hastanesi
Research
Keywords: acute appendicitis, mean platelet volume, total bilirubin, negative appendectomy
Posted Date: December 3rd, 2020
DOI: https://doi.org/10.21203/rs.3.rs-117309/v1
License:
This work is licensed under a Creative Commons Attribution 4.0 International
License.
Read Full License Comparison of Clinical Features Between Patients
with Positive and Negative Appendectomy
Guner Cakmak
(
guner_cakmak@yahoo.com
)
Sakarya Üniversitesi Eğitim Ve Araştırma Hastanesi: Sakarya Universitesi Egitim ve Arastirma Hastanesi
Baris Mantoglu
Sakarya Üniversitesi Eğitim Ve Araştırma Hastanesi: Sakarya Universitesi Egitim ve Arastirma Hastanesi
https://orcid.org/0000-0002-2161-3629
Emre Gonullu
Sakarya Üniversitesi Eğitim Ve Araştırma Hastanesi: Sakarya Universitesi Egitim ve Arastirma Hastanesi
Kayhan Ozdemir
Sakarya Üniversitesi Eğitim Ve Araştırma Hastanesi: Sakarya Universitesi Egitim ve Arastirma Hastanesi
Burak Kamburoglu
Sakarya Üniversitesi Eğitim Ve Araştırma Hastanesi: Sakarya Universitesi Egitim ve Arastirma Hastanesi
Research
Keywords: acute appendicitis, mean platelet volume, total bilirubin, negative appendectomy
Posted Date: December 3rd, 2020
DOI: https://doi.org/10.21203/rs.3.rs-117309/v1
License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read Full License Comparison of Clinical Features Between Patients
with Positive and Negative Appendectomy
Guner Cakmak
(
guner_cakmak@yahoo.com
)
Sakarya Üniversitesi Eğitim Ve Araştırma Hastanesi: Sakarya Universitesi Egitim ve Arastirma Hastanesi
Baris Mantoglu
Sakarya Üniversitesi Eğitim Ve Araştırma Hastanesi: Sakarya Universitesi Egitim ve Arastirma Hastanesi
https://orcid.org/0000-0002-2161-3629
Emre Gonullu
Sakarya Üniversitesi Eğitim Ve Araştırma Hastanesi: Sakarya Universitesi Egitim ve Arastirma Hastanesi
Kayhan Ozdemir
Sakarya Üniversitesi Eğitim Ve Araştırma Hastanesi: Sakarya Universitesi Egitim ve Arastirma Hastanesi
Burak Kamburoglu
Sakarya Üniversitesi Eğitim Ve Araştırma Hastanesi: Sakarya Universitesi Egitim ve Arastirma Hastanesi
Research
Keywords: acute appendicitis, mean platelet volume, total bilirubin, negative appendectomy
Posted Date: December 3rd, 2020
DOI: https://doi.org/10.21203/rs.3.rs-117309/v1
License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read Full License Research License:
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License. Page 1/14 Page 1/14 Page 1/14 Abstract Background: The objective of this study was to retrospectively compare clinical features and prognostic
values between the patients who were referred to the general surgery clinic of our hospital with the
presumed diagnosis of acute appendicitis and underwent positive or negative appendectomy. Methods: Patients were divided into two groups as positive (PA) (n:362) and negative appendectomy
(NA) (n:284) and the data obtained were compared between these two groups. Laboratory investigations were performed in all patients, and white blood cell (WBC), mean platelet
volume (MPV), neutrophils count (NEU), neutrophils (%) (NEU%), C-reactive protein (CRP) and total
bilirubin (TBIL) values were studied. Results: The mean MPV value was found as 7.88 fl in PA groups and 8.09 fl in NA group, and the mean
MPV value was not statistically significantly difference in PA group, compared to NA groups (p=0.012). Laboratory parameters were also compared between genders. Accordingly, the mean MPV value was
statistically significantly higher in female patients compared to male patients in PA group (p = 0.04). The
mean TBIL value was 0.97 mg/dl in PA group and 0.69 mg/dl in NA group, and the mean TBIL value was
statistically significantly higher in PA group (p< 0.001). Finally, TBIL value was statistically significantly
lower in female patients compared to male patients in NA and PA group (p < 0.05). Conclusions: According to the results of our study, MPV and T. BIL values differ in PA and NA groups
depending on gender. Therefore, these values may not be used as specific biomarkers in predicting
positive acute appendicitis. We believe that these results will contribute to the literature and will be
guiding for future studies. Background Appendicitis is the most common emergency surgery, with a lifetime risk for appendicitis estimated as
8.6% in men and 6.7% in women (1). In Europe, 112/100000 persons present to emergency departments
each year due to acute appendicitis (2). The rate of morbidity from acute appendicitis has been reported
between 7%-16% in the general population (3). Appendicitis usually presents with anorexia and
tenderness in the right lower abdominal quadrant (4). However, pain in the right lower quadrant can be
associated with numerous pathologies. Because of the wide spectrum of differential diagnosis and lack
of specific markers for appendicitis, preoperative diagnosis of this disease is quite challenging (5). Today, the incidence of appendicitis is increasing in developing countries (6). Since serious complications
may occur in the case of delayed diagnosis, prompt action is necessary. The management of
appendicitis focuses on the prevention with a timely intervention before an uncomplicated appendicitis
progresses to a complicated one. Appendectomy, which is sometimes performed on a normal
appendicitis is known as “negative appendectomy” (NA) (7). Page 2/14 Page 2/14 Complications of acute appendicitis include perforation, peritonitis and sepsis (8). This can increase the
rate of negative appendectomy operations. Negative appendectomy rate (NAR) is defined as the
incidence of removing appendices that are pathologically normal (9). Negative appendectomy leads to
prolonged hospitalization, morbidity and increased costs. In addition, negative appendectomy may be
associated with severe postoperative complications. Therefore, an accurate preoperative diagnosis is
essential in the cases of suspected acute appendicitis. In the United States of America (USA) over 250000
appendectomy operations are performed yearly, and the rate of negative is approximately 15% in these
operations (10). However, owing to better imaging modalities, a constant decrease is seen in the rate of
NAs (11). Gynecological pathologies in female patients in childbearing period mimic acute appendicitis,
which can cause an increase in the rate of negative NAs (12). According to the current guidelines, the diagnosis of acute appendicitis is established according to
clinical examination and confirmed with imaging investigations and some biomarkers including WBC
count and CRP (13). Computed tomography (CT) and ultrasonography are used as imaging modalities in
order to set a more accurate diagnosis. The objective of this study was to retrospectively compare clinical features and prognostic values
between the patients who were referred to the general surgery clinic of our hospital with the presumed
diagnosis of acute appendicitis and underwent positive or negative appendectomy. Methods Data of a total of 2168 patients who were referred to our general surgery clinic with the presumed
diagnosis of appendicitis and underwent appendectomy between 2010 and 2019 were retrospectively
evaluated. Among these patients, 1522 were excluded from the study because of missing data, and those
who underwent interval appendectomy, and incidental appendectomy, and the patients for whom
appendectomy operation was delayed due to conservative treatment. Finally, data of 646 patients were
obtained and evaluated. Patients aged between 16–90 years. Patients were divided into two groups as
positive (PA) (n:362) and negative appendectomy (NA) (n:284) and the data obtained were compared
between these two groups. Patients’ demographic data such as age and gender, date and type of operations, investigations, clinical
presentation, macroscopic and microscopic findings, imaging and pathological findings were recorded
and analyzed. In addition, laboratory investigations were performed in all patients, and white blood cell
(WBC), mean platelet volume (MPV), neutrophils count (NEU), neutrophils (%) (NEU%), C-reactive protein
(CRP) and total bilirubin (TBIL) values were studied. Page 3/14
Inclusion criteria included all patients operated during the nine years period. Considering clinical
conditions of the patients and in the cases of uncertain diagnosis or where the clinical picture did not
meet physical findings (age, pregnancy, having gynecological examination, comorbidities etc.)
abdominopelvic computed tomography and ultrasonography examinations were performed in some
patients. The diagnosis of appendicitis was established based on macroscopic findings. Histological Page 3/14 diagnosis of appendicitis was set according to the infiltration of muscularis propria with neutrophils
granulocytes. Appendectomies were performed by conventional or laparoscopic methods. diagnosis of appendicitis was set according to the infiltration of muscularis propria with neutrophils
granulocytes. Appendectomies were performed by conventional or laparoscopic methods. Lack of the appendicitis in the pathology reports was considered negative appendectomy. In order to
identify appendectomy cases with negative samples, pathology reports were interpreted by an author
blinded to whether preoperative imaging was performed. Negative appendectomy was defined as a
normal appendix following the resection due to suspected appendicitis or medically unnecessary
appendectomy. Medically unnecessary appendectomy was defined as an appendectomy operation
performed in the case of typically contraindicated surgery. Before the beginning of the study, necessary ethics approval was received from the local
ethics committee of our hospital. Since the study had a retrospective design, receiving informed consent
from the patients was waived. The study was conducted in accordance with the ethical principles of the
Declaration of Helsinki. Statistical Analysis Data obtained in the study were analyzed utilizing SPSS version 23.0 (Statistical Package for Social
Sciences, SPSS, IBM Incusing Chicago, IL, USA) statistical package software. Normality of the variables
was analyzed with the Kolmogorov-Smirnov test. In the comparison of the variables between the groups,
Independent Student t test among was used for the normally distributed variables, and Mann-Whitney U
test for non-normally distributed variables. Continuous variables were expressed with mean ± standard
deviation, while categorical variables were given as frequency and percentage. p < 0.05 values were
considered statistically significant. Results A total of 646 patients were included in the study with 362 (56%) being in PA group and 284 (44%) in NA
group. Of all patients included in the study, 360 (55.7%) were male and 286 (44.3%) were female. A total
of 226 (62.4%) patients were male and 136 (37.6%) were female in PA group. Whereas 134 (47.2%) were
male and 150 (52.8%) were female in the NA group (Table 1). No statistically significant difference was
seen between both groups in terms of gender (p > 0.05). Page 4/14 Table 1
Frequency distribution of patients' gender and age by
type of appendectomy
PA. (n = 362)
NA. (n = 284)
n
%
n
%
Male
226
62.4
134
47.2
Female
136
37.6
150
52,8
Sum
362
100
284
100
Mean Age
28.2 ± 16.65
31.9 ± 17.4
PA: positive appendicitis NA: negative appendicitis Table 1 The mean age of all patients was 30.1 ± 16.9 years, and the mean age was found as 28.2 ± 16.65 years in
PA group and 31.9 ± 17.4 years in NA group (Table 1). There was no statistically significant difference
between PA and NA groups in terms of age (p > 0.05). The mean age of all patients was 30.1 ± 16.9 years, and the mean age was found as 28.2 ± 16.65 years in
PA group and 31.9 ± 17.4 years in NA group (Table 1). There was no statistically significant difference
between PA and NA groups in terms of age (p > 0.05). Appendectomies were performed with laparoscopic method in 199 (54.9%) of the patients in PA group
and in 207 (72.8%) of the patients in NA group. The remaining patients underwent open surgery. Appendectomies were performed with laparoscopic method in 199 (54.9%) of the patients in PA group
and in 207 (72.8%) of the patients in NA group. The remaining patients underwent open surgery. When status of having gynecological examination was examined among the female patients; 105
(77.3%) female patients in PA group and 125 (83.5%) female patients in NA group underwent
gynecological examination. When status of having gynecological examination was examined among the female patients; 105
(77.3%) female patients in PA group and 125 (83.5%) female patients in NA group underwent
gynecological examination. Laboratory outcomes were examined and compared between the two groups. Results Accordingly, the mean WBC
value was found as 9.97 K/mm3 in PA group and 9.51 K/mm3 in NA group, and there was no statistically
significant difference between both groups (p = 0.07). The mean MPV value was found as 7.88 fl in PA
groups and 8.09 fl in NA group, and there was no statistically significant difference between both groups
(p = 0.12). NEU (%) value was found as 76.01 in PA group and 75.69 in NA group. No statistically
significant difference was observed between the two groups in terms of the mean NEU (%) value (p =
0.75). The mean neutrophil count was found as 7.33 K/mm3 in the PA group and 7.50 K/mm3 in NA
group, and no statistically significant difference was found between the two groups (p = 0.48). The mean
CRP value was found as 57.68 mg/dl in PA group and 41.43 mg/dl in NA group. There was a statistically
significant difference between both groups in terms of the mean CRP value (p < 0.001). When total
bilirubin values were examined; the mean TBIL value was 0.97 mg/dl in PA group and 0.69 mg/dl in NA
group, and the mean TBIL value was statistically significantly higher in PA group (p < 0.001) (Table 2). Page 5/14 Page 5/14 Table 2
Comparison results of WBC, MPV, NEU (%), NEU, CRP and TBIL
parameters of patients according to the type of appendectomy
Appendectomy Type
n
x̄
ss
t
p
WBC
Positive
362
9.97
3.44
1.81
.07
Negative
284
9.51
2.96
MPV
Positive
362
7.88
1.65
-1.55
.12
Negative
284
8.09
1.79
NEU%
Positive
362
76.01
12.94
.31
.75
Negative
284
75.69
12.23
NEU
Positive
362
7.33
2.72
− .69
.48
Negative
284
7.50
3.64
CRP
Positive
362
57.68
58.92
3.54
.00
Negative
284
41.43
56.36
TBIL
Positive
362
.97
.57
7.39
.00
Negative
284
.69
.39 In our study, the examined laboratory parameters were also compared between genders. Accordingly, the
mean MPV value was statistically significantly higher in female patients compared to male patients in PA
group (p = 0.04). NEU (%) value was statistically significant between genders both in PA and NA groups. Accordingly, NEU (%) value was statistically significantly lower in male patients compared to female
patients in both PA and NA groups (p < 0.05, p < 0.05; respectively). Neutrophil count was statistically
significantly lower in male patients in NA group (p < 0.05). Results Finally, TBIL value was statistically
significantly lower in female patients compared to male patients in NA and PA group (p < 0.05) (Table 3–
4). 4). Page 6/14 Table 3
Comparison results of WBC, MPV, NEU (%), NEU, CRP and TBIL parameters
according to the gender of the patients' in positive appendectomy group. Type
Variables
Gender
n
x̄
ss
t
p
Positive
Appendectomy
WBC
Male
226
10.08
3.58
.76
.45
Female
136
9.80
3.21
MPV
Male
226
7.74
1.58
-2.09
.04
Female
136
8.12
1.73
NEU%
Male
226
74.98
13.24
-1.96
.04
Female
136
77.73
12.29
NEU
Male
226
7.20
2.78
-1.17
.24
Female
136
7.54
2.61
CRP
Male
226
57.79
57.72
.05
.96
Female
136
57.49
61.07
TBIL
Male
226
1.06
.60
3.98
.00
Female
136
.82
.50 Page 7/14 Table 4
Comparison results of WBC, MPV, NEU (%), NEU, CRP and TBIL parameters
according to the gender of the patients' in negative appendectomy group. Type
Variables
Gender
n
x̄
ss
t
p
Negative
Appendectomy
WBC
Male
134
9.46
2.59
-.28
.78
Female
150
9.55
3.27
MPV
Male
134
7.88
1.59
-1.92
.06
Female
150
8.29
1.95
NEU%
Male
134
73.02
12.72
-3.55
.00
Female
150
78.08
11.29
NEU
Male
134
7.09
1.99
-1.82
.07
Female
150
7.87
4.62
CRP
Male
134
40.99
52.98
− .12
.90
Female
150
41.83
59.39
TBIL
Male
134
.75
.37
-2.57
.01
Female
150
.63
.39 When diameters of appendicitis were evaluated; the mean appendicitis diameter was found as 1.09 cm in
PA group and 0.88 cm in NA group, and the mean appendicitis diameter was statistically significantly
higher in PA group compared to NA group (p < 0.001) (Table 5). Table 5
Comparison results of appendectomy material diameters
according to appendectomy type
Appendectomy
Type
n
x̄
ss
t
p
Diame
Positive
362
1.09
.63
4.17
.00
Negative
284
.88
.63 Table 5
Comparison results of appendectomy material diameters
according to appendectomy type Discussion Surgical intervention of a normal appendicitis exposes patients to unnecessary anesthesia and surgical
complications, and this may be resulted from improper clinical evaluation, and lack of diagnostic
methods. Despite the high incidence of acute appendicitis and use of laboratory markers and imaging Page 8/14 Page 8/14 modalities, the accurate diagnosis remains challenging. Research of the diagnostic process used for
acute appendicitis is highly dynamic, within this context information such as novel inflammatory
biomarkers is constantly reported in the literature (14). As in our country, high rates of appendicitis cases
have been reported in some other countries (15). Therefore, more effort should be made in order to reduc
the incidence of NAR and its complications on patients and hospitals (16). Negative appendectomies
lead to both postoperative complications, increased morbidity and mortality rates and costs. Thus,
investigation of the factors leading to negative appendectomies is of paramount importance. Among
these factors, laboratory parameters take an important place. However, to our knowledge there is no
consensus in the literature on this subject. In our study, we first evaluated laboratory values between
positive and negative appendectomy cases. We performed preoperative computed tomography by excluding elderly patients, pediatric patients and
those with comorbidities. We observed that the rate of negative appendectomy was lower in patients who
underwent CT (24.3%). In a study by Wagner et al., the rate of performing preoperative CT raised to 95%
from 32% within 10 years (17). In our study, 84.6% of female patients were found to have a gynecological examination. In a study by
Joshi et al. 57.1% of female patients were reported to have gynecological examination (16). We believe
that higher gynecological examination rate in our study resulted from the necessity of transvaginal
ultrasound in addition to abdominal ultrasound in cases of suspected gynecological diseases in young
women in order to reduce the incidence of negative appendectomy. Recently, although diagnostic value of laboratory parameters such as MPV, RDW and NAR has been
evaluated in patients with suspected appendicitis, results of these studies are highly controversial (18). MPV is a measurement of thrombocyte size that is obtained as a part of routine complete blood count
and is usually overlooked by clinicians (19). Changes in platelet counts can lead to changes in MPV. The
size and activity of platelets can be influenced by cytokines such as interleukin IL-3 and IL-6. Discussion Elevated
MPV levels have been reported in several diseases including chronic obstructive pulmonary disease
(COPD), myocardial infarction, diabetes mellitus and high altitude (20). Increases in MPV levels are
associated with chronic diseases, while decreases are related to acute diseases (21). In our study, the
mean MPV value was not statistically significantly lower in both the PA group and NA group. Although
there are a few studies about the role of MPV in acute appendicitis, the results of these studies are
variable(20, 22). In a study comparing the healthy control group with patients having acute appendicitis,
MPV level was found to be significantly lower in the acute appendicitis group. In the same study, it was
emphasized that MPV level should not be overlooked in suspected acute appendicitis cases (20). In
another study, a significant reduction was found in MPV level of patients with appendicitis (23). In a
study by Uyanik et al., no statistical significance was observed in MPV levels of patients with acute
appendicitis (22). In another study, higher MPV levels were found in patients with acute appendicitis
compared to the control group (18). In a meta-analysis of five studies including 2101 patients with acute
appendicitis, it was reported that MPV can be used as a biomarker for the diagnosis of positive Page 9/14 Page 9/14 appendicitis and is a rapid and inexpensive indicator (24). In our study, in the gender-based evaluation,
MPV values were statistically significantly lowered only in male patients in the PA group. Based on our
findings, we believe that MPV values under the lower normal range may be affected by gender and
requires further evaluation to be used as a biomarker for positive acute appendicitis. Recent studies have investigated the relationship between hyperbilirubinemia and vermiform of
inflammation, and some of these studies have reported that bilirubin can be used as a specific marker of
appendiceal perforation (25, 26). In our study, TBIL levels were significantly higher in the PA group
compared to the NA group (Table 2). However, in gender-based evaluation, TBIL levels were significantly
higher in male patients both in PA and NA groups. In a study by Akbulut et al., a TBIL cut-off ≥ 0.67 is an
independent factor predicting acute appendicitis (27). Therefore, our study supports the latest literature in
line with our findings. Conclusion According to the results of our study, MPV and T. BIL values differ in PA and NA groups depending on
gender. Therefore, these values may not be used as specific biomarkers in predicting positive acute
appendicitis. We believe that these results will contribute to the literature and will be guiding for future
studies. However, further studies are needed to determine the laboratory parameters that can be used as
diagnostic biomarkers. Discussion In addition, although the high CRP value in acute appendicitis has been reported in
various studies,(13, 28) our outcome of high CRP values in the PA group supports the literature. Although
there are several studies about the use of various laboratory markers in the diagnosis of acute
appendicitis that were mentioned above, there is still no scientific evidence on the use of blood
parameters in predicting acute appendicitis. At this point, it seems possible that NA ratios can be reduced
by correlating more than one laboratory data. In imaging examination, one of the most important findings for the diagnosis of acute appendicitis is
appendicitis diameter. In the present study, appendicitis diameter was statistically significantly higher in
PA group than in NA group (Table 4). Similarly, in a study by Katipoglu et al., the mean appendicitis
diameter was significantly higher in the positive appendectomy cases (29). The main limitations of this study are its retrospective design and being conducted in a single center. However, the number of our patients is relatively higher than the other studies in the literature. In addition,
unlike the other studies in the literature higher MPV (female group) and TBIL values in the positive
appendectomy cases will bring a new projection to the literature. Availability of data and materials There is no additional data available to share with the readers. The datasets used and/or analyzed during
the current study are available from the corresponding author on reasonable request. WBC: White blood count
MPV: Mean platelet volume
NEU: neutrophils count
NEU%: neutrophils count%
CRP: C-Reactive protein
TBIL: Total bilirubin
NAR: Negative appendectomy rate
CT: Computed Tomography WBC: White blood count
MPV: Mean platelet volume
NEU: neutrophils count
NEU%: neutrophils count%
CRP: C-Reactive protein
TBIL: Total bilirubin
NAR: Negative appendectomy rate
CT: Computed Tomography Consent for publication Written informed consent was not received due to the retrospective nature of the study. Written informed consent was not received due to the retrospective nature of the study. Ethics approval and consent to participate Sakarya University Ethics Committee has approved this research project. 71522473/050.01.04/424
Consent for publication Sakarya University Ethics Committee has approved this research project. 71522473/050.01.04/424
Consent for publication Sakarya University Ethics Committee has approved this research project. 71522473/050.01.04/424 Conflict of Interest The authors declared no conflict of interest. The authors declared no conflict of interest. The authors declared no conflict of interest. The authors declared no conflict of interest. PA: Positive appendicitis PA: Positive appendicitis NA: Negative Appendicitis Page 10/14 References 1. Huang TH, Huang YC, Tu CW. Acute appendicitis or not: Facts and suggestions to reduce valueless
surgery. J Acute Med. 2013; 3(4):142–7. 2. Ferris M, Quan S, Kaplan BS, Molodecky N, Ball CG, Chernoff GW, et al. The Global Incidence of
Appendicitis: A Systematic Review of Population-based Studies. Ann Surg. 2017;266(2):237–41. 3. Tsai MC, Kao LT, Lin HC, Chung SD, Lee CZ. Acute appendicitis is associated with peptic ulcers: A
population-based study. Sci Rep. 2015;8;5:18044. 3. Tsai MC, Kao LT, Lin HC, Chung SD, Lee CZ. Acute appendicitis is associated with peptic ulcers: A
population-based study. Sci Rep. 2015;8;5:18044. 4. World Health Organization. Surgical care at the district hospital. 2003; 4. World Health Organization. Surgical care at the district hospital. 2003; 5. Poletti PA, Becker M, Becker CD, Halfon Poletti A, Rutschmann OT, Zaidi H, et al. Emergency
assessment of patients with acute abdominal pain using low-dose CT with iterative reconstruction: a
comparative study. Eur Radiol. 2017; 27(8):3300–9. 5. Poletti PA, Becker M, Becker CD, Halfon Poletti A, Rutschmann OT, Zaidi H, et al. Emergency
assessment of patients with acute abdominal pain using low-dose CT with iterative reconstruction: a
comparative study. Eur Radiol. 2017; 27(8):3300–9. 6. Sartelli M, Baiocchi GL, Di Saverio S, Ferrara F, Labricciosa FM, Ansaloni L, et al. Prospective
Observational Study on acute Appendicitis Worldwide (POSAW). World J Emerg Surg. 2018;13(1):19. 6. Sartelli M, Baiocchi GL, Di Saverio S, Ferrara F, Labricciosa FM, Ansaloni L, et al. Prospective
Observational Study on acute Appendicitis Worldwide (POSAW). World J Emerg Surg. 2018;13(1):19. 7. Ruffolo C, Fiorot A, Pagura G, Antoniutti M, Massani M, Caratozzolo E, et al. Acute appendicitis: What
is the gold standard of treatment? World Journal of Gastroenterology. 2013;19(47):8799–807. 7. Ruffolo C, Fiorot A, Pagura G, Antoniutti M, Massani M, Caratozzolo E, et al. Acute appendicitis: What
is the gold standard of treatment? World Journal of Gastroenterology. 2013;19(47):8799–807. 8. Webb EM, Nguyen A, Wang ZJ, Stengel JW, Westphalen AC, Coakley F V. The negative appendectomy
rate: Who benefits from preoperative CT? Am J Roentgenol. 2011;197(4):861–6. 8. Webb EM, Nguyen A, Wang ZJ, Stengel JW, Westphalen AC, Coakley F V. The negative appendectomy
rate: Who benefits from preoperative CT? Am J Roentgenol. 2011;197(4):861–6. 9. Raja AS, Wright C, Sodickson AD, Zane RD, Schiff GD, Hanson R, et al. Negative appendectomy rate in
the era of CT: An 18-year perspective. Radiology. 2010; 256(2):460–5. 9. Authors’ Contribution GC, BM: Conceived the study design, involved in data collection, performed the statistical analysis,
interpreted data and prepared the manuscript draft. Page 11/14 Page 11/14 Page 11/14 EG: Concept and design of study, analysis and draft formation. EG: Concept and design of study, analysis and draft formation. EG: Concept and design of study, analysis and draft formation. EG: Concept and design of study, analysis and draft formation. KO: Concept and design of study, data collection and analysis. KO: Concept and design of study, data collection and analysis. KO: Concept and design of study, data collection and analysis. KO: Concept and design of study, data collection and analysis. BK: Data collection, analysis and results compilation, Analysis, review and final approval of draft. BK: Data collection, analysis and results compilation, Analysis, review and final approval of draft. All the authors critically reviewed the final version of the manuscript and approved the final version
Acknowledgement All the authors critically reviewed the final version of the manuscript and approved the final version
Acknowledgement References Raja AS, Wright C, Sodickson AD, Zane RD, Schiff GD, Hanson R, et al. Negative appendectomy rate in
the era of CT: An 18-year perspective. Radiology. 2010; 256(2):460–5. 10. Florence M, Flum DR, Jurkovich GJ, Lin P, Steele SR, Symons RG, et al. Negative appendectomy and
imaging accuracy in the washington state surgical care and outcomes assessment program. Ann
Surg. 2008;248(4):557–63. 11. Seetahal SA, Bolorunduro OB, Sookdeo TC, Oyetunji TA, Greene WR, Frederick W, et al. Negative
appendectomy: A 10-year review of a nationally representative sample. Am J Surg. 2011;201(4):433–37. 12. Hatipoglu S, Hatipoglu F, Abdullayev R. Acute right lower abdominal pain in women of reproductive
age: Clinical clues. World J Gastroenterol. 2014;14:4043–9. Page 12/14 Page 12/14 13. Yu CW, Juan LI, Wu MH, Shen CJ, Wu JY, Lee CC. Systematic review and meta-analysis of the
diagnostic accuracy of procalcitonin, C-reactive protein and white blood cell count for suspected
acute appendicitis. . Br J Surg. 2013;100(3):322-9. 14. Sack U, Biereder B, Elouahidi T, Bauer K, Keller T, Tröbs RB. Diagnostic value of blood inflammatory
markers for detection of acute appendicitis in children. BMC Surg. 2006;6(1):15–15. 15. Kirkil C, Karabulut K, Aygen E, Ilhan YS, Yur M, Binnetoǧlu K, et al. Appendicitis scores may be useful
in reducing the costs of treatment for right lower quadrant pain. Ulus Travma ve Acil Cerrahi Derg. 2013;19:13–9. 16. Joshi MK, Joshi R, Alam SE, Agarwal S, Kumar S. Negative Appendectomy: an Audit of Resident-
Performed Surgery. How Can Its Incidence Be Minimized? Indian J Surg. 2015;1(3):913–7. 17. Wagner PL, Eachempati SR, Soe K, Pieracci FM, Shou J, Barie PS. Defining the current negative
appendectomy rate: For whom is preoperative computed tomography making an impact? Surgery. 2008;144:276–82. 18. Narci H, Turk E, Karagulle E, Togan T, Karabulut K. The role of red cell distribution width in the
diagnosis of acute appendicitis: A retrospective case-controlled study. World J Emerg Surg. 2013;8:46. 19. Beyazit Y, Sayilir A, Torun S, Suvak B, Yesil Y, Purnak T, et al. Mean platelet volume as an indicator of
disease severity in patients with acute pancreatitis. Clin Res Hepatol Gastroenterol. 2012;36:162-8. 20. Albayrak Y, Albayrak A, Albayrak F, Yildirim R, Aylu B, Uyanik A, et al. Mean platelet volume: A new
predictor in confirming acute appendicitis diagnosis. Clin Appl Thromb. 2011;17:362–6. 21. Erdem H, Aktimur R, Cetinkunar S, Reyhan E, Gokler C, Irkorucu O, et al. 29. Katipoglu B, Aygun A CH. The effect of appendix diameter on perforation in acute appendicitis cases.
CMJ. 2019;41:392–7. References Evaluation of mean platelet
volume as a diagnostic biomarker in acute appendicitis. Int J Clin Exp Med. 2015;8(1):1291–5. 22. Uyanik B, Kavalci C, Arslan ED, Yilmaz F, Aslan O, Dede S, et al. Role of Mean Platelet Volume in
Diagnosis of Childhood Acute Appendicitis. Emerg Med Int. 2012; 2012:823095. 22. Uyanik B, Kavalci C, Arslan ED, Yilmaz F, Aslan O, Dede S, et al. Role of Mean Platelet Volume in
Diagnosis of Childhood Acute Appendicitis. Emerg Med Int. 2012; 2012:823095. 23. Bilici S, Sekmenli T, Göksu M, Melek M, Avci V. Mean platelet volume in diagnosis of acute
appendicitis in children. Afr Health Sci. 2011;11:427–32. 23. Bilici S, Sekmenli T, Göksu M, Melek M, Avci V. Mean platelet volume in diagnosis of acute
appendicitis in children. Afr Health Sci. 2011;11:427–32. 24. Fan Z, Zhang Y, Pan J, Wang S. Acute appendicitis and mean platelet volume: A systemic review and
meta-analysis. Ann Clin Lab Sci. 2017;47(6):768–72. 24. Fan Z, Zhang Y, Pan J, Wang S. Acute appendicitis and mean platelet volume: A systemic review and
meta-analysis. Ann Clin Lab Sci. 2017;47(6):768–72. 25. D’Souza N, Karim D, Sunthareswaran R. Bilirubin; a diagnostic marker for appendicitis. Int J Surg. 2013;11:1114–7. 25. D’Souza N, Karim D, Sunthareswaran R. Bilirubin; a diagnostic marker for appendicitis. Int J Surg. 2013;11:1114–7. 26. Muller S, Falch C, Axt S, Wilhelm P, Hein D, Königsrainer A, et al. Diagnostic accuracy of
hyperbilirubinaemia in anticipating appendicitis and its severity. Emerg Med J. 2015; 0:1–5. 26. Muller S, Falch C, Axt S, Wilhelm P, Hein D, Königsrainer A, et al. Diagnostic accuracy of
hyperbilirubinaemia in anticipating appendicitis and its severity. Emerg Med J. 2015; 0:1–5. 27. Akbulut S, Koc C, Sahin TT et al. Akut Apandisit ve Perfore Apandisiti öngören faktörlerin
belirlenmesi. Ulus Travma Acil Cerrahi Derg. Ahead of Print: UTD-60344. doi:10.14744/tjtes.2020.60344. 28. Mantoğlu B, Karip B, Mestan M, Işcan Y, Ağca B, Altun H, et al. Should appendectomy be performed
laparoscopically? Clinical prospective randomized trial. Turkish J Surg. 2015;31(4):224-8. 28. Mantoğlu B, Karip B, Mestan M, Işcan Y, Ağca B, Altun H, et al. Should appendectomy be performed
laparoscopically? Clinical prospective randomized trial. Turkish J Surg. 2015;31(4):224-8. 28. Mantoğlu B, Karip B, Mestan M, Işcan Y, Ağca B, Altun H, et al. Should appendectomy be performed
laparoscopically? Clinical prospective randomized trial. Turkish J Surg. 2015;31(4):224-8. Page 13/14 Page 13/14 Page 14/14
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38 38 // JOURNALISTICA · NR. 1 · 2010 AF CHARLOTTE WIEN OG KLAUS LEVINSEN* Moderne massemedier spiller en stor rolle for, hvordan vi opfatter
og forstår ungdommen, og tidligere internationale undersøgelser
har vist, at medierne overvejende formidler et negativt billede af de
unge. På baggrund af kvantitative indholdsanalyser belyser artiklen
hvordan de unge er blevet fremstillet i et udvalg af danske aviser
over de seneste 50 år. Vores undersøgelse viser, at kriminalitet, kul-
tur og sport er de emner, der hyppigst er i spil, men undersøgelsen
kan ikke bekræfte, at der er en dominerende negativ bias i medier-
nes beskrivelser af de unge. Dette kunne hverken bekræftes i forhold
til undersøgelsen af nutidens mediedækning (2003) eller i undersø-
gelsen af langtidsperspektivet (1953-2003). Tvært i mod afslører vo-
res data en udvikling i retning af flere neutrale journalistiske beskri-
velser af unge, og en tendens til, at de unge i dag citeres direkte i
avisartiklerne. Indledning At „ungdommen ikke længere er, hvad den har været“ er et gam-
melt ordsprog og – i logisk henseende – et sandt udsagn: Den
gruppe, der i fortiden var unge rummer i sagens natur ikke de
samme individer som nu, og de vilkår, der gives ungdommen i
dag er ikke de samme som i går. Udsagnet er interessant i vores sammenhæng af andre grunde: JOURNALISTICA · NR. 1 · 2010 // 39 Den første er, at der i ligger en antagelse om, at man i en enkelt
sætning kan sige noget generelt om alle de individer, der tilhører
den sociale gruppe, som vi kalder „ungdommen“. Den anden er,
forestillingen om, at de unges adfærd og holdninger ændrer sig
over tid. Den første er, at der i ligger en antagelse om, at man i en enkelt
sætning kan sige noget generelt om alle de individer, der tilhører
den sociale gruppe, som vi kalder „ungdommen“. Den anden er,
forestillingen om, at de unges adfærd og holdninger ændrer sig
over tid. Ordsproget kan både henvise til „ungdommen“ sådan som den
i virkeligheden er, og til vore forestillinger om ungdommen. Grundantagelsen i denne artikel er, at massemedierne spiller
en stor rolle for begge dele, og formålet er at undersøge, hvordan
„de unge“ og „ungdommen“ i tidens løb er blevet fremstillet i et
udvalg af danske aviser Der findes allerede en række internationale studier af, hvordan
medierne fremstiller ungdommen, men ingen danske. I flere af
disse undersøgelser konkluderes det, at medierne beskriver de
unge negativt, og at dette bl.a. skyldes en tiltagende tabloidise-
ring og kommercialisering af medierne. De mediemæssige be-
tingelser og den journalistiske praksis skaber derved nogle skæv-
vredne billeder af de unge som problematiske samfundsborgere,
der er mere voldelige, ekstravagante og utilpassede end andre
grupper i befolkningen. De fleste af disse studier er imidlertid be-
grænsede af deres relativt korte undersøgelsesperioder, og har
derved vanskeligt ved at sige noget om forandringerne i medier-
nes fremstillinger af de unge. Tidligere studier af hvordan medi-
erne gennem tiden har behandlet „de ældre“, viser, at journali-
stikken betjener sig af stereotype fremstillinger af sociale grup-
per, men også at disse fremstillinger forandrer sig i takt med
tiden (Wien 2005). Med dette udgangspunkt undersøger vi her i artiklen hvordan
danske medier har repræsenteret ungdommen gennem 50 år, og
vil svare på fire spørgsmål, nemlig: • I hvilket omfang indeholder nyhedshistorier om unge hhv. Indledning negative og positive repræsentationer af unge? • Hvilke emner er de foretrukne i nyhedsmediernes dækning af
historier om unge? • Er der forskelle i mediernes dækning af hhv. unge mænd og
unge kvinder? • Hvilken rolle spiller de unge selv i nyhederne? / JOURNALISTICA · NR. 1 · 2010 40 I de følgende afsnit præsenteres først artiklens teoretiske ud-
gangspunkt samt en gennemgang af den tidligere forskning på
området. Herefter følger overvejelser vedrørende data og metode
samt de empiriske analyser. Medierepræsentationer Talrige studier har vist, at medierne påvirker samfundet: Lige fra
den førte politik, vores adfærd og menings- og holdningsdan-
nelser (fx Gamson and Modigliani 1989; Donovan and Leivers
1993; Hurtz and Durkin 2004; Wyn 2005; Paterson 2006; Slater
2007; Bleakney, Hennessy et al. 2008) til vores sprog (Gorham
2006). Og netop derfor kan longitudinale studier af mediernes
repræsentationer af særlige grupper i samfundet tilvejebringe vi-
den om betydningen af strukturelle ændringer i samfundsorde-
nen samt om samfundets udvikling. Begrebet medierepræsentation refererer altså til den måde
hvorpå forskellige grupper baseret på fx alder, køn eller race be-
skrives i medierne og er dermed nært beslægtet med både stu-
diet af nyhedsmediernes fortællemæssige rammer (frames) og
studiet af stereotyper. Reelt mener vi ikke, at der kan lægges et
klart definitorisk snit mellem de tre koncepter: Frames blev op-
rindeligt defineret som “schemata of interpretation” (Goffman
1974:21). Begrebet “schemata” er blevet brugt af socialpsykologer
til at beskrive de kognitive kategorier, som vi bruger, når vi skal
organisere vores hukommelse og kategorisere informationer
(Stangor 2000:6; Tajfel and Forgas 2000:50). Og tilsvarende defi-
nerede Gitlin “media frames” som “persistent patterns of cogni-
tion, by which symbol-handlers routinely organize discourse,
whether verbal or textual” (Gitlin 1980:7).1 At Gitlin anskuede mediernes framing som vedvarende (per-
sistent) kognitive mønstre gør slægtskabet med social psykolo-
gernes stereotyper tydeligt: Ifølge Stangor (Stangor 2000:13) be-
tragtes stereotyper generelt som uforanderlige forestillinger. Men
forskningen heri har vist, at de rent faktisk ændrer sig over tid (fx
(Donovan and Leivers 1993; Devine and Elliott 2000; Wien 2005;
Covert and Dixon 2008)). Med andre ord: Stereotyper er ikke ufor-
anderlige mønstre. Samme konklusion er nået af forskere, der har JOURNALISTICA · NR. 1 · 2010 // 41 41 arbejdet med longitudinale studier af mediernes framing af sær-
lige emner (fx. (Semetko and Valkenburg 2000; Schulz and Zeh
2005; Scheufele and Tewksbury 2007)).2 På samme måde inkluderer studier af medierepræsentationer
ofte analyser af stereotyper (fx (Porteous and Colston 1980; Au-
brun and Grady 2000; Devlin 2000; Heintz-Knowles 2000; Maz-
zarella 2003; Dixon and Maddox 2005; Wyn 2005; Covert and
Dixon 2008).3 I sådanne studier inkluderes også typisk analyser af
kildebrug, mediernes prioritering af et emne, genrer og et antal
kvantitative mål som længde, illustrationer og personlige karak-
teristika (fx køn) af personer omtalt i de enkelte nyhedshistorier
(Porteous and Colston 1980; Falchikov 1986; Devlin 2000; McMa-
nus 2002; Mazzarella 2003; Wyn 2005). Medierepræsentationer Sammenfattende mener vi, at studiet af framing, studiet af
stereotyper og studiet af medierepræsentationer er så nært be-
slægtede, at vi kan hente inspiration til vores analyse fra litteratur
fra alle tre forskningsområder. Men, før vi når så vidt vil vi op-
summere, hvad den tidligere forskning i mediernes repræsenta-
tioner, stereotyper og framing af ungdommen har vist. Tidligere forskning i mediernes repræsentationer af unge Der foreligger som nævnt allerede et antal undersøgelser, der be-
lyser hvordan medierne repræsenterer unge mennesker. Aubrun and Gradys (2000) undersøgte forskellene på de lokale
og nationale amerikanske nyhedsmediers dækning af unge. De-
res undersøgelse viste, at de lokale medier er mere tilbøjelige til
at benytte negative og forsimplede stereotyper om unge. Der er
dog stor forskel på, hvilken vinkel medierne anlægger: De lokale
medier har en tendens til at indtage en slags tilskuerrolle, hvor
det er vanskelligt for læsere, lyttere eller seere at involvere sig fø-
lelsesmæssigt. Det gør de gennem hurtigt klippede øjenvidne-
agtige nyheder, hvor der fokuseres på unge som ofre for eller
årsagen til ulykker eller kriminalitet. De nationale nyhedsmedier
er derimod mere tilbøjelige til at præsentere nyheder om unge
inden for en tematisk ramme, der rummer såvel problemernes
kontekst og proportioner, samt forslag til løsninger (Aubrun and
Grady 2000:2-5). / JOURNALISTICA · NR. 1 · 2010 42 Heintz-Knowles (2000) har undersøgt hvordan unge blev
fremstillet i amerikanske prime-time TV-serier, og resultaterne
viste, at man som seer oftere blev præsenteret for unge kvinder
end for unge mænd, og at der oftere var tale om hvide end om
nogen anden race. Emnemæssigt var fokus på kærlighed, ven-
skab og familie og på emner som narkotika, mens alkohol og
kriminalitet kun meget sjældent indgik. Alligevel blev de unge of-
test beskrevet som årsagen til et eller andet problem, som „de
voksne“ skulle ordne (Heintz-Knowles 2000:6). Hendes resultater
adskiller sig dog markant fra de undersøgelser der er foretaget af
nyhedsmediernes dækning af de unge. Nichols and Good (2004) undersøger, hvordan mediernes
dækning af vold, sex, tobak, alkohol, narkotika osv. har ændret
sig over tid. I den forbindelse diskuterer de også mediernes re-
præsentationer af unge i sådanne historier. De mener, at ame-
rikanernes holdning til de unge bliver mere og mere negativ
(Nichols & Good 2004:5), og forklarer årsagerne hertil med en
kombination af ændringer i familiemønstre og øget medieindfly-
delse. I deres perspektiv er det især underholdnings- og nyheds-
medierne, der bærer skylden, fordi der er en skævvredet dæk-
ning med alt for stort fokus på negative historier om børn og
unge. Ifølge dem er det den hastige vækst på medieområdet
gennem de seneste 50 år, der er årsagen til, at medierne leverer
en nyhedsdækning, hvor fokus er på historier, der involverer
unge som ofre for ulykker eller kriminalitet eller som årsagen der-
til. Tidligere forskning i mediernes repræsentationer af unge Ifølge dem er det det, der får mediepublikummet til at “[see]
youth in problematic terms”(Nichols & Good 2004:9). Deres arbejde er interessant i vores sammenhæng fordi de når
frem til et antal hypoteser, hvoraf vi gerne vil teste to: Den første
er, at nyhedshistorier om unge overvejende er negative, og den
anden er, at denne tendens forstærkes over tid. Amundson, Lichter et al. fokuserer ligesom Aubrun and Grady
(Aubrun and Grady 2000) på forskelle mellem nationale og lokale
nyhedsmedier. De konkluderer, at “local news stories about youth
tend to treat adolescence as a source of personal problems and so-
cial disruptions that need to be addressed by adults”. De hævder
videre, at “the operant principle among the journalists seemed to
be “if it bleeds it leads”[…].”, når nyheder handler om unge JOURNALISTICA · NR. 1 · 2010 // 43 (Amundson, Lichter et al. 2005:25). Den eneste formildende om-
stændighed i nyhedsmediernes dækning af unge er ifølge dem, at
nationale nyhedsmedier prioriterer emner relateret til uddan-
nelse højt, men at de så til gengæld har en langt mindre intensiv
dækning af de unge end af andre grupper i samfundet. Sammenfattende kan man sige, at mediernes repræsentatio-
ner af unge mennesker varierer afhængigt af genre, medie og
mediets dækning (lokal eller national). Da vi her ønsker at dække
en 50-årig periode i Danmark, vælger vi at fokusere på aviserne,
fordi disse som de eneste medier er systematisk registreret og ar-
kiveret over en tilstrækkelig lang periode. Dermed er det også re-
levant at se nærmere på den del af forskningen, som hovedsa-
gelig har baseret sig på avisernes fremstilling af de unge. Stensaas (1961) analyserer en række amerikanske aviser og
konkluderer, at nyhedshistorier om unge mennesker generelt er
negative og sætter de unge i et dårligt lys. Eneste undtagelse fra
dette mønster er ifølge Stensaas, at positive historier om unge
mennesker til gengæld oftest at finde på forsiderne. Tilsvarende analyserer Porteous og Colston (1980) en række
britiske aviser og finder, at de foretrukne emner for nyhedshisto-
rier om unge er sport eller kriminalitet og at de unge sædvanlig-
vis bliver fremstillet som ofre for – eller årsagen til – ulykker og
ulovligheder. Tidligere forskning i mediernes repræsentationer af unge Porteous og Colstons undersøgelse er ikke i sig selv
et longitudinalt studium, men da Falchikov (1986) efterfølgende
i 1985 foretog en tilsvarende undersøgelse viste det sig, at der i de
mellemliggende fem år var sket en række ændringer i mediernes
beskrivelser af de unge. I 1985 var de fire foretrukne emner om
unge i den skrevne presse: De unge som ofre, kriminalitet, sport
eller arbejdsløshed. Falchikovs undersøgelse viste dermed, at det
er muligt at spore mindre ændringer i mediedækningen af en
social gruppe – fx de unge – over blot en femårig periode. Siden årtusindskiftet er der foretaget yderligere tre undersø-
gelser af mediedækningen af de unge: En irsk undersøgelse viser,
at hovedparten af de irske nyhedshistorier portrætterer unge
mennesker som enten “being a problem or as having problems”:
Næsten 85 % af nyhedshistorierne om unge handler om de unge
som årsag til eller ofre for kriminalitet og vold, eller beskriver
unge som en gruppe, der er særligt udsat for skadelige påvirk- // JOURNALISTICA · NR. 1 · 2010 44 ninger (Devlin 2000:47). Ifølge denne undersøgelse stemmer me-
diedækningen godt overnes med en generel samfundsmæssig
tendens til at præsentere unge mennesker som “[…] either ac-
tively “deviant” or passively “at risk”, and sometimes both simul-
taneously” (Devlin 2000, citing Griffin 1997:17-18). McManus (2002) foretager en indholdsanalyse af en række
californiske aviser og deres dækning af unge og vold. Han kon-
kluderer, at der er signifikante forskelle i hvordan medierne
beskriver og repræsenterer de unge i rutinejournalistikken og i
efterspillet efter signifikante begivenheder og deraf følgende fo-
kusjournalistik (Lund 2002:9). McManus illustrerer denne pointe
ved hjælp af en analyse af mediedækningen efter Columbine
High-massakren. Han viser, at journalisterne i forbindelse med
fokusjournalistik i højere grad benytter en tematisk framing end
en episodisk, og at de viser større villighed til at diskutere proble-
mets årsag, oprindelse og mulige løsninger på det end i forbin-
delse med rutinejournalistikken. Tilsvarende viser hans analyse
af rutinejournalistikken, at den fokuserer langt mindre på årsager
og løsninger end på de problemer, der beskrives. Han konklude-
rer derfor, at der er meget stor kvalitetsforskel på fokusjournali-
stikkens og rutinejournalistikkens nyhedsdækning af de unge. Mediedækningen af de unge er senest blevet undersøgt af
Wyn (2005). Hun sammenligner mediernes repræsentationer af
unge i USA, Australien, New Zealand og Storbritannien baseret
på en række udvalgte aviser og TV-stationers nyhedsdækning. Tidligere forskning i mediernes repræsentationer af unge Hun konkluderer, at mediernes repræsentationer af de unge sy-
nes at afspejle to konkurrerende diskurser: På den ene side be-
skrives unge som en trussel mod samfundsordenen, og på den
anden side beskrives de unge som en kilde til håb for fremtiden
(Wyn 2005:31-32). Sammenfattende viser den tidligere forskning, at mediernes
repræsentationer af unge mennesker varierer afhængigt af genre,
medie og mediets dækning (lokal eller national). Men den viser
imidlertid også, at det er de negative emner og repræsentationer
af ungdommen som dominerer. JOURNALISTICA · NR. 1 · 2010 // 45 Metode At undersøge udviklingen i mediebilledet af de unge over 50 år i
alle danske mediers samlede dækning er en ganske uoverkom-
melig opgave. Det er derfor nødvendigt at udvælge enkelte ny-
hedsmedier. Eftersom der ikke var etableret regionale eller lokale elektro-
niske medier i Danmark for 50 år siden, og eftersom der ikke fin-
des fuldstændige arkiver over de elektroniske mediers nyheds-
dækning gennem de seneste 50 år, må undersøgelsen – som
nævnt – basere sig på de skrevne nyhedsmedier. Aviserne er placeret før de elektroniske medier i den journali-
stiske fødekæde: TV har med andre ord tendens til at bringe de
samme nyheder, som har været bragt i aviserne (Lund 2000:145). Resultaterne i denne undersøgelse kan derfor i et vist omfang
forventes også at gøre sig gældende for fremstillingen af unge i
f.eks. nyhederne på TV. Gennem de seneste 50 år har der eksisteret et utal af dagblade
på regionalt, lokalt og nationalt niveau. Det er derfor nødvendigt
at afgrænse empirivalget yderligere. For det første er det nødven-
digt, at de analyserede dagblade har eksisteret i hele undersøgel-
sesperioden. For andet har vi ønsket at sikre, at vores materiale
omfattede både større dagblade, tabloidaviser, lokalaviser, regio-
nalaviser og landsdækkende aviser. Følgende aviser er valgt for at
give så stor spredning som muligt: • Politiken • Kristeligt Dagblad
• B.T. • Fyens Stiftstidende • Næstved Tidende Men selv med blot fem aviser er det en uoverkommelig opgave at
analysere alle redaktionelle enheder gennem 50 år. Derfor er der
foretaget en række punktnedslag, som begrænser datamængden. En del af den danske og internationale forskning i journalistik
baserer sig på den såkaldte nyhedsuge (Lund 2000; Petersen
2003): Uge 46. Denne uge er valgt, fordi den i nyhedsmæssig sam-
menhæng skønnes at være en gennemsnitlig uge.4 Men selv med blot fem aviser er det en uoverkommelig opgave at
analysere alle redaktionelle enheder gennem 50 år. Derfor er der
foretaget en række punktnedslag, som begrænser datamængden. En del af den danske og internationale forskning i journalistik
baserer sig på den såkaldte nyhedsuge (Lund 2000; Petersen
2003): Uge 46. Denne uge er valgt, fordi den i nyhedsmæssig sam-
menhæng skønnes at være en gennemsnitlig uge.4 JOURNALISTICA · NR. 1 · 2010 46 Da analysen sigter på at identificere udviklingen i mediernes
repræsentationer af de unge over tid, er analysen samtidig be-
grænset til seks punktnedslagsår: 1953, 1963, 1973, 1983, 1993 og
2003. Metode Hver enkelt avis blev gennemlæst af en forskningsassistent,
der udvalgte alle artikler, som handlede om unge. Der blev an-
vendt en kombination af to udvalgskriterier: Alle artikler, der en-
ten omtalte mennesker mellem 14-24 år5 eller rummede ord som
unge, ungdom, teenager eller andre synonymer herfor, blev ud-
valgt. I alt blev 1174 artikler udvalgt.6 Vores kodeark omfatter dels en række objektive baggrundsva-
riable som dato, placering, længde og kildebrug. Desuden har vi
kodet for emne, ud fra polinfos emneordsliste.7 Endelig omfatter
kodearket en variabel, som er udviklet specielt til denne under-
søgelse: En gennemlæsning af materialet afslørede, at det var re-
lativt let at identificere om artiklernes overordnede fortællemæs-
sige ramme var positiv eller negativ overfor de unge. Vi beslut-
tede derfor at kode artikler, der beskrev unge som en ressource,
som socialt engagerede, sympatiske, smukke, pligtopfyldende,
samvittighedsfulde e.l. som „positive“, mens artikler, der beskrev
de unge som utilpassede, passive, kriminelle, aggressive, fjendt-
lige eller som en trussel mod samfundsordenen e.l. som „nega-
tive“.8 Tre assistenter kodede materialet og tastede alle data ind i en
SPSS datafil.9 En beregning af Krippendorf’s alpha for samtlige
variable viste et resultat, der svingede mellem 0.82 til 1.0 og er så-
ledes inden for et acceptabelt reliabilitetsniveau. Tabel 1 1953
1963
1973
1983
1993
2003
Alle år
Politiken
35
32
52
38
50
58
265
B.T. 33
36
58
38
40
59
264
Næstved
Tidende
30
36
56
37
31
28
218
Kr. Dagblad
19
22
40
15
13
25
134
Fyns
Stiftstidende
31
28
64
39
57
74
293
N
148
154
270
167
191
244
1174 I vores materiale findes det største antal artikler om unge i 1973. Dette tal mener vi at kunne forklare som et resultat af ung-
domsoprøret og dets efterspil: Medierne synes ganske enkelt at
have rettet et stigende fokus mod de unge og at have givet dem
en stigende prioritering i deres nyhedsdækning. Samtlige aviser har nogenlunde lige mange artikler om unge
over hele perioden (218-293) eneste undtagelse herfra er Kr. Dag-
blad med 134 artikler. Dette resultat er ikke overraskende efter-
som avisen henvender sig til et ældre publikum, og eftersom den
rummer færre og længere artikler end de øvrige. Resultater Det samlede antal artikler i de udvalgte aviser er gengivet i tabel
1. Over årene 1953-1973 fandt vi en bemærkelsesværdig stigning
på 80 % i antallet af artikler om unge. Vi skal dog i den forbindelse
bemærke, at der generelt er sket en stigning i antallet af artikler i
aviserne og at stigningen i antallet af artikler om unge derfor ikke
nødvendigvis afspejler, at den samlede andel af artikler om ung-
dommen er vokset. JOURNALISTICA · NR. 1 · 2010 // 47 Stigende negativ bias? Vores data kan ikke bekræfte en stigende tendens i retning af et
stadigt mere negativt mediebillede af de unge, sådan som den
tidligere forskning på området har tydet på. Som det fremgår af
tabel 2, er der snarere tale om, at medierne i stigende omfang
balancerer mellem positive, negative og neutrale repræsentatio-
ner af ungdommen. Faktisk er antallet af „neutrale“ repræsen-
tationer af de unge signifikant højere fra 1983-2003 end i perioden
før. Der er formentlig flere årsager hertil: Dels medførte partipres-
sens sammenbrud i slutningen af 1960erne at aviserne i højere og
højere grad har stræbt mod en neutral dækning af nyhederne, / JOURNALISTICA · NR. 1 · 2010 48 Tabel 2
Negative og positive beskrivelser af de unge
1953
1963
1973
1983
1993
2003
Alle år
Positive
83
86
127
29
55
78
458
56,1%
55,8%
47,0%
17,4%
28,8%
32,0%
39,0%
Negative
38
37
90
69
66
78
378
25,7%
24,0%
33,3%
41,3%
34,6%
32,0%
32,2%
Neutrale
27
31
52
69
70
88
337
18,2%
20,1%
19,3%
41,3%
36,6%
36,1%
28,7%
N
148
154
270
167
191
244
1174 Tabel 2
Negative og positive beskrivelser af de unge dels er journalister og journalistikken blevet stadig mere og mere
professionaliseret gennem perioden. Ifølge hypotesen om den øgede kommercialisering af medi-
erne ville vi desuden have forventet, at tabloidavisen havde den
højeste andel af negative bias i artikler om unge. Men en nær-
mere analyse af tallene afslører, at det rent faktisk er den regio-
nale avis, der er mest negativ. Dette resultat er interessant af to
årsager: Den første er, at dette resultat dermed bekræfter de tidli-
gere undersøgelser, der viste, at lokale medier er mere tilbøjelige
til at beskrive unge mennesker negativt (Aubrun and Grady 2000;
Amundson, Lichter et al. 2005). Den anden er, at undersøgelsen
af ældrebilledet i medierne viste, at når det drejede sig om be-
skrivelserne af ældre mennesker var regionalavisen langt mere
positiv end de landsdækkende aviser (Wien, 2005). Tidligere forskning i mediestereotyper har påvist signifikante
forskelle i den måde unge mænd og unge kvinder blev beskrevet
i medierne og i hyppigheden af hvor ofte de to køn var repræsen-
teret. (Falchikov 1986; Devlin 2000; Mazzarella 2003). Vores data
bekræfter også disse forskelle: Mere end en tredjedel af artiklerne
handlede udelukkende om unge mænd og det gælder for alle år
jfr. tabel 4. Tabel 3 1953
1963
1973
1983
1993
2003
Alle år
Kriminalitet
(retsvæsen)
23
19
22
30
28
16
22
Kultur
24
34
9
9
13
16
16
Sport
8
10
14
11
7
19
12
Uddannelse
8
9
14
14
12
8
11
Landspolitik
3
2
9
7
17
5
8
Socialforhold
4
6
9
3
5
9
6
Etik, filosofi
og religion
5
3
4
4
2
2
3
Ulykker
8
4
2
1
3
2
3
Arbejds-
marked
8
4
2
1
3
2
3
Samliv
2
3
1
7
1
3
3
Udlandet
1
2
4
1
3
3
3
Sundhed
2
1
3
4
2
3
3
Andet
7
5
6
7
5
11
7
N
148
154
270
167
191
244
1174 sitive og negative historier om unge fordelt på køn. Materia-
let afslører, at der er en klar tendens til, at de negative historier
handler om unge mænd, mens de positive historier er domineret
af kvinder. Vi finder en tilsvarende kønsskævhed når vi krydstabulerer
køn med citater (tabellen er ikke medtaget): Unge mænd citeres
meget hyppigere end unge kvinder. Stigende negativ bias? Vi ser dog tegn i retning af en ændring i dette mønster:
I 1953-1993 var kvinder kun repræsenteret i 12-15 % af artiklerne, i
2003 gjaldt det for 22 % af artiklerne. Vores data bekræfter også, at der er forskel på hvilken måde
medierne omtaler de to køn: Tabel 5 viser fordelingen af hhv. po- JOURNALISTICA · NR. 1 · 2010 // 49 49 Tabel 3
Klassifikation af artikler om unge efter emne pct. Hvilke emner? Som allerede omtalt viste Porteous og Colston (1980) og Falchikov
(1986), at de dominerende emner i medierne vedrørende unge
var kriminalitet, sport, arbejdsløshed og unge som ofre. Vores
materiale viser et lidt anderledes billede, sådan som det ses i ta- // JOURNALISTICA · NR. 1 · 2010 50 Tabel 4 1953
1963
1973
1983
1993
2003
Alle år
Mand/mænd
66
52
103
65
83
82
451
44,6%
33,8%
38,1%
38,9%
43,5%
33,6%
38,4%
Kvinde/kvinder
21
23
33
26
29
52
184
14,2%
14,9%
12,2%
15,6%
15,2%
21,3%
15,7%
Begge køn
12
14
40
13
12
24
115
8,1%
9,1%
14,8%
7,8%
6,3%
9,8%
9,8%
Ikke omtalt
49
65
94
63
67
86
424
33,1%
42,2%
34,8%
37,7%
35,1%
35,2%
36,1%
N
148
154
270
167
191
244
1174 bel 3. Her har vi klassificeret de enkelte artikler i et antal katego-
rier inddelt efter emne. Den hyppigst forekommende kategori i vores materiale er kri-
minalitet (22 % af alle artikler). Men både i 1953, 1963 og 2003 er
der en ligeså stor eller større andel af artikler, der handler om
kultur. Blandt de øvrige fremtrædende kategorier er „kultur“ (16
%), „sport“ (12 %) og „uddannelse“ (11 %). Og lægger man ande-
lene af artikler, der handler om kultur, sport og uddannelse sam-
men udgør artikler om disse emner en langt større andel end
andelen af artikler om kriminalitet. Vores undersøgelse kan heller ikke bekræfte, at flere og flere hi-
storier om unge handler om ulykker: Her er kun tale om en lille
andel af artikler, som over perioden viser en faldende tendens. I forhold til den tidligere forskning på området adskiller vores
resultater sig således markant: Vi finder ikke, at historier om
unge i medierne hovedsageligt handler om kriminalitet og ulyk-
ker. Tvært i mod: Mediernes foretrukne emner, når det handler
om de unge er kultur, sport og uddannelse. Hvilken rolle spiller de unge? Forskningslitteraturen om mediernes stereotypificering lægger
vægt på en tendens til at beskrive sociale grupper og gruppead-
færd snarere end at beskrive individer. Dette gælder særligt, når JOURNALISTICA · NR. 1 · 2010 // 51 51 Tabel 5 Mand/mænd
Kvinde/kvinder
Begge køn
NA
Total
Positiv
170
107
57
124
458
37,7%
58,2%
49,6%
29,2%
39,0%
Negativ
223
32
26
97
378
49,4%
17,4%
22,6%
22,9%
32,2%
Neutral
58
45
32
202
337
12,9%
24,5%
27,8%
47,6%
28,7%
N
451
184
115
424
1174 Mand/mænd
Kvinde/kvinder
Begge køn
NA
Total man taler om „ungdommen“ som en særlig gruppe med særlige
sociale og kulturelle karakteristika: Da man begyndte at forske i
børn og unge i begyndelsen af det 20ende århundrede og da
moralsk panik over ungdomskulturen blev et centralt emne for
både den offentlige og den politiske debat blev „ungdommen“ en
særlig gruppe. I medierne blev unge mennesker – og især unge
mænd – beskrevet som tilhørende grupper, der forstyrrede den
offentlige orden fx som medlemmer af (kriminelle) bander, eller
som nogen, der introducerede særlige nye kulturer båret fx af
musik (Thompson 1998; Welch 2002). For at undersøge i hvilket omfang unge mennesker beskrives
som tilhørende en generel gruppe („ungdommen“) eller en som
tilhørende særlige ungdomsgrupper („læderjakker“, „punkere“)
eller som individer, har vi kodet materialet i tre forskellige kate-
gorier efter hvordan de unge omtales. Tabel 6 viser, at artikler, hvor unge mennesker portrætteres
som gruppemedlemmer, var hyppigst i 1953 (n=53), 1963 (n=62)
og 1973 (n=112). Den næsten modsatte tendens gælder avisernes
beskrivelser af de unge med en samlebetegnelse som fx „ung-
dommen“. En fjerdedel af artiklerne fra 1983, 1993 og 2003 inde-
holdt en samlebetegnelse for de unge. Når man krydstabulerer
dette med de enkelte artiklers emne viser det sig, at 25-30 % af
disse artikler havde de dominerende emner: sport, kriminalitet
og kultur. Det var altså disse artikler, som havde gruppeperspek-
tivet og ikke de negative historier om kriminalitet og ulykker. // JOURNALISTICA · NR. 1 · 2010 52 Tabel 6
Unge som individer eller som gruppemedlemmer? 1953
1963
1973
1983
1993
2003
Alle år
Individ
(fx navngiven
person)
71
60
104
95
111
128
569
48,0%
39,0%
38,5%
56,9%
58,1%
52,5%
48,5%
Gruppe
(fx „læder-
jakker“)
53
62
112
8
25
34
294
35,8%
40,3%
41,5%
4,8%
13,1%
13,9%
25,0%
Samle-
betegnelse (fx
„ungdommen“)
23
27
35
45
43
60
233
15,5%
17,5%
13,0%
26,9%
22,5%
24,6%
19,8%
Ved ikke
1
5
19
19
12
22
78
0,7%
3,2%
7,0%
11,4%
6,3%
9,0%
6,6%
N
148
154
270
167
191
244
1174 Tabel 6
Unge som individer eller som gruppemedlemmer? Vores data viser altså, at ca. Tabel 5 halvdelen af de artikler, der beskri-
ver unge mennesker, beskriver dem som individer. Derfor mener
vi ikke det er rimeligt at hævde, at unge hovedsageligt fremstil-
les i grove og generaliserende gruppebetegnelser. I Grossbergs analyse af politisk ungdomskultur kalder han
ungdommen for “the most silenced generation” (Grossberg 1994,
citet hos Mazzarella 2003: 236). Når unge mennesker beskrives i
medierne er det – ifølge ham – oftere ved hjælp af citater fra eks-
perter end de unge selv. Problemet ved det er iflg. Grossberg, at
når sådanne eksperter som psykologer, sociologier og læger får
ordet har de en tendens til at generalisere enkeltstående proble-
matiske episoder til generelle sociale problemer. Når de unge
ikke får lov til at tale for sig selv, bliver de “also subjected to me-
dia’s attempts to categorize them” (Mazzarella 2003:236). Vores
data giver os mulighed for at undersøge, om dette pessimistiske
postulat faktisk stemmer overens med virkeligheden. Vores analyse af hvor ofte de unge selv udtaler sig viser, som det
fremgår af tabel 7, at kun en lille del af artiklerne indeholder di-
rekte citater af unge mennesker. Men når vi ser på udviklingen JOURNALISTICA · NR. 1 · 2010 // 53 53 Tabel 7 1953
1963
1973
1983
1993
2003
Alle år
Citeret
8
22
39
38
49
57
213
5,4%
14,3%
14,4%
22,8%
25,7%
23,4%
18,1%
Ikke
citeret
140
131
230
129
141
187
958
94,6%
85,1%
85,2%
77,2%
73,8%
76,6%
81,6%
Ved ikke
0
1
1
0
1
0
3
,0%
,6%
,4%
,0%
,5%
,0%
,3%
N
148
154
270
167
191
244
1174 over tid, så viser det sig, at der faktisk bliver flere – både i andele
og i procent – hvor unge citeres direkte. I 1953 var det således kun i 5 % af artiklerne. I 1963 og 1973 var
det 14 % og i 1983, 1993 og 2003 var dette steget til 23-26 %. Med an-
dre ord tyder vores undersøgelse ikke på, at der er en stigende
tendens til at gøre ungdommen til en “silenced generation” i me-
dierne. Vi er dog ikke gået dybere ned i analysen af på hvilken
måde og til hvad journalisterne bruger citaterne af de unge og
om denne stigning i direkte citater er større end for andre grup-
per i samfundet. En forklaring herpå kunne jo netop være at jour-
nalistisk praksis har ændret sig. Vores analyse omfatter også brugen af eksperter i artiklerne. Analysen viser, at kun i 5 % af artiklerne fra 1953 optrådte der en
ekspert. I resten af årene fra 1963-2003 varierer dette mellem 12-
17 %. En mulig forklaring på dette er den stigende professionali-
sering af de forskellige sektorer i velfærdsstaten, som har børn og
unge som deres ansvarsområde. Så dermed ser vi i lighed med
Grossberg at eksperter inddrages i stigende omfang, men vi er
dog knapt så pessimistiske som han, da vi parallelt hermed ser,
at de unge i stigende omfang også selv kommer til orde. // JOURNALISTICA · NR. 1 · 2010 54 Konkluderende bemærkninger Sammenfattende kan vi sige, at vores undersøgelse ikke kan be-
kræfte, at der er en dominerende negativ bias i mediernes beskri-
velser af de unge. Dette kunne hverken bekræftes i forhold til
undersøgelsen af nutidens mediedækning (2003) eller i under-
søgelsen af langtidsperspektivet herpå (1953-2003) Tvært i mod
afslører vores data en udvikling i retning af flere og flere neutrale
beskrivelser af unge. Vores undersøgelse viser, at „kriminalitet“, „kultur“ og „sport“
er de emner, der hyppigst er i spil, i artikler om unge. Vores ma-
teriale tyder således også her på en afvigelse i forhold til den tid-
ligere forskning på området. Når vi ser nærmere på, hvilken rolle de unge spiller i nyheds-
dækningen finder vi en tendens til, at medierne citerer de unge
direkte. Denne tendens synes at være stærkest i årene efter ung-
domsoprøret (1973 og frem). Desuden er der nogle interessante
forskelle i avisernes repræsentationer af unge mænd og kvinder:
Vores undersøgelse tyder på, at unge kvinder spiller en stigende
rolle i medierne. Men krydstabuleringer med øvrige variable i
materialet tyder dog fortsat på, at beskrivelserne af de unge
mænd er langt mere varierede, selvom unge mænd dog har den
tvivlsomme ære af overvejende at blive beskrevet i negative hi-
storier. På dette tidspunkt kan vi ikke tilbyde udtømmende forklarin-
ger på disse observationer eller på forskellene mellem vores data
og den tidligere forskning. Vi vil dog tillade os at fremsætte en
række mulige forklaringer til diskussion. En mulig forklaring på afvigelserne er forskelle i dataindsam-
lingsteknikkerne: Vores data er indsamlet ved hjælp af stratifice-
rede tilfældigt udvalgte stikprøver over en 50-årig periode. Hvert
enkelt punktnedslag dækker dog en forholdsvis kort periode på
kun en uge. Den tidligere forskning på området er baseret på
stikprøver, der er udtaget over længere perioder (to til fire uger). Vi må derfor erkende, at faren for at de udsving vi ser, er tilfældige
er relativt stor. Vi imødegår den fare efter bedste evne ved ude-
lukkende at tolke meget tydelige tendenser, som strækker sig
over flere punktnedslag. JOURNALISTICA · NR. 1 · 2010 // 5 55 Yderligere indsamlede Porteous og Colston (1980) og Falchikov
(1986) deres materiale over hhv. en måned i Juni (1979) og to uger
i oktober (1984). Ingen af dem præsenterer nogen forklaring på
hvorfor netop disse perioder er valgt. Konkluderende bemærkninger Der kan argumenteres for,
at det er yderst problematisk at indsamle datamateriale til analy-
ser af mediedækningen i sommerperioden, hvis man ønsker at
generalisere resultaterne til resten af året, sådan som Porteous og
Colston gør. Og der kan yderligere argumenteres for, at det er pro-
blematisk at sammenligne data indsamlet over sommerperioden
med data indsamlet i efteråret, sådan som Falchikov gør. Vores
data er indsamlet på samme tid af året i alle seks punktnedslag. Det er også muligt, at forskelle i mediestystemerne spiller en
rolle: Både Porteous og Colston (1980) og Falchikov (1986) ind-
samlede deres materiale i en britisk kontekst. Det vil ifølge Hal-
lin og Mancini (2004) sige, at data er indsamlet fra nyhedsmedier
under „den liberale model“. Vores data er indsamlet fra danske
aviser, som kan siges at tilhøre „den demokratiske korporativi-
stiske model“, hvor såvel det politiske system som markedsme-
kanismerne er forskellige. Yderligere kan forskelle i graden af ta-
bloidisering mellem danske og britiske medier forklare en del af
forskellen (Esser 1999). N O T E R *
Vi ønsker at især at takke studentermedhjælperne Therese Dyrby,
Julie Nørrelund, Bo Jessen, Kristian D. Knudsen og Saba Khalaj for
assistance i firbindelse med dataindsamling og kodning, samt vore
kolleger Christian Elmelund-Præstekær og David Nicolas Hopmann
for hjælp og nyttige kommentarer til tidligere versioner af artiklen. 1. Herved bliver det nære slægtskab mellem medieforskningen i fra-
ming og social psykologernes arbejde med stereotyper klart: Eksem-
pelvis hævder Stangor og Schaller (Stangor and Schaller 2000:64), at
stereotyper er det materiale som samfundet er vævet af: Stereoty-
perne deles af individerne inden for en given kulturkreds. De udgør
en slags fælles informationsbase som alle i kulturkredsen refererer til
(Stangor and Schaller 2000:68). Tilsvarende hævder Hinton, at ste- // JOURNALISTICA · NR. 1 · 2010 56 reotyper er en slags kognitive mønstre, som gør tænkning “quick and
easy” (Hinton 2000:22-23). 2. Vi finder yderligere støtte for at studiet af framing og af stereotyper er
nært beslægtede hos Entmann, der i hans hyppigt citerede artikel fra
1993 oplister fem faktorer, som han mener, bør indgå i analyser af me-
diernes fortællemæssige rammer. En af disse er stereotyper (Entman
1993:53). Entman definerer stereotype som ‘mentally stored clusters
of ideas that guide the individuals’ processing of information’ (Ent-
man 1993:53). De fire øvrige kategorier er “certain keywords, stock
phrases, stereotyped images, sources of information and sentences
that provide thematically reinforcing clusters of facts or judgements”
(Entman 1993:52) 3. I nogle af undersøgelserne bruges begrebet ‘image’ som synonym for
stereotype (e.g. Porteous and Colston 1980; Falchikov 1986; Wyn 2005) 3. I nogle af undersøgelserne bruges begrebet ‘image’ som synonym for
stereotype (e.g. Porteous and Colston 1980; Falchikov 1986; Wyn 2005) 4. Det skyldes en antagelse om, at der ikke er særlige årligt tilbageven-
dende begivenheder i den uge, som kan påvirke nyhedsstrømmen. Valget af nyhedsugen er imidlertid ikke uproblematisk. For det før-
ste er antagelsen om gennemsnitlighed stort set ubegrundet. For det
andet indeholder brugen af nyhedsugen den ubegrundede og ofte
usagte antagelse, at man ved analyse af én uge kan udtale sig gene-
relt om alle uger i det pågældende år. Alligevel anvendes „nyheds-
ugen“ i dette projekt i mangel af bedre. Det skal dog bemærkes, at
også andre perioder er blevet anvendt i beslægtede undersøgelser. Pedersen & Horst (2000) anvender perioden fra den 1.-7. februar i tre
punktnedslagsår for at dække udviklingen i den politiske journalistik
fra 1958 til 1998. Albæk et al. N O T E R (2002) anvender 15. januar til 14. februar
i tiårsintervaller i en analyse af dagbladendes brug af ekspertkilder. 4. Det skyldes en antagelse om, at der ikke er særlige årligt tilbageven-
dende begivenheder i den uge, som kan påvirke nyhedsstrømmen. Valget af nyhedsugen er imidlertid ikke uproblematisk. For det før-
ste er antagelsen om gennemsnitlighed stort set ubegrundet. For det
andet indeholder brugen af nyhedsugen den ubegrundede og ofte
usagte antagelse, at man ved analyse af én uge kan udtale sig gene-
relt om alle uger i det pågældende år. Alligevel anvendes „nyheds-
ugen“ i dette projekt i mangel af bedre. Det skal dog bemærkes, at
også andre perioder er blevet anvendt i beslægtede undersøgelser. Pedersen & Horst (2000) anvender perioden fra den 1.-7. februar i tre
punktnedslagsår for at dække udviklingen i den politiske journalistik
fra 1958 til 1998. Albæk et al. (2002) anvender 15. januar til 14. februar
i tiårsintervaller i en analyse af dagbladendes brug af ekspertkilder. 5. Svarende til FNs definition af unge. http://www.un.org/esa/soc-
dev/unyin/qanda.htm#1, 24. september, 2007 6. McManus’s (2002) konkluderer, at ca. 14 % af nyhedsartiklerne om-
handler unge. Vi har ikke optalt samtlige artikler, men vi mener, at
tallet for de danske aviser næppe adskiller sig signifikant herfra. 7. Se http://www.db.dk/binaries/polinfoclassic_2001.doc. Vi har dog
kun benyttet emneordslistens øverste niveau og kun indekseret med
en enkelt term pr. artikel 8. Kodearket kan ses på https://e-learn.sdu.dk/bbcswebdav/orgs/bbc
omm477/Tekster/Diverse_faelles_dok JOURNALISTICA · NR. 1 · 2010 // 57 9. Datafilen kan downloades fra https://e-learn.sdu.dk/bbcswebdav/
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Peter Lang: New York. y , J (
5)
yp
y
g p
p
,
in A. Williams and C. Thurlow, (Eds). Communication in Adolescence:
Perspectives on Language and Social Interaction in the Teenage Years,
Peter Lang: New York. JOURNALISTICA · NR. 1 · 2010 // 135 135 Media representations of youth over 50 years:
A content analysis of Danish newspapers from 1953 to 2003
CHARLOTTE WIEN AND KLAUS LEVINSEN The modern mass media affects how we perceive and under-
stand the youth; and previous international studies have shown
that the media mostly conveys a negative image of young people. Based on a quantitative content analysis, this article examines
how young people are presented in at selection of Danish new-
spapers over the past 50 years. Our study shows that crime, cul-
ture and sports are the most frequent topics in the newspapers
relating to young people, but the study could not confirm the ex-
istence of a dominant negative bias in the newspaper stories. This could neither be confirmed in relation to the study of con-
temporary media coverage (2003) or in the examination of the
long-term perspective (1953-2003). Quite the contrary, our data
reveals a trend towards more and more neutral journalistic des-
criptions of young people, and a tendency to quote young people
in newspaper articles.
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https://digital.csic.es/bitstream/10261/230177/1/Clustering_Zhong_PV_Art2020.pdf
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English
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Clustering of Tir during enteropathogenic E. coli infection triggers calcium influx–dependent pyroptosis in intestinal epithelial cells
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PLoS biology
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cc-by
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PLOS BIOLOGY PLOS BIOLOGY PLOS BIOLOGY RESEARCH ARTICLE OPEN ACCESS Citation: Zhong Q, Roumeliotis TI, Kozik Z,
Cepeda-Molero M, Ferna´ndez LA´, Shenoy AR, et al. (2020) Clustering of Tir during enteropathogenic E. coli infection triggers calcium influx–dependent
pyroptosis in intestinal epithelial cells. PLoS Biol
18(12): e3000986. https://doi.org/10.1371/journal. pbio.3000986 Academic Editor: Hans-Uwe Simon, University of
Bern, SWITZERLAND Received: June 3, 2020
Accepted: November 24, 2020
Published: December 30, 2020
Copyright: © 2020 Zhong et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited. Received: June 3, 2020
Accepted: November 24, 2020
Published: December 30, 2020
Copyright: © 2020 Zhong et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited. Received: June 3, 2020
Accepted: November 24, 2020
Published: December 30, 2020 Copyright: © 2020 Zhong et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited. Data Availability Statement: Data are available
from the Pride Database, the accession number is
PXD018763. Funding: T.I.R and J.S.C are supported by the
Cancer Research UK Centre grant C309/A25144. GF is supported a Wellcome Investigator Award
107057/Z/15/Z. QZ is supported by Imperial
College President’s PhD Scholarship. GF is
supported by a Wellcome Investigator Award
(107057/Z/15/Z). T.I.R and J.S.C were funded by Abstract Clustering of the enteropathogenic Escherichia coli (EPEC) type III secretion system
(T3SS) effector translocated intimin receptor (Tir) by intimin leads to actin polymerisation
and pyroptotic cell death in macrophages. The effect of Tir clustering on the viability of
EPEC-infected intestinal epithelial cells (IECs) is unknown. We show that EPEC induces
pyroptosis in IECs in a Tir-dependent but actin polymerisation-independent manner, which
was enhanced by priming with interferon gamma (IFNγ). Mechanistically, Tir clustering
triggers rapid Ca2+ influx, which induces lipopolysaccharide (LPS) internalisation, followed
by activation of caspase-4 and pyroptosis. Knockdown of caspase-4 or gasdermin D
(GSDMD), translocation of NleF, which blocks caspase-4 or chelation of extracellular Ca2+,
inhibited EPEC-induced cell death. IEC lines with low endogenous abundance of GSDMD
were resistant to Tir-induced cell death. Conversely, ATP-induced extracellular Ca2+ influx
enhanced cell death, which confirmed the key regulatory role of Ca2+ in EPEC-induced pyr-
optosis. We reveal a novel mechanism through which infection with an extracellular patho-
gen leads to pyroptosis in IECs. Academic Editor: Hans-Uwe Simon, University of
Bern, SWITZERLAND RESEARCH ARTICLE
Clustering of Tir during enteropathogenic E.
coli infection triggers calcium influx–
dependent pyroptosis in intestinal epithelial
cells Qiyun ZhongID1, Theodoros I. RoumeliotisID2, Zuza Kozik2, Massiel Cepeda-Molero1, Luis
A´ ngel Ferna´ndez3, Avinash R. Shenoy4, Chris BakalID5, Gad FrankelID1*, Jyoti
S. ChoudharyID2* a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 1 Centre for Molecular Bacteriology and Infection, Department of Life Sciences, Imperial College, London,
United Kingdom, 2 Functional Proteomics Group, Chester Beatty Laboratories, The Institute of Cancer
Research, London, United Kingdom, 3 Department of Microbial Biotechnology, Centro Nacional de
Biotecnologı´a, Consejo Superior de Investigaciones Cientı´ficas (CNB-CSIC), Campus UAM-Cantoblanco,
Madrid, Spain, 4 Centre for Molecular Bacteriology & Infection, Department of Infectious Disease, Imperial
College, London, United Kingdom, 5 Dynamical Cell Systems, Chester Beatty Laboratories, The Institute of
Cancer Research, London, United Kingdom * g.frankel@imperial.ac.uk (GF); jyoti.choudhary@icr.ac.uk (JSC) * g.frankel@imperial.ac.uk (GF); jyoti.choudhary@icr.ac.uk (JSC) PLOS BIOLOGY PLOS BIOLOGY EPEC Tir triggers calcium influx-dependent pyroptosis they hijack multiple cell signalling pathways, including those regulating cytoskeletal dynamics,
inflammation, vesicle trafficking, and cell survival [4]. Multiple T3SS effectors trigger or antag-
onise host immune responses, including those mediated by nuclear factor kappa B (NF-κB)
(e.g., translocated intimin receptor (Tir), NleF, NleC, NleD and NleE) [5–8], caspase-1 (NleA)
[9], and caspase-4 (NleF) [10,11]. T3SS effector translocation is a highly regulated process
which involves the transmembrane gatekeeper effector EspZ [12]; uncontrolled effector trans-
location is cytotoxic [12]. the CRUK Centre grant (C309/A25144). "The
funders had no role in study design, data collection
and analysis, decision to publish, or preparation of
the manuscript". Competing interests: "The authors have declared
that no competing interests exist". Competing interests: "The authors have declared
that no competing interests exist". PLOS BIOLOGY Abbreviations: A/E, attaching and effacing; AGC,
automatic gain control; ANOVA, analysis of
variance; APOB, apolipoprotein B; APOE,
apolipoprotein E; ASC, apoptosis-associated
speck-like protein containing a CARD; BSA, bovine
serum albumin; CANX, calnexin; CASP4, caspase-
4; CFU, colony forming unit; CID, collision-induced
dissociation; CREB1, cAMP responsive element
binding protein 1; DAMP, damage-associated
molecular pattern; DMEM, Dulbecco’s Modified
Eagle Medium; DMTU, N,N0-dimethylthiourea;
EPEC, enteropathogenic Escherichia coli; ER,
endoplasmic reticulum; FBS, foetal bovine serum;
FDR, false discovery rate; GAPDH, glyceraldehyde
3-phosphate dehydrogenase; GBP2, guanylate-
binding protein 2; GEO, Gene Expression Omnibus;
GOBP, Gene Ontology Biological Process; GSDMD,
gasdermin D; GSDME, gasdermin E; HCD, higher-
energy collisional dissociation; HEPES, N-2-
hydroxyethylpiperazine-N-2-ethane sulphonic acid;
HPLC, high-performance liquid chromatograph;
IAA, iodoacetamide; IEC, intestinal epithelial cell;
IFNGR, interferon gamma receptor; IFNγ, interferon
gamma; IL, interleukin; IPTG, isopropyl β-d-1-
thiogalactopyranoside; IRF, interferon regulatory
factor; IT, injection time; ITPR2, inositol 1,4,5-
trisphosphate receptor type 2; JAK, Janus kinase;
JAK-STAT, Janus kinase/signal transducers and
activators of transcription; LBP, LPS-binding
protein; LC–MS, liquid chromatography–mass
spectrometry; LPS, lipopolysaccharide; MOI,
multiplicity-of-infection; Nec1, necrostatin-1; NF-
κB, nuclear factor kappa B; NLR, NOD leucine-rich
repeat protein; NLRP3, NLR family pyrin domain
containing 3; NOD, nucleotide-binding
oligomerisation domain; NSA, necrosulfonamide;
OCR, oxygen consumption rate; OMV, outer
membrane vesicle; PAMP, pathogen-associated
molecular pattern; PARP1, poly [ADP-ribose]
polymerase 1; PBS, phosphate buffered saline;
PBST, phosphate buffered saline, 0.05% Tween 20;
PCA, principal component analysis; PI, propidium
iodide; PI3K, phosphatidylinositol 3-kinase; PMA,
phorbol 12-myristate 13-acetate; PRIDE,
PRoteomics IDEntifications database; PVDF,
polyvinylidene difluoride; qRT-PCR, real-time
quantitative PCR; RIPA, radioimmunoprecipitation;
RIPK, receptor-interacting serine/threonine-protein
kinase; ROS, reactive oxygen species; RP,
reversed-phase; RPE, retinal pigment epithelium; EPEC colonise intestinal epithelial cells (IECs) via a mechanism known as attaching and
effacing (A/E) lesions, which are characterised by intimate bacterial attachment and effacing
of the brush border microvilli [13]. Intimate EPEC attachment is mediated by avid interactions
between the bacterial outer membrane adhesin intimin (encoded by the eae gene) and the
effector Tir [14,15]. Binding to intimin leads to Tir clustering, phosphorylation of Tir tyrosine
residue Y474 by host non-receptor tyrosine kinases [16,17], and recruitment of the host adap-
tor protein Nck, which, in turn, recruits N-WASP that activates Arp2/3 [18–20]. In addition,
Tir Y454 forms a complex with phosphatidylinositol 3-kinase (PI3K) [21] and binds the host
adaptors IRTKS/IRSp53, scaffold proteins that regulate actin organisation [22,23]. PLOS BIOLOGY While the
signalling downstream of Y474 cascade leads to robust actin polymerisation and formation of
actin-rich pedestal-like structures at the site of bacterial attachment [18–20], pathway down-
stream of Y454 Triggers weak actin polymerisation activity [17,24]. Mutating both tyrosine
residues to alanine or phenylalanine abolishes Tir phosphorylation and recruitment of host
proteins involved in actin polymerisation [17,25]. Recently, in order to determine whether intimin–Tir interaction is necessary and sufficient
for colonisation of the gut mucosa, we systematically deleted effector genes from the prototype
EPEC strain E2348/69, generating EPEC-0 missing all the effector genes, EPEC-1 expressing
only Tir and EPEC-2 that expresses only Tir and EspZ [26]. EPEC-0, 1, and 2 express intimin
and a functional T3SS, while only EPEC-1 and EPEC-2 can trigger actin polymerisation in
infected cells [26]. We have recently shown that intimin-induced Tir clustering, phosphorylation, and actin
polymerisation trigger inflammasome-mediated pyroptotic cell death in EPEC-infected mac-
rophages [25]. The inflammasome is a multi-protein complex which canonically involves cas-
pase-1, the adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC)
and the nucleotide-binding oligomerisation domain (NOD) leucine-rich repeat proteins
(NLRs), which act as sensors for pathogen/damage-associated molecular patterns (PAMPs/
DAMPs) such as extracellular ATP, reactive oxygen species (ROS), bacterial toxins, and secre-
tion system/flagella subunits [27–29]. The caspase-1 inflammasome cleaves and activates gas-
dermin D (GSDMD), a pore-forming protein that executes pyroptotic cell death [30,31] and
secretion of interleukin (IL)-1β and IL-18 [25]. The noncanonical inflammasome comprises
caspase-4, which is activated by direct binding to bacterial lipopolysaccharide (LPS) [30–32],
known to enter the cytosol during bacterial invasion [33], via outer membrane vesicles
(OMVs) [34,35] or via specific cellular receptors [36]. Recent studies have shown that the
receptor-interacting serine/threonine-protein kinase 1 (RIPK1)/caspase-8 axis plays an impor-
tant role in regulating apoptotic, necroptotic, and pyroptotic cell death pathways in IECs [37]. Our current understanding on the mechanisms of EPEC-induced cell death is mainly based
on studies performed in macrophages. Nonetheless, pyroptosis can also be triggered in non-
phagocytic IECs during bacterial infection [38]. Deep proteomic analysis of a panel of 50 intes-
tinal cancer cell lines shows that caspase-4 and GSDMD are constitutively expressed, while
NLR family pyrin domain containing 3 (NLRP3), gasdermin E (GSDME), and caspase-5 are
undetectable under standard growth conditions [39]. Introduction The extracellular pathogen enteropathogenic Escherichia coli (EPEC) causes persistent infan-
tile diarrhoea [1] and has been found to preferentially colonise colorectal cancer tissue in adult
patients [2]. EPEC infection of the intestinal mucosa is mediated by a type III secretion system
(T3SS) [3], a molecular syringe that injects 21 effectors directly into the host cell cytosol where PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986
December 30, 2020 1 / 31 The interaction of EPEC with the colorectal epithelial cell line SNU-C5 We aimed to investigate whether EPEC could trigger cell death in IECs and the potential role
of IFNγ in this process. Most studies of EPEC infection have used either HeLa or Caco-2 cell
lines; however, the latter contains several mutations in genes involved in IFNγ signalling path-
way, including interferon gamma receptor 2 (IFNGR2) and Janus kinase 1 (JAK1). For this
reason, we first sought to identify a suitable intestinal cell model for this study. Using publicly
available proteomics data for a panel of 50 colorectal cancer cell lines [39], we analysed the
abundance levels of proteins mapping in a characteristic immune response module, selected
based on a weighted correlation network analysis (WGCNA) [42]. We ranked all 50 cell lines
by the median scaled abundance of the immune response module [39] (Fig 1A) and selected
the SNU-C5 cell line as a representative of moderate abundance of the proteins in this module. In addition, SNU-C5 cell line has no mutations in the genes involved in IFNγ signalling and
noncanonical inflammasome: IFNGR1, IFNGR2, JAK1, JAK2, signal transducer and activator
of transcription 1 (STAT1), interferon regulatory factor 1 (IRF1), IRF2, caspase-4 (CASP4),
and GSDMD, according to the Colorectal Cancer Atlas [43]. We did not consider the varia-
tions of the canonical inflammasome components due to the undetectable protein expression
levels of all known pyroptosis-related NLRs [39]. Additionally, the increased expression of
guanylate-binding protein 2 (GBP2), an interferon-induced gene, in SNU-C5 primed with
IFNγ for 24 h was validated by real-time quantitative PCR (qRT-PCR) (S1A Fig), confirming
that SNU-C5 cells respond to IFNγ. To further evaluate the cell model, we examined whether SNU-C5 cells are susceptible to
EPEC infection. We used Tir-induced actin polymerisation and NF-κB activation as indicators
of successful infection. SNU-C5 cells were infected with EPEC wild-type (WT), EPEC-1 (Tir
only), and EPEC-0 (effectorless). While all 3 strains adhered to >98% of SNU-C5 cells (Fig 1B
and 1C), robust actin polymerisation was detected underneath EPEC-WT and EPEC-1, but
not EPEC-0 (Fig 1D). Immunofluorescence staining of NF-κB p65 subunit showed that
EPEC-WT did not trigger NF-κB activation up to 4 h postinfection (Fig 1E and 1F), consistent
with the notion that it expresses multiple effectors that inhibit the NF-κB pathway [6–8]. PLOS BIOLOGY The expression level of some of these
pyroptotic proteins may be elevated via NF-κB or Janus kinase/signal transducers and PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986
December 30, 2020 2 / 31 PLOS BIOLOGY EPEC Tir triggers calcium influx-dependent pyroptosis rpm, revolutions per minute; RPMI, Roswell Park
Memorial Institute; SCARB1, scavenger receptor
class B member; SDC, sodium deoxycholate; SDS-
PAGE, sodium dodecyl sulphate polyacrylamide gel
electrophoresis; SERCA, sarco/endoplasmic
reticulum Ca2+; shRNA, short-hairpin RNA; siRNA,
small interfering RNA; SPS, synchronous
precursor selection; STAT1, signal transducer and
activator of transcription 1; STING, stimulator of
interferon genes; STS, staurosporine; T3SS, type
III secretion system; TBST, tris buffered saline,
0.1% Tween 20; TCEP, tris-2-carboxyethyl
phosphine; TEAB, triethylammonium bicarbonate;
Th, Thomson unit; Tir, translocated intimin
receptor; TMEM173, transmembrane protein 173;
TMT, tandem mass tag; TRP, transient receptor
potential; WGCNA, weighted correlation network
analysis; WT, wild-type; YFP, yellow fluorescent
protein. activators of transcription (JAK-STAT) signalling pathways during a process called priming,
via pretreatment with LPS or interferon gamma (IFNγ), respectively [28]. Although priming is
most extensively used for phagocytic cells, IFNγ induces expression of caspase-1 and caspase-5
in the colonic epithelial cell line HT-29 [40]. Moreover, EPEC infection of the colorectal cancer
cell line Caco-2 triggers caspase-4–dependent release of IL-18 [41], suggesting that EPEC may
also induce pyroptosis in IECs. The aim of this study was to investigate whether EPEC causes
pyroptosis in an IEC line model and to decipher its molecular mechanism. The interaction of EPEC with the colorectal epithelial cell line SNU-C5 In
contrast, EPEC-0, lacking these effectors, provoked modest NF-κB activation, which is likely
due to the activity of the T3SS apparatus [44], while infection with EPEC-1 triggered signifi-
cant NF-κB activation above EPEC-0 (Fig 1E and 1F). These results suggest that in the absence
of the anti-inflammatory effectors and the gatekeeper EspZ [12], high-level Tir translocation
in EPEC-1 triggers strong pro-inflammatory responses. Together, these data indicate that
SNU-C5 responds as expected to EPEC infection and thus provides a robust model to study
EPEC infection of IECs. EPEC-induced lytic cell death in SNU-C5 is enhanced by IFNγ priming We next investigated whether EPEC infection induces death in SNU-C5 cells in a manner
dependent on its effectors, using propidium iodide (PI) uptake as a reporter for membrane PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986
December 30, 2020 3 / 31 PLOS BIOLOGY EPEC Tir triggers calcium influx-dependent pyroptosis Fig 1. EPEC induces Tir-dependent actin polymerisation and NF-κB activation in SNU-C5 cells. (A) Ranking of 50
colorectal cell lines based on the scaled abundance of proteins in the immune response module according to WGCNA
[39]. (B) Adhesion of EPEC WT, EPEC-0, and EPEC-1 to SNU-C5. Means ± SEM from n = 3 independent biological
repeats. (C, D) Immunofluorescence labelling of SNU-C5 cells infected with EPEC WT, EPEC-0 and EPEC-1 or 4 h. (C) Percentage of infected cells. (D) Immunofluorescent images. DAPI: blue; EPEC: green; Phalloidin: red. Representative images from n = 3 independent biological repeats. Scale bar: 5 μm. Example bacteria are marked with
white arrows. (E) Immunofluorescence staining of p65 in UI and EPEC-infected cells, analysed by high content
imaging. Representative images from n = 3 independent biological repeats. Scale bar: 20 μm. (F) Nuclear/cytosolic
ratio of the NF-κB p65 immunofluorescence intensity calculated by the Columbus-2 software. Statistical significance
was determined using 1-way ANOVA with Tukey posttest. ns, nonsignificant; p 0.05; p 0.01; p 0.001. The underlying data for this figure can be found in S1 Data. ANOVA, analysis of variance; DAPI, 40,6-diamidino-
2-phenylindole; EPEC, enteropathogenic Escherichia coli; NF-κB, nuclear factor kappa B; SEM, standard error of the Fig 1. EPEC induces Tir-dependent actin polymerisation and NF-κB activation in SNU-C5 cells. (A) Ranking of 50
colorectal cell lines based on the scaled abundance of proteins in the immune response module according to WGCNA
[39]. (B) Adhesion of EPEC WT, EPEC-0, and EPEC-1 to SNU-C5. Means ± SEM from n = 3 independent biological
repeats. (C, D) Immunofluorescence labelling of SNU-C5 cells infected with EPEC WT, EPEC-0 and EPEC-1 or 4 h. (C) Percentage of infected cells. (D) Immunofluorescent images. DAPI: blue; EPEC: green; Phalloidin: red. Representative images from n = 3 independent biological repeats. Scale bar: 5 μm. Example bacteria are marked with
white arrows. (E) Immunofluorescence staining of p65 in UI and EPEC-infected cells, analysed by high content
imaging. Representative images from n = 3 independent biological repeats. Scale bar: 20 μm. PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986
December 30, 2020 EPEC-induced lytic cell death in SNU-C5 is enhanced by IFNγ priming (F) Nuclear/cytosolic
ratio of the NF-κB p65 immunofluorescence intensity calculated by the Columbus-2 software. Statistical significance
was determined using 1-way ANOVA with Tukey posttest. ns, nonsignificant; p 0.05; p 0.01; p 0.001. The underlying data for this figure can be found in S1 Data. ANOVA, analysis of variance; DAPI, 40,6-diamidino-
2-phenylindole; EPEC, enteropathogenic Escherichia coli; NF-κB, nuclear factor kappa B; SEM, standard error of the Fig 1. EPEC induces Tir-dependent actin polymerisation and NF-κB activation in SNU-C5 cells. (A) Ranking of 50
colorectal cell lines based on the scaled abundance of proteins in the immune response module according to WGCNA
[39]. (B) Adhesion of EPEC WT, EPEC-0, and EPEC-1 to SNU-C5. Means ± SEM from n = 3 independent biological
repeats. (C, D) Immunofluorescence labelling of SNU-C5 cells infected with EPEC WT, EPEC-0 and EPEC-1 or 4 h. (C) Percentage of infected cells. (D) Immunofluorescent images. DAPI: blue; EPEC: green; Phalloidin: red. Representative images from n = 3 independent biological repeats. Scale bar: 5 μm. Example bacteria are marked with
white arrows. (E) Immunofluorescence staining of p65 in UI and EPEC-infected cells, analysed by high content
imaging. Representative images from n = 3 independent biological repeats. Scale bar: 20 μm. (F) Nuclear/cytosolic
ratio of the NF-κB p65 immunofluorescence intensity calculated by the Columbus-2 software. Statistical significance
was determined using 1-way ANOVA with Tukey posttest. ns, nonsignificant; p 0.05; p 0.01; p 0.001. The underlying data for this figure can be found in S1 Data. ANOVA, analysis of variance; DAPI, 40,6-diamidino-
2-phenylindole; EPEC, enteropathogenic Escherichia coli; NF-κB, nuclear factor kappa B; SEM, standard error of the PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986
December 30, 2020 4 / 31 PLOS BIOLOGY EPEC Tir triggers calcium influx-dependent pyroptosis mean; Tir, translocated intimin receptor; UI, uninfected; WGCNA, weighted correlation network analysis; WT, wild-
type. mean; Tir, translocated intimin receptor; UI, uninfected; WGCNA, weighted correlation network analysis; WT, wild-
type. https://doi.org/10.1371/journal.pbio.3000986.g001 damage and necrotic cell lysis. This revealed that EPEC-WT induced 19% increase in PI
uptake after 8 h of infection compared with uninfected control cells, while EPEC-0 increased
PI uptake by 11% (Fig 2A, S1B Fig). Notably, EPEC-1 induced 35% increase in PI uptake, sig-
nificantly higher than that of EPEC-WT and EPEC-0 (Fig 2A). damage and necrotic cell lysis. EPEC-induced lytic cell death in SNU-C5 is enhanced by IFNγ priming This revealed that EPEC-WT induced 19% increase in PI
uptake after 8 h of infection compared with uninfected control cells, while EPEC-0 increased
PI uptake by 11% (Fig 2A, S1B Fig). Notably, EPEC-1 induced 35% increase in PI uptake, sig-
nificantly higher than that of EPEC-WT and EPEC-0 (Fig 2A). y
g
g
In order to determine the effect of IFNγ priming on EPEC-induced cell death, we pre-
treated the SNU-C5 monolayer with IFNγ 24 h prior to infection. IFNγ priming alone did
not induce an increase in PI uptake (S1C Fig). Infecting the primed cells with EPEC-0 for 8 h
showed no increase in PI uptake (12%), compared with unprimed cells. In contrast, IFNγ
priming increased PI uptake to 27% and 51% following infection with EPEC-WT and EPEC-1,
respectively (Fig 2A and 2B). The time course of PI uptake during EPEC infection revealed
that cell death first started to increase at around 1.5 h and plateaued at 6 to 8 h postinfection
(Fig 2B). To confirm that Tir is responsible for enhanced cell death, a control infection with
EPECΔtir revealed markedly reduced cell death compared with EPEC WT in primed cells (Fig
2C). Collectively, these results show that, similar to macrophages [25], Tir triggers cell death in
an epithelial cell model and that this activity is amplified by IFNγ. We therefore decided to
investigate EPEC-induced cell death mechanisms in IFNγ-primed cells. PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986
December 30, 2020 Tir-induced cell death is dependent on caspase-4 and GSDMD To test the involvement of caspases in Tir-induced cell death in IECs, we pretreated SNU-C5
cells with either a pan-caspase inhibitor z-VAD-fmk (zVAD) or the pyroptotic caspase-1/4/5
inhibitor z-YVAD-fmk (YVAD). Both zVAD and YVAD significantly reduced cell death
caused by EPEC-1 at 8 h postinfection, indicating the involvement of pyroptosis, while the low
level of Tir-independent cell death in EPEC-0 was unaffected by caspase inhibition (Fig 2D). Furthermore, western blot of infected cells showed no cleavage of the apoptosis marker poly
[ADP-ribose] polymerase 1 (PARP1), ruling out apoptosis in Tir-dependent cell death (S2A
Fig). In contrast to macrophage [25], Tir-dependent cell death in SNU-C5 was not inhibited
by the NLRP3 inhibitor MCC950 (S2B and S2C Fig). As Yersinia, a related Gram-negative
pathogen carrying T3SS, has recently been shown to trigger macrophage pyroptosis via
RIPK1- and caspase-8-dependent cleavage of GSDMD [45, 46, 47], we infected SNU-C5 cells
in the presence of the RIPK1 inhibitor Nec1 (S2D–S2F Fig) or with caspase-8 small interfering
RNA (siRNA) silencing (S2G and S2H Fig), both of which revealed no inhibition of cell death. The pan-caspase inhibition did not completely abolish Tir-dependent cell death, with ca. 15% of EPEC-1-infected cells sill exhibiting PI uptake compared with EPEC-0-infected cells
(Fig 2D). As necroptosis can be activated during pyroptosis inhibition [48], we investigated if
the residual cell death is due to necroptosis. For this, we used necrostatin-1 (Nec1) to inhibit
RIPK1, necrosulfonamide (NSA) to inhibit MLKL and siRNA to silence RIPK3. While control
experiments have shown that these treatments prevented necroptosis induced by staurospor-
ine (STS) and zVAD co-stimulation [49], they did not significantly affect Tir-dependent cell
death, either on their own or in combination with zVAD or caspase-4 silencing (S2D–S2K
Fig). Taken together, these results implicate the pyroptotic caspases as the main contributor
in EPEC-induced cell death, while the mechanism leading to the residual cell death remains
elusive. As neither caspase-1 nor caspase-5 is detected in SNU-C5 [39], we next investigated the
involvement of caspase-4 which is detected in the proteome. Western blotting of cell lysates
and supernatants collected at 8 h postinfection revealed cleavage of the approximately 45 kDa PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986
December 30, 2020 5 / 31 PLOS BIOLOGY EPEC Tir triggers calcium influx-dependent pyroptosis Fig 2. IFNγ enhances Tir- and caspase-4-dependent SNU-C5 cell death. (A) PI uptake into SNU-C5 cells infected
with EPEC WT, EPEC-0, and EPEC-1 for 8 h. Tir-induced cell death is dependent on caspase-4 and GSDMD Cells were either unprimed or primed with 10 ng/ml of IFNγ. PI uptake
results were normalised to uninfected cells. Means ± SEM from n = 5 independent biological repeats. (B) Time-course
PI uptake into primed SNU-C5 cells throughout the 8 h of infection period. Measurements were taken every 10 min. Means ± SEM from n = 5 independent biological repeats are shown. (C) PI uptake into primed SNU-C5 cells infected
with EPEC WT and Δtir. Means ± SEM from n = 3 independent biological repeats. (D) PI uptake into primed SNU-C5
cells treated with zVAD and/or YVAD 30 min before infection with EPEC WT and EPEC-1. Means ± SEM from n = 3
(EPEC WT & EPEC-0) and 7 (EPEC-1) independent biological repeats. (E) Caspase-4 western blot of lysates and
supernatants from primed cells infected by EPEC WT, EPEC-0, and EPEC-1. Representative blot from n = 3
independent biological repeats. (F) Caspase-4 western blot of lysates from primed cells transfected with caspase-4
siRNA for 2 days. Representative blot from n = 3 independent biological repeats are shown. (G) Control and caspase-4
siRNA transfection into primed SNU-C5 cells 2 days before infection. Shown is PI uptake following infection with Fig 2. IFNγ enhances Tir- and caspase-4-dependent SNU-C5 cell death. (A) PI uptake into SNU-C5 cells infected
with EPEC WT, EPEC-0, and EPEC-1 for 8 h. Cells were either unprimed or primed with 10 ng/ml of IFNγ. PI uptake
results were normalised to uninfected cells. Means ± SEM from n = 5 independent biological repeats. (B) Time-course
PI uptake into primed SNU-C5 cells throughout the 8 h of infection period. Measurements were taken every 10 min. Means ± SEM from n = 5 independent biological repeats are shown. (C) PI uptake into primed SNU-C5 cells infected
with EPEC WT and Δtir. Means ± SEM from n = 3 independent biological repeats. (D) PI uptake into primed SNU-C5
cells treated with zVAD and/or YVAD 30 min before infection with EPEC WT and EPEC-1. Means ± SEM from n = 3
(EPEC WT & EPEC-0) and 7 (EPEC-1) independent biological repeats. (E) Caspase-4 western blot of lysates and
supernatants from primed cells infected by EPEC WT, EPEC-0, and EPEC-1. Representative blot from n = 3
independent biological repeats. (F) Caspase-4 western blot of lysates from primed cells transfected with caspase-4
siRNA for 2 days. PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986
December 30, 2020 Tir-induced cell death is dependent on caspase-4 and GSDMD Representative blot from n = 3 independent biological repeats are shown. (G) Control and caspase-4
siRNA transfection into primed SNU-C5 cells 2 days before infection. Shown is PI uptake following infection with PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986
December 30, 2020 6 / 31 PLOS BIOLOGY EPEC Tir triggers calcium influx-dependent pyroptosis EPEC WT and EPEC-1 or transfection with LPS. Means ± SEM from n = 4 (LPS & EPEC WT) and 7 (EPEC-1)
independent biological repeats. (H) PI uptake into primed SNU-C5 cells infected with EPEC-1 and EPEC-1-NleF. Means ± SEM from n = 3 independent biological repeats. (I) GSDMD western blot of lysates from primed cells
infected by EPEC WT, EPEC-0 and EPEC-1. Lysate of THP-1 cells after LPS/Nigericin treatment were used as positive
control for GSDMD activation. Representative blot from n = 3 independent biological repeats. (J) GSDMD western
blot of lysates from primed cells transfected with GSDMD siRNA for 3 days. Representative blot from n = 3
independent biological repeats are shown. (K) GSDMD western blot of lysates from primed GSDMD stable
knockdown cells. Representative blot from n = 3 independent biological repeats are shown. (L) Control and GSDMD
siRNA transfection into primed SNU-C5 cells 3 days before infection. Shown is PI uptake following infection with
EPEC WT and EPEC-1 or transfection with LPS. Means ± SEM from n = 4 independent biological repeats. (M)
Control and GSDMD stable knockdown cells were primed and transfected by LPS, infected by EPEC WT or EPEC-1. Means ± SEM from n = 3 independent biological repeats. (N) Left panel: the scaled endogenous abundance of caspase-
4, GSDMD, caspase-1, 5 and 8 in the 4 cell lines [39]. N/A: undetectable. Right panel: PI uptake into primed SNU-C5,
HT-29, SNU-C2B, and COLO-320-HSR cells infected with EPEC-0 and EPEC-1. Means ± SEM from n = 3
independent biological repeats. Statistical significance was determined using 2-way ANOVA with Bonferroni posttest
(A, D, G, L, M, N) or 2-tailed t test (C, H). p 0.05; p 0.01; p 0.001. The underlying data for this figure can
be found in S1 Data. ANOVA, analysis of variance; EPEC, enteropathogenic Escherichia coli; GSDMD, gasdermin D;
IFNγ, interferon gamma; LPS, lipopolysaccharide; PI, propidium iodide; SEM, standard error of the mean; siRNA,
small interfering RNA; Tir, translocated intimin receptor; WT, wild-type; YVAD, z-YVAD-fmk; zVAD, z-VAD-fmk. Tir-induced cell death is dependent on caspase-4 and GSDMD https://doi.org/10.1371/journal.pbio.3000986.g002 pro-caspase-4 into the active form approximately 30 kDa caspase-4 large subunit in EPEC-
1-infected cells (Fig 2E). To verify its functional role, we silenced caspase-4 by siRNA, which
reduced cell death when LPS transfection was used as a positive control (Fig 2F and 2G). Upon
infection with either EPEC WT or EPEC-1, cell death was suppressed by caspase-4 siRNA (Fig
2G), suggesting that Tir-induced cytotoxicity is mediated by caspase-4-driven pyroptosis. Indeed, ectopic expression of NleF, which inhibits caspase-4 [10,11], in EPEC-1 resulted in
strong inhibition of Tir-induced cell death (Fig 2H). Moreover, EPEC-1-infected cells exhib-
ited higher levels of cleaved N-terminal GSDMD fragment that is responsible for the pyropto-
tic pores (Fig 2I). Silencing GSDMD in SNU-C5 by siRNA only mildly reduced EPEC-
1-induced cell death (Fig 2J and 2L). Consequently, we constructed GSDMD stable knock-
down SNU-C5 cell line using the microRNA30E short-hairpin RNA (shRNA) strategy [25,50]. GSDMD shRNA strongly reduced cell death by the positive control LPS transfection as well as
both EPEC WT and EPEC-1, confirming that GSDMD is required for Tir-dependent cell
death (Fig 2K and 2M). g
To further verify the dependence of cell death on GSDMD, we selected 3 additional IEC
lines, HT-29, SNU-C2B, and COLO-320-HSR, in which the abundance of GSDMD is 1.9,
15.5, and 36.7 times lower compared with SNU-C5, while the abundance ratios for caspase-4
of these cell lines over SNU-C5 are 1.1, 0.7, and 0.1, respectively (Fig 2N, left panel). These cell
lines display no mutations in either caspase-4 or GSDMD genes. Caspase-5 was undetectable,
and caspase-8 was expressed in all cell lines, while caspase-1 was only detected at a low level in
HT-29 and SNU-C2B cells (Fig 2N, left panel). Adhesion of EPEC WT, EPEC-0, and EPEC-1
and Tir translocation was confirmed in all cell lines (S3 Fig). Infections of IFNγ-primed
SNU-C5 and HT-29 with EPEC-1 resulted in higher cell death compared with those with
EPEC-0 (Fig 2N, right panel). In contrast, EPEC-0 caused similar lower level of cell death
upon infection of COLO-320-HSR and SNU-C2B (Fig 2N, right panel). These results reveal a
direct and specific correlation between the endogenous level of GSDMD and Tir-dependent
cell death. Tir clustering triggers epithelial cell death Binding of intimin to Tir leads to its clustering and activation of a robust actin polymerisation
cascade, which is essential for triggering cell death in human macrophages [25]. In order to
determine whether Tir-induced actin polymerisation is also necessary for epithelial cell death, PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986
December 30, 2020 7 / 31 PLOS BIOLOGY EPEC Tir triggers calcium influx-dependent pyroptosis we generated a TirY454A/Y474A mutant in EPEC-1 (EPEC-1-TirAA), which should not induce
actin polymerisation. As controls, we confirmed that EPEC-1-TirAA adhered to SNU-C5 cells
similar to EPEC-1 (Fig 3A and 3B), did not induce detectable actin polymerisation (Fig 3C),
and triggered p65 nuclear translocation at a level comparable with EPEC-1 (Fig 3D and 3E). Importantly, contrary to the observation in human macrophages [25], EPEC-1-TirAA still
induced pyroptosis following infection of SNU-C5 cells (Fig 3F). Both YVAD and ectopic
expression of NleF markedly reduced EPEC-TirAA-induced cell death, suggesting that like WT
Tir, TirAA activates caspase-4 in this model (Fig 3G and 3H). To further confirm that actin
polymerisation is dispensable for cell death, we pretreated SNU-C5 cells with the actin poly-
merisation inhibitor cytochalasin D, which prevented actin pedestal formation but not cell
death (Fig 3I and 3J). To assess the role of Tir clustering, we used a deletion mutant lacking
intimin from EPEC-1 (EPEC-1Δeae) [26]. The infection of SNU-C5 cells with EPEC-1Δeae
resulted in significant reduction of cell death (Fig 3K). This implied that while intimin-
induced Tir clustering is essential, the actin polymerisation function of Tir is dispensable for
induction of cell death in IECs. EspZ has been shown to limit the level of Tir translocation [12]. In order to determine if the
magnitude of cell death is impacted by the level of Tir translocation, we measured cell death
induced by EPEC-2 (EPEC-1 expressing EspZ) and EPEC-1-TirAA ectopically expressing EspZ
(EPEC-1-TirAA-EspZ). EPEC-2 and EPEC-1-TirAA-EspZ induced significantly less cell death
compared with EPEC-1 and EPEC-1-TirAA, respectively (Fig 3F). Notably, the relatively high
level of cell death in EPEC-1-TirAA- and EPEC-1-TirAA-EspZ-infected cells compared with
EPEC-1- and EPEC-2-infected cells, respectively (Fig 3F), as well as the mild increase in cell
death caused by cytochalasin D, suggests that the loss of actin polymerisation ability enhances
cell death. Based on these data, we conclude that the dosage of translocated Tir impacts on cell
death. Tir clustering triggers epithelial cell death Moreover, it shows a strain-dependent gradient of EPEC-induced cell death in both
primed and unprimed cells: EPEC-0 < EPEC-2 < EPEC-1-TirAA-EspZ < EPEC-1 < EPEC-
1-TirAA (Fig 3F, S3C Fig). PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986
December 30, 2020 Tir affects the abundance of Ca2+ transport and response proteins (F–H) PI uptake into primed
SNU-C5 cells infected with EPEC-0, EPEC-2, EPEC-1-TirAA-EspZ (F), EPEC-1 (F, I), EPEC-1-TirAA (F–H), and
EPEC-1-TirAA-NleF (H). The effector compositions in EPEC-0, EPEC-2, EPEC-1-TirAA-EspZ, EPEC-1, and EPEC-
1-TirAA are listed (F). Primed SNU-C5 cells were treated by YVAD and infected with EPEC-1-TirAA and used for PI
uptake assays. Means ± SEM from n = 5 (F) and 3 (G-H) independent biological repeats. (I) Immunofluorescence
labelling of SNU-C5 cells infected with EPEC-1 in the presence of absence of cytochalasin D treatment. DAPI: blue;
EPEC: green; Phalloidin: red. Representative images from n = 4 independent biological repeats. Scale bar: 5 μm. Example bacteria are marked with white arrows. (J) PI uptake into EPEC-1-infected SNU-C5 cells in the presence of
absence of cytochalasin D. Means ± SEM from n = 3 independent biological repeats. (K) PI uptake into SNU-C5 cells
infected by EPEC-1 or EPEC-1 Δeae. Means ± SEM from n = 3 independent biological repeats. Statistical significance
was determined using 2-tailed t test (A, D, G, H, J, K) or 1-way ANOVA with Tukey posttest (F). p 0.05;
p 0.01; p 0.001. The underlying data for this figure can be found in S1 Data. ANOVA, analysis of variance;
DAPI, 40,6-diamidino-2-phenylindole; EPEC, enteropathogenic Escherichia coli; NF-κB, nuclear factor kappa B; PI,
propidium iodide; SEM, standard error of the mean; Tir, translocated intimin receptor; YVAD, z-YVAD-fmk. Fig 3. Clustering by intimin is essential for Tir-induced cell death. (A) Infection rates of EPEC-1 and EPEC-1-TirAA
on SNU-C5 cells. Means ± SEM from n = 3 independent biological repeats. (B, C) Immunofluorescence labelling of
SNU-C5 cells infected with EPEC-1 and EPEC-1-TirAA for 2 h. (B) Percentage of infected cells. (C) DAPI: blue; EPEC:
green; Phalloidin: red. Representative images from n = 3 independent biological repeats. Scale bar: 5 μm. Example
bacteria are marked with white arrows. (D, E) Nuclear/cytosolic ratio of the NF-κB p65 immunofluorescence intensity
calculated by the Columbus-2 software (D) and representative images (E) from n = 3 independent biological repeats of
unprimed SNU-C5 cells infected with EPEC-1 and EPEC-1-TirAA. Scale bar: 20 μm. (F–H) PI uptake into primed
SNU-C5 cells infected with EPEC-0, EPEC-2, EPEC-1-TirAA-EspZ (F), EPEC-1 (F, I), EPEC-1-TirAA (F–H), and
EPEC-1-TirAA-NleF (H). The effector compositions in EPEC-0, EPEC-2, EPEC-1-TirAA-EspZ, EPEC-1, and EPEC-
1-TirAA are listed (F). Tir affects the abundance of Ca2+ transport and response proteins In order to gain mechanistic insights of Tir-induced caspase-4 activation, we performed prote-
omics analysis of SNU-C5 cells following infection of unprimed and IFNγ-primed cells with
EPEC-0, EPEC-2, EPEC-1-TirAA-EspZ, EPEC-1, and EPEC-1-TirAA, using isobaric labelling
(S5A Fig). We selected 2 h postinfection for the analysis representing the time point where
phenotypic signs of cell death are evident but with limited cell loss (Fig 2B, S1B Fig). We quan-
tified a total of 8,888 human proteins, of which 1,752 were found differentially regulated
between the different infections (analysis of variance [ANOVA], adjusted P < 0.05). Principal
component analysis (PCA) using all proteins showed a strong separation by IFNγ priming as
well as by the strain-dependent gradient of cell death, with the largest variation observed
between EPEC-0/EPEC-2 and EPEC-1-TirAA for both primed and unprimed cells (S5B Fig). Hierarchical clustering revealed groups of differentially regulated proteins either positively or
negatively correlating with the cell death gradient as well as clusters of proteins regulated by
IFNγ-priming in an infection-independent manner (Fig 4A). Enrichment analysis identified a
diverse range of signalling pathways and biological processes with up- or down-regulation
trends across infections (Fig 4B). As expected, proteins involved in antigen presentation and
response to IFNγ were highly up-regulated in all primed samples (Fig 4B). A heatmap of the
106 differentially regulated proteins upon IFNγ priming, including caspase-4 and GSDMD, is
shown in S5 Fig. Several basic cellular processes including, proteolysis, spliceosome, cell cycle,
and cytoskeleton proteins showed a decreased abundance that is correlated with Tir-induced PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986
December 30, 2020 8 / 31 PLOS BIOLOGY EPEC Tir triggers calcium influx-dependent pyroptosis Fig 3. Clustering by intimin is essential for Tir-induced cell death. (A) Infection rates of EPEC-1 and EPEC-1-TirAA
on SNU-C5 cells. Means ± SEM from n = 3 independent biological repeats. (B, C) Immunofluorescence labelling of
SNU-C5 cells infected with EPEC-1 and EPEC-1-TirAA for 2 h. (B) Percentage of infected cells. (C) DAPI: blue; EPEC:
green; Phalloidin: red. Representative images from n = 3 independent biological repeats. Scale bar: 5 μm. Example
bacteria are marked with white arrows. (D, E) Nuclear/cytosolic ratio of the NF-κB p65 immunofluorescence intensity
calculated by the Columbus-2 software (D) and representative images (E) from n = 3 independent biological repeats of
unprimed SNU-C5 cells infected with EPEC-1 and EPEC-1-TirAA. Scale bar: 20 μm. PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986
December 30, 2020 https://doi.org/10.1371/journal.pbio.3000986.g003 Tir affects the abundance of Ca2+ transport and response proteins (F) Hierarchical clustering of proteins selected by creb1_gpl1261_gds3660_down (TF-LOF Expression from GEO)
and Calcium (Keywords). The underlying data for this figure can be found in S1 Data. ANOVA, analysis of variance; CREB1, cAMP responsive element
binding protein 1; EPEC, enteropathogenic Escherichia coli; FDR, false discovery rate; IFNγ, interferon gamma; GEO, Gene Expression Omnibus; TF-LOF
transcription factor loss of function; Tir, translocated intimin receptor. Fig 4. Proteomic analysis of SNU-C5 infected with EPEC variants inducing variable levels of cell death. (A) Hierarchical clustering (Euclidean
distance) of differentially regulated proteins (ANOVA, FDR < 0.05) between EPEC-0, EPEC-2, EPEC-1-TirAA-EspZ, EPEC-1, and EPEC-1-TirAA-infected
cells with and without INFNγ priming. Data represent log2 ratios versus the uninfected sample and 2 independent biological repeats were performed per
infection. Individual repeats are labelled in the heatmap (R1 and R2). (B) Representative enriched pathways shown as a heatmap of the enrichment scores
(Benjamini–Hochberg FDR < 0.05). (C) Box plot of changes in Ca2+ transporter proteins selected using Keywords across the different samples. The boxes
are color-coded according to the enrichment scores shown in panel B. (D) Transcription factor enrichment was performed using the “TF-LOF Expression
from GEO” library. (E) Box plot of protein changes of a gene set with known reduced expression upon CREB1 perturbation (therefore positively regulated
by CREB1). These proteins tend to be up-regulated in our data set, suggesting the activation of CREB1. The boxes are color-coded according to the
enrichment scores shown in panel D. (F) Hierarchical clustering of proteins selected by creb1_gpl1261_gds3660_down (TF-LOF Expression from GEO)
and Calcium (Keywords). The underlying data for this figure can be found in S1 Data. ANOVA, analysis of variance; CREB1, cAMP responsive element
binding protein 1; EPEC, enteropathogenic Escherichia coli; FDR, false discovery rate; IFNγ, interferon gamma; GEO, Gene Expression Omnibus; TF-LOF,
transcription factor loss of function; Tir, translocated intimin receptor. htt
//d i
/10 1371/j
l bi 3000986 004 Fig 4. Proteomic analysis of SNU-C5 infected with EPEC variants inducing variable levels of cell death. (A) Hierarchical clustering (Euclidean
distance) of differentially regulated proteins (ANOVA, FDR < 0.05) between EPEC-0, EPEC-2, EPEC-1-TirAA-EspZ, EPEC-1, and EPEC-1-TirAA-infected
cells with and without INFNγ priming. Data represent log2 ratios versus the uninfected sample and 2 independent biological repeats were performed per
infection. Individual repeats are labelled in the heatmap (R1 and R2). (B) Representative enriched pathways shown as a heatmap of the enrichment scores
(Benjamini–Hochberg FDR < 0.05). Tir affects the abundance of Ca2+ transport and response proteins Primed SNU-C5 cells were treated by YVAD and infected with EPEC-1-TirAA and used for PI
uptake assays. Means ± SEM from n = 5 (F) and 3 (G-H) independent biological repeats. (I) Immunofluorescence
labelling of SNU-C5 cells infected with EPEC-1 in the presence of absence of cytochalasin D treatment. DAPI: blue;
EPEC: green; Phalloidin: red. Representative images from n = 4 independent biological repeats. Scale bar: 5 μm. Example bacteria are marked with white arrows. (J) PI uptake into EPEC-1-infected SNU-C5 cells in the presence of
absence of cytochalasin D. Means ± SEM from n = 3 independent biological repeats. (K) PI uptake into SNU-C5 cells
infected by EPEC-1 or EPEC-1 Δeae. Means ± SEM from n = 3 independent biological repeats. Statistical significance
was determined using 2-tailed t test (A, D, G, H, J, K) or 1-way ANOVA with Tukey posttest (F). p 0.05;
p 0.01; p 0.001. The underlying data for this figure can be found in S1 Data. ANOVA, analysis of variance;
DAPI, 40,6-diamidino-2-phenylindole; EPEC, enteropathogenic Escherichia coli; NF-κB, nuclear factor kappa B; PI,
propidium iodide; SEM, standard error of the mean; Tir, translocated intimin receptor; YVAD, z-YVAD-fmk. https://doi.org/10.1371/journal.pbio.3000986.g003 9 / 31 PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986
December 30, 2020 PLOS BIOLOGY EPEC Tir triggers calcium influx-dependent pyroptosis Fig 4. Proteomic analysis of SNU-C5 infected with EPEC variants inducing variable levels of cell death. (A) Hierarchical clustering (Euclidean
distance) of differentially regulated proteins (ANOVA, FDR < 0.05) between EPEC-0, EPEC-2, EPEC-1-TirAA-EspZ, EPEC-1, and EPEC-1-TirAA-infected
cells with and without INFNγ priming. Data represent log2 ratios versus the uninfected sample and 2 independent biological repeats were performed per
infection. Individual repeats are labelled in the heatmap (R1 and R2). (B) Representative enriched pathways shown as a heatmap of the enrichment scores
(Benjamini–Hochberg FDR < 0.05). (C) Box plot of changes in Ca2+ transporter proteins selected using Keywords across the different samples. The boxes
are color-coded according to the enrichment scores shown in panel B. (D) Transcription factor enrichment was performed using the “TF-LOF Expression
from GEO” library. (E) Box plot of protein changes of a gene set with known reduced expression upon CREB1 perturbation (therefore positively regulated
by CREB1). These proteins tend to be up-regulated in our data set, suggesting the activation of CREB1. The boxes are color-coded according to the
enrichment scores shown in panel D. https://doi.org/10.1371/journal.pbio.3000986.g004 Tir affects the abundance of Ca2+ transport and response proteins (C) Box plot of changes in Ca2+ transporter proteins selected using Keywords across the different samples. The boxes
are color-coded according to the enrichment scores shown in panel B. (D) Transcription factor enrichment was performed using the “TF-LOF Expression
from GEO” library. (E) Box plot of protein changes of a gene set with known reduced expression upon CREB1 perturbation (therefore positively regulated
by CREB1). These proteins tend to be up-regulated in our data set, suggesting the activation of CREB1. The boxes are color-coded according to the
enrichment scores shown in panel D. (F) Hierarchical clustering of proteins selected by creb1_gpl1261_gds3660_down (TF-LOF Expression from GEO)
and Calcium (Keywords). The underlying data for this figure can be found in S1 Data. ANOVA, analysis of variance; CREB1, cAMP responsive element
binding protein 1; EPEC, enteropathogenic Escherichia coli; FDR, false discovery rate; IFNγ, interferon gamma; GEO, Gene Expression Omnibus; TF-LOF,
transcription factor loss of function; Tir, translocated intimin receptor. https://doi.org/10.1371/journal.pbio.3000986.g004 PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986
December 30, 2020 10 / 31 PLOS BIOLOGY EPEC Tir triggers calcium influx-dependent pyroptosis death profile (Fig 4B). Strikingly, Ca2+ transport, protein folding in endoplasmic reticulum
(ER), and mitochondrial metabolism were among the most up-regulated pathways by EPEC-1
and EPEC-1-TirAA, and to a lesser extent by EPEC-1-TirAA-EspZ, compared with EPEC-2 and
EPEC-0 (Fig 4B). In particular, Ca2+ transport proteins showed a clear trend of up-regulation
correlating with the cell death gradient (Fig 4C). To investigate whether any of the observed proteomic changes could be explained by early
activation or inhibition of upstream factors, we performed enrichment analysis using expres-
sion gene sets from transcription factor perturbations from the Gene Expression Omnibus
(GEO) database [51]. This analysis identified cAMP responsive element binding protein
(CREB1) as the most significantly enriched transcription factor with enrichment scores posi-
tively correlating with the cell death gradient (Fig 4D and 4E). CREB1 is activated by Ca2+
influx and subsequently regulates the downstream expression of Ca2+ transport proteins as a
feedback response [52,53]. Proteins related to Ca2+ signalling, including the ER Ca2+ trans-
porters ATP2A2 (1 of the sarco/endoplasmic reticulum Ca2+ ATPases (SERCA); the main ER
Ca2+ importer) and inositol 1,4,5-trisphosphate receptor type 2 (ITPR2, ER Ca2+ release chan-
nel), Ca2+ response protein calnexin (CANX) which senses and regulates Ca2+ oscillations, and
several other Ca2+-binding proteins, were also predicted downstream of CREB1 signalling and
show changes in expression similar to the cell death gradient (Fig 4F). Tir affects the abundance of Ca2+ transport and response proteins In addition, ER and mitochondria are both major Ca2+ storage sites and modulated by Ca2+
signalling. Many enzymes involved in protein folding in ER and mitochondrial metabolism
require Ca2+ binding and are regulated by Ca2+ homeostasis [54,55]. Therefore, it is likely that
the up-regulation of ER protein folding and mitochondrial metabolism proteins during EPEC
infection resulted from enhanced Ca2+ signalling. Importantly, caspase-4 can be activated by
increased intracellular Ca2+ level, for example, ER Ca2+ release mediated by transmembrane
protein 173 (TMEM173) (also known as stimulator of interferon genes (STING)) [56] or the
SERCA inhibitor thapsigargin [57]. Based on our current findings and published data, we
hypothesised that a change in Ca2+ concentration in the cytosol modulates the viability of the
infected cells. Ca2+ influx mediates Tir-induced cell death Based on the changes in the abundance of the plasma membrane Ca2+ influx transporters and
the ER Ca2+ importer ATP2A2 along the cell death gradient (Fig 4), we hypothesised that
EPEC infection leads to Ca2+ influx. To test this hypothesis, we used the Fluo-4 Ca2+ reporter
to measure cytosolic-free Ca2+ levels. We found that 40 min postinfection with EPEC-1 and
EPEC-1-TirAA, SNU-C5 cells exhibit increased cytosolic Ca2+ levels compared with EPEC-
0-infected and uninfected cells (Fig 5A, S6A Fig). In addition, Tir-dependent Ca2+ influx was
detected in unprimed cells infected with either EPEC-1 or EPEC-1-TirAA at similar level to
primed cells, suggesting IFNγ treatment has negligible effect on Ca2+ influx (S7B Fig). Although mitochondrial proteins were up-regulated and ROS generated from respiration
as a result of Ca2+ influx could lead to cell death [58], we found that the ROS scavenger and the
respiration inhibitor oligomycin had no significant effect on Tir-induced cell death (S8 Fig). In order to investigate the source of Ca2+ influx in cell death, we pretreated SNU-C5 cells
with extracellular Ca2+ chelating agent EGTA. This completely diminished the increase in
cytosolic Ca2+ and resulted in the attenuation of pyroptosis induced by both EPEC-1 and
EPEC-1-TirAA (Fig 5C and 5D; S7A and S7B Fig), without affecting EPEC-1-mediated actin
polymerisation (Fig 5B). Moreover, cell death induced by EPEC-1 was significantly lower
when SNU-C5 cells were grown in Ca2+-free, compared with complete DMEM media (Fig
5E). Enumeration of CFU showed similar bacterial attachment in Ca2+-free medium (Fig 5F). Based on the changes in the abundance of the plasma membrane Ca2+ influx transporters and
the ER Ca2+ importer ATP2A2 along the cell death gradient (Fig 4), we hypothesised that
EPEC infection leads to Ca2+ influx. To test this hypothesis, we used the Fluo-4 Ca2+ reporter
to measure cytosolic-free Ca2+ levels. We found that 40 min postinfection with EPEC-1 and
EPEC-1-TirAA, SNU-C5 cells exhibit increased cytosolic Ca2+ levels compared with EPEC-
0-infected and uninfected cells (Fig 5A, S6A Fig). In addition, Tir-dependent Ca2+ influx was
detected in unprimed cells infected with either EPEC-1 or EPEC-1-TirAA at similar level to
primed cells, suggesting IFNγ treatment has negligible effect on Ca2+ influx (S7B Fig). PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986
December 30, 2020 Ca2+ influx mediates Tir-induced cell death The Fluo-4 emission at 40 min postinfection was plotted. Means ± SEM from n = 3 independent
biological repeats. (H) PI uptake into SNU-C5 cells grown in complete or Ca2+-free DMEM transfected with LPS with
or without 30 min pretreatment with YVAD. Means ± SEM from n = 3 independent biological repeats. Statistical
significance was determined using 2-way ANOVA with Bonferroni posttest (A, G, H) or 2-tailed t test (C-F).
p 0.05; p 0.01; p 0.001. The underlying data for this figure can be found in S1 Data. ANOVA, analysis of
variance; DAPI, 40,6-diamidino-2-phenylindole; EGTA, ethylene glycol-bis(β-aminoethyl ether)-N,N,N0,N0-tetraacetic
acid; EPEC, enteropathogenic Escherichia coli; PI, propidium iodide; SEM, standard error of the mean; Tir,
translocated intimin receptor; YVAD, z-YVAD-fmk. Fig 5. Tir-induced extracellular Ca2+ influx promotes cell death. (A) Fluo-4 assay of primed SNU-C5 cells infected
with EPEC-0, EPEC-1 and EPEC-1-TirAA with or without 30 min pretreatment with EGTA. The Fluo-4 emission at 40
min postinfection was plotted. Means ± SEM from n = 3 independent biological repeats. (B) Immunofluorescence
labelling of SNU-C5 cells infected with EPEC-1 for 2 h with and without EGTA pretreatment. DAPI: blue; EPEC:
green; Phalloidin: red. Representative images from 3 independent biological repeats are shown. Error bar: 5 μm. (C, D)
PI uptake into SNU-C5 cells infected with EPEC-1 (C) or EPEC-1-TirAA (D) with or without 30 min pretreatment
with EGTA. Means ± SEM from n = 3 independent biological repeats. (E) PI uptake into SNU-C5 cells grown in
complete DMEM or Ca2+-free DMEM infected with EPEC-1. Means ± SEM from n = 4 independent biological
repeats. (F) Adhesion of EPEC-1 to SNU-C5 cells grown in complete or Ca2+-free DMEM. Means ± SEM from n = 3
independent biological repeats. (G) Fluo-4 of SNU-C5 cells infected with EPEC-1 with or without 30 min pretreatment
with YVAD. The Fluo-4 emission at 40 min postinfection was plotted. Means ± SEM from n = 3 independent
biological repeats. (H) PI uptake into SNU-C5 cells grown in complete or Ca2+-free DMEM transfected with LPS with
or without 30 min pretreatment with YVAD. Means ± SEM from n = 3 independent biological repeats. Statistical
significance was determined using 2-way ANOVA with Bonferroni posttest (A, G, H) or 2-tailed t test (C-F).
p 0.05; p 0.01; p 0.001. The underlying data for this figure can be found in S1 Data. Ca2+ influx mediates Tir-induced cell death Means ± SEM from n = 3 independent biological repeats. (B) Immunofluorescence
labelling of SNU-C5 cells infected with EPEC-1 for 2 h with and without EGTA pretreatment. DAPI: blue; EPEC:
green; Phalloidin: red. Representative images from 3 independent biological repeats are shown. Error bar: 5 μm. (C, D
PI uptake into SNU-C5 cells infected with EPEC-1 (C) or EPEC-1-TirAA (D) with or without 30 min pretreatment
with EGTA. Means ± SEM from n = 3 independent biological repeats. (E) PI uptake into SNU-C5 cells grown in
complete DMEM or Ca2+-free DMEM infected with EPEC-1. Means ± SEM from n = 4 independent biological
repeats. (F) Adhesion of EPEC-1 to SNU-C5 cells grown in complete or Ca2+-free DMEM. Means ± SEM from n = 3
independent biological repeats. (G) Fluo-4 of SNU-C5 cells infected with EPEC-1 with or without 30 min pretreatmen
with YVAD. The Fluo-4 emission at 40 min postinfection was plotted. Means ± SEM from n = 3 independent
biological repeats. (H) PI uptake into SNU-C5 cells grown in complete or Ca2+-free DMEM transfected with LPS with
or without 30 min pretreatment with YVAD. Means ± SEM from n = 3 independent biological repeats. Statistical
significance was determined using 2-way ANOVA with Bonferroni posttest (A, G, H) or 2-tailed t test (C-F). Fig 5. Tir-induced extracellular Ca2+ influx promotes cell death. (A) Fluo-4 assay of primed SNU-C5 cells infected
with EPEC-0, EPEC-1 and EPEC-1-TirAA with or without 30 min pretreatment with EGTA. The Fluo-4 emission at 40
min postinfection was plotted. Means ± SEM from n = 3 independent biological repeats. (B) Immunofluorescence
labelling of SNU-C5 cells infected with EPEC-1 for 2 h with and without EGTA pretreatment. DAPI: blue; EPEC:
green; Phalloidin: red. Representative images from 3 independent biological repeats are shown. Error bar: 5 μm. (C, D)
PI uptake into SNU-C5 cells infected with EPEC-1 (C) or EPEC-1-TirAA (D) with or without 30 min pretreatment
with EGTA. Means ± SEM from n = 3 independent biological repeats. (E) PI uptake into SNU-C5 cells grown in
complete DMEM or Ca2+-free DMEM infected with EPEC-1. Means ± SEM from n = 4 independent biological
repeats. (F) Adhesion of EPEC-1 to SNU-C5 cells grown in complete or Ca2+-free DMEM. Means ± SEM from n = 3
independent biological repeats. (G) Fluo-4 of SNU-C5 cells infected with EPEC-1 with or without 30 min pretreatment
with YVAD. Ca2+ influx mediates Tir-induced cell death Although mitochondrial proteins were up-regulated and ROS generated from respiration
as a result of Ca2+ influx could lead to cell death [58], we found that the ROS scavenger and the
respiration inhibitor oligomycin had no significant effect on Tir-induced cell death (S8 Fig). In order to investigate the source of Ca2+ influx in cell death, we pretreated SNU-C5 cells
with extracellular Ca2+ chelating agent EGTA. This completely diminished the increase in
cytosolic Ca2+ and resulted in the attenuation of pyroptosis induced by both EPEC-1 and
EPEC-1-TirAA (Fig 5C and 5D; S7A and S7B Fig), without affecting EPEC-1-mediated actin
polymerisation (Fig 5B). Moreover, cell death induced by EPEC-1 was significantly lower
when SNU-C5 cells were grown in Ca2+-free, compared with complete DMEM media (Fig
5E). Enumeration of CFU showed similar bacterial attachment in Ca2+-free medium (Fig 5F). PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986
December 30, 2020 11 / 31 PLOS BIOLOGY EPEC Tir triggers calcium influx-dependent pyroptosis Fig 5. Tir-induced extracellular Ca2+ influx promotes cell death. (A) Fluo-4 assay of primed SNU-C5 cells infected
with EPEC-0, EPEC-1 and EPEC-1-TirAA with or without 30 min pretreatment with EGTA. The Fluo-4 emission at 40
min postinfection was plotted. Means ± SEM from n = 3 independent biological repeats. (B) Immunofluorescence
labelling of SNU-C5 cells infected with EPEC-1 for 2 h with and without EGTA pretreatment. DAPI: blue; EPEC:
green; Phalloidin: red. Representative images from 3 independent biological repeats are shown. Error bar: 5 μm. (C, D)
PI uptake into SNU-C5 cells infected with EPEC-1 (C) or EPEC-1-TirAA (D) with or without 30 min pretreatment
with EGTA. Means ± SEM from n = 3 independent biological repeats. (E) PI uptake into SNU-C5 cells grown in
complete DMEM or Ca2+-free DMEM infected with EPEC-1. Means ± SEM from n = 4 independent biological
repeats. (F) Adhesion of EPEC-1 to SNU-C5 cells grown in complete or Ca2+-free DMEM. Means ± SEM from n = 3
independent biological repeats. (G) Fluo-4 of SNU-C5 cells infected with EPEC-1 with or without 30 min pretreatment
with YVAD. The Fluo-4 emission at 40 min postinfection was plotted. Means ± SEM from n = 3 independent Fig 5. Tir-induced extracellular Ca2+ influx promotes cell death. (A) Fluo-4 assay of primed SNU-C5 cells infected
with EPEC-0, EPEC-1 and EPEC-1-TirAA with or without 30 min pretreatment with EGTA. The Fluo-4 emission at 4
min postinfection was plotted. PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986
December 30, 2020 Tir induces Ca2+-dependent pyroptosis on a primary epithelial cell line To determine if Tir-induced cell death is restricted to tumorigenic epithelial cells, we pro-
ceeded to use the primary human retinal pigment epithelium (RPE) cell, a non-tumorigenic
model cell line that has been previously used in epithelial cell death studies, including caspase-
4-dependent cell death [61]. Infection of RPE cells by EPEC WT and EPEC-1, but not EPEC-0,
showed actin pedestal formation, confirming EPEC attachment (Fig 7A). In IFNγ-primed
RPE cells, EPEC-1 induced a significantly higher level of cell death compared with both EPEC
WT and EPEC-0 (Fig 7B). Similar to SNU-C5, cell death induced by EPEC-1 in RPE cells was
inhibited by the pyroptosis inhibitor YVAD as well as by the chelation of extracellular Ca2+
(Fig 7C and 7D). This suggests that the ability of Tir to induce Ca2+-dependent pyroptosis is
conserved across the tumorigenic and primary epithelial cell lines. Ca2+ influx mediates Tir-induced cell death ANOVA, analysis of
variance; DAPI, 40,6-diamidino-2-phenylindole; EGTA, ethylene glycol-bis(β-aminoethyl ether)-N,N,N0,N0-tetraacetic
acid; EPEC, enteropathogenic Escherichia coli; PI, propidium iodide; SEM, standard error of the mean; Tir,
translocated intimin receptor; YVAD, z-YVAD-fmk. https://doi.org/10.1371/journal.pbio.3000986.g005 https://doi.org/10.1371/journal.pbio.3000986.g005 12 / 31 PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986
December 30, 2020 PLOS BIOLOGY EPEC Tir triggers calcium influx-dependent pyroptosis While the GSDMD pore has been previously shown to induce Ca2+ influx [59], YVAD did
not affect Tir-induced Ca2+ influx, suggesting that this event lies upstream of caspase-4 and
GSDMD cleavage (Fig 5G, S7C Fig). Notably, cell death caused by LPS transfection was not
reduced in Ca2+-free DMEM (Fig 5H), indicating that extracellular Ca2+ influx is not necessary
for activation of caspase-4 by cytosolic LPS. To further substantiate the role of Ca2+ in cell death, we used ATP, an agonist of the
Ca2+-permeable purinergic channels [60], to pharmacologically induce Ca2+ influx before
EPEC infection. First, to confirm the effect of ATP on Ca2+ influx on SNU-C5 cells, we per-
formed Fluo-4 assay on cells treated with 0.5 mM ATP, which revealed increased Ca2+ influx
within 2 min after ATP treatment (Fig 6A). We then performed PI uptake assays on EPEC-0
and EPEC-1-infected cells pretreated with ATP for 30 min. ATP treatment significantly
enhanced cell death by EPEC-1 but not EPEC-0, while ATP alone did not affect the viability of
uninfected cells (Fig 6B). Therefore, although Ca2+ augments Tir-dependent cell death, it can-
not substitute Tir. Furthermore, treating cells with EGTA before the addition of ATP reduced
the rapid Ca2+ uptake and Tir-induced cell death (Fig 6C and 6D). To study whether ATP-
induced Ca2+ influx enhances Tir-dependent cell death via caspase-4, we treated the cells with
YVAD which by itself did not inhibit Ca2+ influx (Fig 6C). YVAD prevented ATP-dependent
increase in Tir-induced cell death, suggesting that increased Ca2+ influx cannot bypass the
need of caspases (Fig 6D). In support of this, we also found that ATP failed to promote Tir-
induced cell death in the presence of NleF (Fig 6E). Importantly, ATP pretreatment did not
elevate cell death caused by LPS transfection (Fig 6F). Hence, Ca2+ influx specifically promotes
Tir-induced cell death. PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986
December 30, 2020 Tir induces Ca2+-dependent LPS internalisation Our data thus far show that Tir-induced cell death is dependent on Ca2+ influx and caspase-4
activation. In order to mechanistically integrate these observations, we investigated if Ca2+
influx could promote LPS internalisation. To this end, E. coli LPS conjugated with Alexa-488
was added as a tracer before infection. Fluorescent LPS was detected as clusters surrounding
the perinuclear region in approximately a third of EPEC-1- and EPEC-1-TirAA-infected cells,
which was significantly higher than EPEC-0-infected cells (Fig 8A and 8B), suggesting that Tir
promotes the internalisation of LPS independent of actin polymerisation. Depletion of extra-
cellular Ca2+ reduced LPS perinuclear localisation (Fig 8A), indicating the role of Ca2+ in the
LPS internalisation process. The requirement of both Ca2+ and Tir for LPS internalisation mir-
rors the Tir-induced cell phenotype. Taken together, these results show a novel cascade leading
to EPEC-induced cell death, starting with Tir translocation and clustering and induction of
Ca2+ influx, which promote LPS internalisation and activation of caspase-4. PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986
December 30, 2020 13 / 31 PLOS BIOLOGY EPEC Tir triggers calcium influx-dependent pyroptosis Fig 6. Pharmacologically induced Ca2+ influx promotes Tir-dependent cell death upstream of pyroptotic caspase
activation. (A) Fluo-4 assay of SNU-C5 cells with or without 2 min pretreatment with ATP. Means ± SEM from n = 3
independent biological repeats. (B) PI uptake into SNU-C5 cells infected with EPEC-0 or EPEC-1, with or without 30
min pretreatment with ATP. Means ± SEM from n = 6 (UI and EPEC-1) and 3 (EPEC-0) independent biological
repeats. (C, D) PI uptake into SNU-C5 cells infected with EPEC-1 with 30 min pretreatment with ATP, EGTA, or
YVAD or 45 min pretreatment with EGTA or YVAD, followed by ATP for 30 min. The order of treatment in each
sample is indicated by the arrow. Fluo-4 assay was performed on the uninfected cells with the same treatment at 2 min
after ATP addition. Means ± SEM from n = 5 (EPEC-1 and EPEC-1 + ATP in D) and 3 (C, D) independent biological
repeats. (E) PI uptake into SNU-C5 cells infected with EPEC-1 and EPEC-1-NleF with or without 30 min pretreatment
with ATP. Means ± SEM from n = 3 independent biological repeats. (F) PI uptake into SNU-C5 cells transfected with
LPS with or without pretreatment of ATP for 30 min. Means ± SEM from n = 4 independent biological repeats are
shown. PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986
December 30, 2020 Tir induces Ca2+-dependent LPS internalisation https://doi.org/10.1371/journal.pbio.3000986.g006 14 / 31 PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986
December 30, 2020 PLOS BIOLOGY EPEC Tir triggers calcium influx-dependent pyroptosis Fig 7. EPEC induces Tir- and Ca2+-dependent pyroptosis in RPE cells. (A) Immunofluorescence labelling of RPE
cells infected with EPEC WT, EPEC-0 and EPEC-1 for 2 h. DAPI: blue; EPEC: green; Phalloidin: red. Representative
images from n = 3 independent biological repeats. Scale bar: 5 μm. Example bacteria are marked with white arrows. (B)
PI uptake into primed RPE cells infected with EPEC WT, EPEC-0, and EPEC-1 for 8 h. Means ± SEM from n = 3
independent biological repeats. (C) PI uptake into RPE cells in the presence or absence of YVAD and infected with
EPEC-1. Means ± SEM from n = 3 independent biological repeats. (D) PI uptake into RPE cells infected with EPEC-1
in complete DMEM or Ca2+-free DMEM. Means ± SEM from n = 3 independent biological repeats. Statistical
significance was determined using 1-way ANOVA with Tukey posttest (B) or 2-tailed t test (C, D) ns, nonsignificant;
p 0.05; p 0.01. The underlying data for this figure can be found in S1 Data. ANOVA, analysis of variance;
DMEM, Dulbecco’s Modified Eagle’s medium; EGTA, ethylene glycol-bis(β-aminoethyl ether)-N,N,N0,N0-tetraacetic
acid; EPEC, enteropathogenic Escherichia coli; PI, propidium iodide; SEM, standard error of the mean; Tir,
translocated intimin receptor; YVAD, z-YVAD-fmk. Fig 7. EPEC induces Tir- and Ca2+-dependent pyroptosis in RPE cells. (A) Immunofluorescence labelling of RPE
cells infected with EPEC WT, EPEC-0 and EPEC-1 for 2 h. DAPI: blue; EPEC: green; Phalloidin: red. Representative
images from n = 3 independent biological repeats. Scale bar: 5 μm. Example bacteria are marked with white arrows. (B)
PI uptake into primed RPE cells infected with EPEC WT, EPEC-0, and EPEC-1 for 8 h. Means ± SEM from n = 3
independent biological repeats. (C) PI uptake into RPE cells in the presence or absence of YVAD and infected with
EPEC-1. Means ± SEM from n = 3 independent biological repeats. (D) PI uptake into RPE cells infected with EPEC-1
in complete DMEM or Ca2+-free DMEM. Means ± SEM from n = 3 independent biological repeats. Statistical
significance was determined using 1-way ANOVA with Tukey posttest (B) or 2-tailed t test (C, D) ns, nonsignificant;
p 0.05; p 0.01. The underlying data for this figure can be found in S1 Data. Tir induces Ca2+-dependent LPS internalisation Statistical significance was determined using 2-way ANOVA with Bonferroni posttest (A), 2-tailed t test (B, E,
F) or 1-way ANOVA with Tukey posttest (C, D). ns, nonsignificant; p 0.05; p 0.01; p 0.001. The
underlying data for this figure can be found in S1 Data. ANOVA, analysis of variance; ATP, adenosine triphosphate;
EGTA, ethylene glycol-bis(β-aminoethyl ether)-N,N,N0,N0-tetraacetic acid; EPEC, enteropathogenic Escherichia coli;
PI, propidium iodide; SEM, standard error of the mean; Tir, translocated intimin receptor; YVAD, z-YVAD-fmk. https://doi org/10 1371/journal pbio 3000986 g006 Fig 6. Pharmacologically induced Ca2+ influx promotes Tir-dependent cell death upstream of pyroptotic caspase
activation. (A) Fluo-4 assay of SNU-C5 cells with or without 2 min pretreatment with ATP. Means ± SEM from n = 3
independent biological repeats. (B) PI uptake into SNU-C5 cells infected with EPEC-0 or EPEC-1, with or without 30
min pretreatment with ATP. Means ± SEM from n = 6 (UI and EPEC-1) and 3 (EPEC-0) independent biological
repeats. (C, D) PI uptake into SNU-C5 cells infected with EPEC-1 with 30 min pretreatment with ATP, EGTA, or
YVAD or 45 min pretreatment with EGTA or YVAD, followed by ATP for 30 min. The order of treatment in each
sample is indicated by the arrow. Fluo-4 assay was performed on the uninfected cells with the same treatment at 2 min
after ATP addition. Means ± SEM from n = 5 (EPEC-1 and EPEC-1 + ATP in D) and 3 (C, D) independent biological
repeats. (E) PI uptake into SNU-C5 cells infected with EPEC-1 and EPEC-1-NleF with or without 30 min pretreatment
with ATP. Means ± SEM from n = 3 independent biological repeats. (F) PI uptake into SNU-C5 cells transfected with
LPS with or without pretreatment of ATP for 30 min. Means ± SEM from n = 4 independent biological repeats are
shown. Statistical significance was determined using 2-way ANOVA with Bonferroni posttest (A), 2-tailed t test (B, E,
F) or 1-way ANOVA with Tukey posttest (C, D). ns, nonsignificant; p 0.05; p 0.01; p 0.001. The
underlying data for this figure can be found in S1 Data. ANOVA, analysis of variance; ATP, adenosine triphosphate;
EGTA, ethylene glycol-bis(β-aminoethyl ether)-N,N,N0,N0-tetraacetic acid; EPEC, enteropathogenic Escherichia coli;
PI, propidium iodide; SEM, standard error of the mean; Tir, translocated intimin receptor; YVAD, z-YVAD-fmk. Tir induces Ca2+-dependent LPS internalisation ANOVA, analysis of variance;
DMEM, Dulbecco’s Modified Eagle’s medium; EGTA, ethylene glycol-bis(β-aminoethyl ether)-N,N,N0,N0-tetraacetic
acid; EPEC, enteropathogenic Escherichia coli; PI, propidium iodide; SEM, standard error of the mean; Tir,
translocated intimin receptor; YVAD, z-YVAD-fmk. PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986
December 30, 2020 Discussion In this study, we investigate if EPEC can trigger cell death in IECs. We show that similar to
macrophages (both THP1 and primary human monocyte-derived macrophages) [25], infec-
tion of SNU-C5 cells induced PI uptake that was dependent on Tir, with the magnitude of cell
death directly correlated with the extent of Tir translocation. Importantly, in contrast to mac-
rophages, EPEC-induced cell death in IECs was independent of both actin polymerisation and
the canonical inflammasome (i.e., NLRP3 and caspase-1). This study provides the first evi-
dence for caspase-4-dependent pyroptosis in the absences of NLRP3 and caspase-1 in IECs
upon EPEC infection. The cell death of SNU-C5 was independent of caspase-8 or caspase-5 (which was undetect-
able) or necroptosis but, similar to macrophages [25], dependent on caspase-4 and GSDMD. Tir-induced pyroptosis was enhanced by IFNγ, which triggered expression of caspase-4 and
GSDMD in SNU-C5, while cell lines endogenously expressing low level of GSDMD were
refractory to EPEC-induced cell death. These results demonstrate not only that the genetic
background of host cells affects susceptibility to infection, but also that GSDMD is the rate lim-
iting factor impacting on EPEC-induced cell death. PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986
December 30, 2020 15 / 31 PLOS BIOLOGY EPEC Tir triggers calcium influx-dependent pyroptosis Fig 8. Tir promotes Ca2+-dependent LPS internalisation. (A) Alexa-488 conjugated LPS (or non-fluorescent LPS as
negative control for autofluorescence) and EGTA was added to primed SNU-C5 cells 30 min before infection. Cells
were infected by EPEC-0, EPEC-1 and EPEC-1-TirAA for 2 h. DAPI: blue; LPS: green; EPEC: purple. Representative
images from n = 4 biological repeats are shown. Scale bar: 5 μm. Enlarged images of the bacteria (autofluorescence)
and perinuclear regions of infected cells are shown with blue and red borders, respectively. (B) The percentage of cells
with perinuclear LPS clusters. Means ± SEM from n = 4 independent biological repeats are shown. Statistical
significance was determined using 1-way ANOVA with Tukey posttest. p 0.05. The underlying data for this figure
can be found in S1 Data. ANOVA, analysis of variance; DAPI, 40,6-diamidino-2-phenylindole; EGTA, ethylene glycol-
bis(β-aminoethyl ether)-N,N,N0,N0-tetraacetic acid; EPEC, enteropathogenic Escherichia coli; LPS, lipopolysaccharide;
SEM, standard error of the mean; Tir, translocated intimin receptor. https://doi.org/10.1371/journal.pbio.3000986.g008 Fig 8. Tir promotes Ca2+-dependent LPS internalisation. (A) Alexa-488 conjugated LPS (or non-fluorescent LPS as
negative control for autofluorescence) and EGTA was added to primed SNU-C5 cells 30 min before infection. PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986
December 30, 2020 Discussion Cells
were infected by EPEC-0, EPEC-1 and EPEC-1-TirAA for 2 h. DAPI: blue; LPS: green; EPEC: purple. Representative
images from n = 4 biological repeats are shown. Scale bar: 5 μm. Enlarged images of the bacteria (autofluorescence)
and perinuclear regions of infected cells are shown with blue and red borders, respectively. (B) The percentage of cells
with perinuclear LPS clusters. Means ± SEM from n = 4 independent biological repeats are shown. Statistical
significance was determined using 1-way ANOVA with Tukey posttest. p 0.05. The underlying data for this figure
can be found in S1 Data. ANOVA, analysis of variance; DAPI, 40,6-diamidino-2-phenylindole; EGTA, ethylene glycol-
bis(β-aminoethyl ether)-N,N,N0,N0-tetraacetic acid; EPEC, enteropathogenic Escherichia coli; LPS, lipopolysaccharide;
SEM, standard error of the mean; Tir, translocated intimin receptor. https://doi.org/10.1371/journal.pbio.3000986.g008 EPEC-induced cell death is not limited to tumorigenic cells, as the primary cell line RPE
showed similar susceptibility. Induction of pyroptosis during EPEC infection could benefit the
host, by means of secretion of pro-inflammatory cytokines (e.g., IL-18) [11] and specific elimi-
nation of infected cells, as cell death is contact dependent (i.e., intimin–Tir interactions). Indeed, the EPEC-like murine pathogen Citrobacter rodentium [62,63] lacking EspZ is highly
attenuated [64], which is consistent with our findings that uncontrolled Tir translocation leads
to removal of infected cells by pyroptosis. On the pathogen side, as intimin–Tir interactions,
which are essential for colonisation, lead to unintentional cell death, EPEC injects NleF, an
effector expressed in the majority of typical EPEC strains [65], to mitigate the impact by inhib-
iting caspase-4 and blocking pyroptosis. NleF was also found to be associated more frequently
with diarrhoeagenic cases compared with asymptomatic cases in atypical EPEC isolates
recently found in Brazil [66], indicating the importance of cell death inhibition in bacterial vir-
ulence. Moreover, our data suggest that Tir-induced actin polymerisation also contributes to
the moderation of cell death in IECs. This would benefit the pathogen as it could preserve the
colonisation site and sustain the infection. PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986
December 30, 2020 16 / 31 PLOS BIOLOGY EPEC Tir triggers calcium influx-dependent pyroptosis To mechanistically link plasma membrane-associated Tir with cytosolic caspase-4, we per-
formed proteomic analysis to identify proteins and pathways correlated with the cell death lev-
els induced by the different EPEC variants. This revealed the remodelling of diverse cellular
processes upon IFNγ treatment and EPEC infection. Discussion Several pathways and proteins strongly
correlated with cell death levels were induced by the different EPEC strains. Others showed a
negative correlation with cell death level, including cell cycle, cell division, and protein degra-
dation machinery, which have also been demonstrated in previous researches to be released by
pyroptotic cells [67]. Notably, Ca2+-related cellular processes, including activation of the tran-
scription factor CREB1, clearly correlated with the cell death gradient. While infection resulted
in higher abundance of Ca2+ transporters, the precise route of Ca2+ influx remains unclear. The rapid onset of Ca2+ influx independent of priming suggests the involvement of an IFNγ-
independent posttranslational mechanism. Intimin-mediated Tir clustering has been shown to
cause plasma membrane curvature, resulting in localised membrane disruption that could
facilitate the influx of Ca2+. This hypothesis is consistent with the correlation between the level
of Tir translocation and the magnitude of pyroptosis. Tir clustering may trigger the activation
of Ca2+ channels in the plasma membrane. For example, members of the transient receptor
potential (TRP) cation channel family that responds to mechanical changes in the local mem-
brane [68,69] could be activated by such stimulus. In addition, E. coli LPS can intercalate into
the plasma membrane leading to membrane compression which activates certain members of
the TRP family [69–72]. TRPV2 and TRPM7, expressed in SNU-C5 [39], can facilitate Ca2+
influx in response to both membrane stretch [73–75] and LPS exposure [76,77]. The ability of
TRPV2 to promote LPS-dependent NF-κB activation [76] also mirrors our finding during
EPEC-1 and EPEC-1-TirAA infection. While extracellular Ca2+ is essential for Tir-induced pyroptosis, it is dispensable for cell
death by caspase-4 in another scenario, for example, the delivery of LPS by transfection. Indeed, our results indicate that the role of Ca2+ influx is in promoting LPS internalisation. Increased intracellular Ca2+ concentration can activate diverse cell signalling events, while
Ca2+ overload in mitochondria can lead to opening of the mitochondrial permeability tran-
sition pore, release of cytochrome c, and ROS production [58,78]. However, we have shown
that neither ROS nor apoptosis are involved in EPEC-induced cell death in SNU-C5 cells. While inhibiting Ca2+ influx by extracellular Ca2+ chelation and growing SNU-C5 cells in
Ca2+-free medium inhibited Tir-induced cell death, inducing a sharp preinfection Ca2+
influx by ATP increased the level of Tir-induced pyroptosis, while not affecting LPS-
induced cell death. PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986
December 30, 2020 Discussion Together, these findings strongly suggest that Ca2+ influx plays a
key role upstream of cell death. Notably, Ca2+ influx alone did not induce cell death in the
absence of Tir, suggesting that Ca2+ synergies but does not replace Tir-dependent down-
stream processes. Recent studies have shown that E. coli and Streptococcus pneumoniae induce cytosolic Ca2+
up-regulation in murine macrophages, which was dependent on the cooperation between
the mitochondrial proteins TMEM173 and ITPR1 (the main ER Ca2+ release channel) [56]. Moreover, TMEM173-mediated Ca2+ release from intracellular stores promoted cleavage of
GSDMD by caspase-1/11 or caspase-8 in response to E. coli or S. pneumoniae, respectively
[56]. Conversely, the overexpression of ATP2A2, which was found to be up-regulated by Tir
and predicted to be downstream of CREB1 in our study, limited E. coli and S. pneumoniae-
induced GSDMD N-terminus fragment formation [56], suggesting the presence of negative
feedback during Tir-induced cell death. Intracellular compartmentalisation of LPS was detected in Tir- and Ca2+-dependent man-
ners. LPS uptake has been attributed to multiple plasma membrane proteins, including the
LPS-binding protein (LBP) and TLR4 [79,80]. Although TLR4-dependent LPS uptake also PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986
December 30, 2020 17 / 31 PLOS BIOLOGY EPEC Tir triggers calcium influx-dependent pyroptosis Fig 9. A model portraying the pathway of EPEC-induced cell death. EPEC infection is initiated with Tir
translocation. Binding of intimin leads to intimate attachment, Tir clustering, and local membrane curvature, which
could activate Ca2+ transporters and influx. Ca2+ influx leads to LPS entry, which can lead to the activation of caspase-4
and pyroptosis, while NleF can inhibit caspase-4 activation. IFNγ priming induces expression of caspase-4 and
GSDMD, enhancing cell death. EPEC, enteropathogenic Escherichia coli; GSDMD, gasdermin D; IFNγ, interferon
gamma; LPS, lipopolysaccharide; Tir, translocated intimin receptor. https://doi.org/10.1371/journal.pbio.3000986.g009 Fig 9. A model portraying the pathway of EPEC-induced cell death. EPEC infection is initiated with Tir
translocation. Binding of intimin leads to intimate attachment, Tir clustering, and local membrane curvature, which
could activate Ca2+ transporters and influx. Ca2+ influx leads to LPS entry, which can lead to the activation of caspase-4
and pyroptosis, while NleF can inhibit caspase-4 activation. IFNγ priming induces expression of caspase-4 and
GSDMD, enhancing cell death. EPEC, enteropathogenic Escherichia coli; GSDMD, gasdermin D; IFNγ, interferon
gamma; LPS, lipopolysaccharide; Tir, translocated intimin receptor. https://doi.org/10.1371/journal.pbio.3000986.g009 https://doi.org/10.1371/journal.pbio.3000986.g009 https://doi.org/10.1371/journal.pbio.3000986.g009 requires extracellular Ca2+ influx [77], TLR4 is not detected in the SNU-C5 proteome [39]. Discussion Scavenger receptor-mediated LPS uptake and the subsequent perinuclear compartmentalisa-
tion have been characterised in HeLa cell overexpressing the scavenger receptor class B mem-
ber 1 (SCARB1) [81]. SCARB1 and the LPS-binding apolipoproteins apolipoprotein B
(APOB) and apolipoprotein E (APOE) are up-regulated upon EPEC-1 and EPEC-1-TirAA
infections correlating with the cell death gradient, suggesting a possible role of these proteins
in LPS uptake. Intimin-mediated Tir clustering has been shown to cause plasma membrane curvature,
which could facilitate the influx of both Ca2+ and LPS. This hypothesis is consistent with the
correlation between the level of Tir translocation and the magnitude of pyroptosis. As Ca2+
has been shown to stabilise LPS at the bacterial outer membrane [82,83], it may also facilitate
adhesion of free LPS to the plasma membrane of infected cells, which likely increases its
chance of entry via Tir-induced local membrane permeability. Overall, our results converge to
a novel pathway, which relies on influx of Ca2+ through the plasma membrane during infec-
tion with an extracellular pathogen, in a Tir-dependent manner, leading to LPS internalisation
followed by caspase-4-mediated GSDMD cleavage (Fig 9). Materials and methods Bacterial strains and cell lines Infection of IECs Cells were seeded 1 day before infection. EPEC strains were primed by diluting the overnight
cultures 50× in non-supplemented DMEM (low glucose) and growing for 3 h static at 37˚C
with 5% CO2. Isopropyl β-d-1-thiogalactopyranoside (IPTG) (Sigma-Aldrich) at 1 mM was
added to the bacterial culture 30 min before infection when required. Infection was carried out
at a multiplicity-of-infection (MOI) of 50:1. Spent medium was replaced with fresh serum-free
RPMI (SNU-C5, HT-29, SNU-C2B, COLO-320-HSR and THP-1) or DMEM medium (RPE) 1
h before infection. SNU-C5 and RPE cells for infection in Ca2+-free condition were grown
either in Ca2+-free DMEM (high glucose) (Gibco) (supplemented with 1 mM sodium pyruvate
to the same level as Ca2+-containing DMEM) or Ca2+-containing DMEM (high glucose)
(Sigma-Aldrich) as control from 1 h before infection. Infected cells were centrifuged at 700 g
for 10 min and incubated for 2 h static at 37˚C, 5% CO2. For over 2 h of infection, 200 μg/ml
gentamicin (Sigma-Aldrich) or 250 μg/ml kanamycin (Sigma-Aldrich) (for EPEC-1-TirAA
strain) was added 2 h postinfection. Generating of EPEC-1-TirAA strain Genomic DNA from WT EPEC was extracted using the DNeasy Blood & Tissue Kit (Qiagen,
Hilden, Germany). All primers were ordered from Merck (Darmstadt, Germany). Primers
1.1.1 and 1.1.2 were used to amplify tir gene from the genomic DNA. The tir gene and the pSE-
VA612S plasmid were digested by BamHI and HindIII and ligated to make pSEVA612S-tir fol-
lowed by transformation into E. coli CC118λpir. Primers 1.3.2 and 1.4.2 were used to mutate
the Y454 and Y474 in pSEVA612S-tir to alanine. The resulting linear PCR product was
digested by DpnI and ligated to create pSEVA612S-tirAA plasmid and then transformed into E. coli CC118λpir. Mutations were confirmed by plasmid sequencing (Eurofins Genomics, Con-
stance, Germany) using primers M13-FP, M13-RP, 1.7, and 1.8. pSEVA612S-tirAA plasmid was conjugated from E. coli CC118λpir pSEVA612S-tirAA into
EPEC-0 (containing pACBSR plasmid) using the helper strain E. coli 1047 pRK2013. Colonies
that grew on agar plates with both gentamicin and streptomycin were selected, and chromo-
somal integration of pSEVA612S-tirAA was confirmed by colony PCR using primers 1.5
and 1.6. All plasmid descriptions and primer sequences are listed in S1B and S1C Table,
respectively. Bacterial strains and cell lines EPEC E2348/69 strains (S1A Table) were grown in Luria Bertani (LB) (Sigma-Aldrich,
St. Louis, Missouri, United States of America) broth or agar. Overnight bacterial cultures were 18 / 31 PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986
December 30, 2020 PLOS BIOLOGY EPEC Tir triggers calcium influx-dependent pyroptosis grown at 37˚C 180 revolutions per minute (rpm) shaking (liquid) or static (agar) and primed
in Dulbecco’s Modified Eagle Medium (DMEM) as described below for infections. SNU-C5, HT-29, SNU-C2B, COLO-320-HSR, and THP-1 cell lines were cultured in Ros-
well Park Memorial Institute (RPMI) medium (Sigma-Aldrich) with 10% (v/v) foetal bovine
serum (FBS) (Gibco, Carlsbad, California, USA), 2 mM Glutamax (Gibco), 1 mM sodium
pyruvate (Sigma-Aldrich), 10 mM N-2-hydroxyethylpiperazine-N-2-ethane sulphonic acid
(HEPES) (Sigma-Aldrich), and 2,500 μg/ml glucose (Sigma-Aldrich). THP-1 cells were differ-
entiated using 100 ng/ml phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich) for 48 h
and PMA withdrawn for 24 h. RPE cell line was cultured in DMEM/F-12 medium (Sigma-
Aldrich) with 10% (v/v) FBS, 2 mM Glutamax, and 0.26% sodium bicarbonate (Gibco). HEK293E cell line was cultured in DMEM (high glucose, 4,500 mg/L) (Sigma-Aldrich) with
10% (v/v) FBS, 1 mM sodium pyruvate, and 10 mM HEPES (pH 7.4). siRNA transfection A total of 1.25 × 104 cells/well were seeded in black clear-bottom 96-well plates 3 days prior to
infection. In addition, 50 mM siRNA (0.25 μl) (Dharmacon, Lafayette, Colorado, USA) (S1D
Table) mixed with 0.3 μl of TransIT-X2 transfection reagent (Mirus Bio, Madison, Wisconsin,
USA) in 9 μl Opti-MEM were added to the cells 2 days before infection (caspase-4 siRNA) or
6 h after seeding, i.e., 3 days before infection (GSDMD, caspase-8 and RIPK3 siRNA). The
medium was replaced with IFNγ-containing RPMI 1 day before infection. Immunofluorescence staining A total of 1.5 × 105 cells/well were seeded in 24-well plates on glass coverslips for imaging with
Zeiss AxioImager Z1 microscope (Carl Zeiss, Jena, Germany). A total of 1.5 × 104 cells/well
were seeded in black clear-bottom 96-well plates for imaging with Opera Cell::Explorer auto-
mated spinning-disk confocal microscope (Perkin-Elmer, Waltham, Massachusetts, USA). Infection experiments were carried out as described. Alexa-488-conjugated E. coli O55:B5 LPS
(Thermo Fisher Scientific, Waltham, Massachusetts, USA) was added to the cells at 5 μg/ml 30
min before infection when required. Cells were fixed by 4% paraformaldehyde for 15 min,
washed by 3× PBS, permeabilised by 0.2% Triton X-100 (Sigma-Aldrich) for 4 min, washed
again and blocked with 1% bovine serum albumin (BSA) for 10 min before being incubated
with primary antibodies (S1E Table) for 45 min. Cells were re-blocked with 1% BSA for 10
min and incubated with secondary antibodies (S1E Table) for 30 min. For imaging using Z1
microscope using 100× oil lens, coverslips were mounted on glass slides (VWR, Radnor, Penn-
sylvania, USA) with Gold-Pro-Long-Anti-fade (Invitrogen). Cytokine and drug treatment A total of 10 ng/ml human recombinant-IFNγ (R&D Systems, Minneapolis, Minnesota, USA)
was added to the cells 24 h before infection. Moreover, 50 μM z-VAD-fmk (zVAD) (R&D 19 / 31 PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986
December 30, 2020 PLOS BIOLOGY EPEC Tir triggers calcium influx-dependent pyroptosis Systems), 50 μM z-YVAD-fmk (YVAD) (R&D Systems), 25 μM necrostatin-1 (Nec1) (Santa
Cruz Biotechnology, Dallas, Texas, USA), 5 μM necrosulfonamide (NSA) (Tocris), 200 nM cyto-
chalasin D (Sigma-Aldrich), 1 mM EGTA (Sigma-Aldrich), 0.5 mM ATP (Sigma-Aldrich), 5 μM
MCC950 (Tocris), 200 μM Tiron (Sigma-Aldrich), 50 mM N,N0-dimethylthiourea (DMTU)
(Sigma-Aldrich), 10 μM oligomycin (Sigma-Aldrich) or 2 μM staurosporine (STS) (Calbiochem,
San Diego, California, USA) were added to the cells 30 min before infection. For co-treatment,
ATP was added 30 min before infection, while EGTA or YVAD were added 45 min before infec-
tion. Ultrapure E. coli O111:B4 LPS (Invivogen, San Diego, California, USA) transfection was
performed using Lipofectamine 2000 (Invitrogen, Waltham, Massachusetts, USA) at 5 μg/ml. GSDMD stable silencing Retroviral plasmid pMX-CMV-YFP-LacZ-miRNA30E and pMX-CMV-YFP-GSDMD-miR-
NA30E (S1B and S1D Table) [25, 50, 84] were packaged in HEK293E cells with the packaging
plasmid pCMV-MMLV-Gag/Pol and pCMV-VSV-G, in a 5:4:1 ratio, using Lipofectamine
2000. After 48 h of viral transfection, the medium containing the virus was filtered through a
0.45-μm filter and transferred to pre-seeded SNU-C5 cells. Puromycin (5 μg/ml) (Gibco,
Carlsbad, California, USA) was added to the SNU-C5 cells 48 h after viral transduction. Cells
were sorted by FACS Aria III Flow Sorter in CMBI high throughput single-cell analysis facility
(HTSCAF) for yellow fluorescent protein (YFP) expression. Puromycin treatment was contin-
ued after sorting to maintain a stable YFP-expressing population. Infection rate measurement A total of 1.5 × 105 cells/well were seeded in 24-well plates. Infection was carried out 1 day
post-seeding as described for 2 h (no antibiotic). Infected cells were washed by 3× PBS and
treated by 0.5% porcine pancreas trypsin and 0.02% EDTA in PBS (Sigma-Aldrich) for count-
ing by haemocytometer (Hecht Glaswarenfabrik, Rho¨n, Germany). Counted cells were lysed
with 0.1% Triton X-100, serially diluted and plated. Colony forming units (CFUs) were
counted after overnight incubation at 37˚C. Infection rate was calculated by CFU per cell. High-content image acquisition and analysis Imaging was performed using an Opera Cell::Explorer automated spinning-disk confocal
microscope using 40× lens (Perkin-Elmer). Twenty-five evenly distributed fields were imaged
per well. Columbus-2 System (Perkin-Elmer) was used for image analysis. Nucleus and cyto-
solic segmentation was performed using the DAPI and phalloidin staining, respectively. NF-κB activation was represented by the p65 nuclear-to-cytosolic ratio measured and cal-
culated by Columbus-2. The nucleus region for p65 intensity measurement was 2 pixels
inward from the nuclear–cytosolic boundary defined by DAPI to avoid segmentation errors
at the boundary. The cytosolic region was a 2-pixel-ring further 2-pixel-outward from the
nuclear–cytosolic boundary to avoid measurement skewing by cell shape [85]. Western blotting Cells were lysed with lysis buffer containing radioimmunoprecipitation (RIPA) buffer (50 mM
Tris-HCl, pH 8, 2 mM EDTA, 300 mM NaCl, 2% NP-40, 1% sodium deoxycholate) and 1× PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986
December 30, 2020 20 / 31 PLOS BIOLOGY EPEC Tir triggers calcium influx-dependent pyroptosis protease inhibitor (Pierce, Waltham, Massachusetts, USA) and mixed with 1× Laemmli sample
buffer (Bio-Rad, Hercules, California, USA) and 5% β-mercaptoethanol (Sigma-Aldrich). The
lysates were run on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)
gels. Western blotting was performed using a TransBlot Turbo Transfer System (Bio-Rad) to
transfer the protein bands to the polyvinylidene difluoride (PVDF) membrane. Membranes
were blocked with 5% milk in phosphate buffered saline, 0.05% Tween 20 (PBST) or tris buff-
ered saline, 0.1% Tween 20 (TBST) for 1 h at room temperature and probed with primary anti-
bodies (S1F Table) overnight at 4˚C. The secondary antibodies (S1F Table) were added for 1 h
at room temperature. Membranes were developed using the ECL Western Blotting Reagents
(GE, Amersham, UK) and imaged using the ChemiDoc MP imaging system (Bio-Rad). PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986
December 30, 2020 Agilent Seahorse metabolic assays A total 1.5 × 104 cells/well were seeded in XFp Cell Culture Miniplates (Agilent Technologies,
Santa Clara, California, USA) 1 day prior to measurement. Cells were incubated at 37˚C in
non-CO2 incubator for 45 min in XF RPMI base medium with 2 mM glutamine, 1 mM
sodium pyruvate, and 10 mM glucose, with pH 7.4. Measurements of oxygen consumption
rate (OCR) were carried out in a Seahorse XFp Analyzer (Agilent Technologies). After mea-
surements, OCR was normalised by the number of cells counted by haemocytometer after
trypsin digestion. qRT-PCR A total of 5 × 105 cells/well were seeded in 12-well plates 1 day prior to RNA extraction. When
required, IFNγ treatments were applied as described previously at the time of seeding. RNA
extraction using the RNeasy kit (Qiagen) and DNase digestion using the RQ1 RNase-free
DNase (Qiagen) were performed following the manufacturer’s instructions. A total of 0.5 μl
each of oligo-dT and random primers (Promega, Madison, Wisconsin, USA) were added to
every 1 μg RNA and incubated at 70˚C for 5 min. M-MLV reverse transcriptase (Promega)
was used to perform the RT reaction at 42˚C for 60 min following the manufacturer’s instruc-
tions. q-PCR reactions were performed on 10 ng cDNA/sample using gene-specific primers
and PowerUp SyBr-Green Mastermix in a StepOne Real-Time PCR system (Thermo). Data
analysis was performed using StepOne Software v2.3. Glyceraldehyde 3-phosphate dehydroge-
nase (GAPDH) cDNA level was used as an internal control. Data used for statistical analysis
were then normalised by log 2 transformation of the fold change to untreated control. PI uptake assay A total of 5 × 104 cells/well were seeded in black clear-bottom 96-well plates 1 day prior to
infection and IFNγ and inhibitor treatment were applied as described previously. Alterna-
tively, 1.25 × 104 cells/well were seeded in 3 days prior to infection for siRNA transfection as
described before. Bacteria were primed in phenol-red-free DMEM (low glucose). Cells were
incubated in phenol-red-free RPMI or DMEM, for different cell lines as described previously,
supplemented with 5 μg/ml PI (Sigma-Aldrich) 1 h prior to infection and throughout the
infection. Cell-free medium-only wells were prepared as blank. Positive control wells were pre-
pared by cell lysis using RPMI with 5 μg/ml PI and 0.05% Triton X-100 (Sigma-Aldrich) 10
min prior to infection. Infections were carried out as described before. Time course measure-
ments were carried out from 10 min to 8 h postinfection with 10-min intervals, measuring
620-nm emission with 520-nm excitation. The percentage of PI uptake of each well was calcu-
lated by dividing each blank-normalised reading over blank-normalised positive control read-
ing at the same time point. The increase in percentage PI uptake of the infected cells compared
with uninfected was then calculated by subtraction of the percentage PI uptake in uninfected
samples (with the same drug/siRNA treatments). 21 / 31 PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986
December 30, 2020 PLOS BIOLOGY EPEC Tir triggers calcium influx-dependent pyroptosis Fluo-4 assay A total of 5 × 104 cells/well were seeded in black clear-bottom 96-well plates 1 day prior to
infection, and IFNγ was applied as described. Cells were incubated in Fluo-4 Direct reagent
(Molecular Probes, Eugene, Oregon, USA) diluted 2-fold in phenol-red-free RPMI for 30 min
before the first measurement. Fluorescence readings were performed in Omega microplate
reader measuring 520-nm emission with 485-nm excitation. All drugs were added 30 min
before infection, unless specified otherwise. ATP-treated samples were followed by a second
measurement at 2 min after treatment. Cell-free medium-only wells were prepared as blank. When required, time course measurements were carried out from 20 min before to 1 h after
infection with 10-min intervals. Raw measurements of Fluo-4 fluorescence of each well were
normalised by subtraction of the fluorescence of the blank well. PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986
December 30, 2020 High-pH reversed-phase peptide fractionation and LC–MS analysis Offline high-pH reversed-phase (RP) peptide fractionation and desalting were performed
using the Waters (Milford, Massachusetts, USA) XBridge C18 column (2.1 × 150 mm, 3.5
mm) on a Dionex Ultimate 3000 (Thermo Fisher Scientific) high-performance liquid chro-
matograph (HPLC) system. Mobile phase A was 0.1% ammonium hydroxide, and mobile
phase B was acetonitrile and 0.1% ammonium hydroxide. The TMT-labelled peptides were
reconstituted in 100 μl of mobile phase A and fractionated using a multistep gradient elution
method at 200 μl/min as follows: 5 min at 5% phase B, for 35 min up to 35% phase B, up to
80% phase B in 5 min, isocratic for 5 min and re-equilibration to 5% phase B. Fractions were
collected in a 96-well plate every 42 s to a total of 65 fractions, then concatenated into 28 frac-
tions and vacuum dried. Liquid chromatography–mass spectrometry (LC–MS) analysis was
performed on the Dionex Ultimate 3000 system coupled with the Orbitrap Fusion Lumos
mass spectrometer (Thermo Fisher Scientific). Each peptide fraction was reconstituted in 50 μl
0.1% formic acid, and 10 μl were loaded to the Acclaim PepMap 100, 100 μm × 2 cm, 5 μm,
100 Å C18 trapping column (Thermo Fisher Scientific) at a 10 μl/min flow rate. The sample
was then analysed with the EASY-Spray C18 capillary column (75 μm × 50 cm, 2 μm) (Thermo
Fisher Scientific) at 45˚C. Mobile phase A was 0.1% formic acid, and mobile phase B was 80%
acetonitrile and 0.1% formic acid. The gradient separation at a flow rate of 300 nl/min was gra-
dient for 90 min from 5% to 38% phase B and for 10 min up to 95% phase B, isocratic for 5
min at 95% B, re-equilibrated to 5% phase B in 5 min, and isocratic for 10 min at 5% phase B. Precursors between 375 and 1,500 m/z were selected with mass resolution of 120,000, auto-
matic gain control (AGC) of 4 × 105, and injection time (IT) of 50 ms, with the top speed
mode in 3 s, and were isolated for collision-induced dissociation (CID) fragmentation with a
quadrupole isolation width of 0.7 Th (Thomson unit). Collision energy was set at 35%, with
AGC at 1 × 104 and IT at 50 ms. MS3 quantification was obtained with higher-energy colli-
sional dissociation (HCD) fragmentation of the top 5 most abundant CID fragments isolated
with synchronous precursor selection (SPS). High-pH reversed-phase peptide fractionation and LC–MS analysis Quadrupole isolation width was set at 0.7 Th, col-
lision energy was applied at 65%, and the AGC setting was at 1 × 105 with IT at 105 ms. The
HCD MS3 spectra were acquired for the mass range of 100 to 500 m/z with a resolution of
50,000. Targeted precursors were dynamically excluded for further isolation and activation for
45 s with 7 ppm mass tolerance. The raw mass spectrometry files have been deposited to PRo-
teomics IDEntifications database (PRIDE accession: PXD018763). Sample preparation and TMT labelling Cell pellets were solubilised in lysis buffer (100 mM triethylammonium bicarbonate (TEAB),
1% sodium deoxycholate (SDC), 10% isopropanol, 50 mM NaCl) supplemented with halt pro-
tease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, Catalog Number 78442),
assisted with pulsed probe sonication. The samples were subsequently boiled for 5 min at 90˚C
and then subjected to a second round of sonication. The protein concentration was measured
with the Coomasie Plus assay (Thermo Fisher Scientific) according to manufacturer’s instruc-
tions. Aliquots containing 100 μg of protein were prepared for trypsin digestion. Samples were
reduced with 5 mM tris-2-carboxyethyl phosphine (TCEP) and alkylated with 10 mM iodoace-
tamide (IAA). Proteins were then digested by adding trypsin at the 75 ng/μl final concentra-
tion and incubating the samples for 18 h at room temperature, shaking at 300 rpm. The
resultant peptides were diluted up to 100 μl with 100 mM TEAB buffer and labelled with PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986
December 30, 2020 22 / 31 PLOS BIOLOGY EPEC Tir triggers calcium influx-dependent pyroptosis tandem mass tags (TMTs; TMT11plex) multiplex reagents (Thermo Fisher Scientific) accord-
ing to manufacturer’s instructions. The quenching reaction was performed by adding hydrox-
ylamine to a final concentration of 0.27% (v/v) and incubating the samples for 15 min at room
temperature. Finally, 11 samples per batch were combined in equal amounts to a single tube. Formic acid was added to a final concentration of 2% (v/v), and samples were centrifuged for 5
min at 10,0000 rpm to precipitate SDC. The supernatant containing TMT-labelled peptides
was moved to a new tube and dried with a centrifugal vacuum concentrator. Database search and protein quantification The acquired mass spectra were searched for protein identification and quantification in Pro-
teome Discoverer 2.3 (Thermo Fisher Scientific) with the SEQUEST-HT search engine. The
spectra were searched for fully tryptic peptides with maximum of 2 missed-cleavages, 20 ppm
precursor mass tolerance, and 0.5 Da fragment ion mass tolerance. The search considered
static carbamidomethylation of Cys residues and TMT6plex modification of peptide N-term
and Lys residues, as well as dynamic oxidation of Met and deamidation of Asp and Glu 23 / 31 PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986
December 30, 2020 PLOS BIOLOGY EPEC Tir triggers calcium influx-dependent pyroptosis residues. The Percolator node was used to estimate the confidence of peptide identifications at
1% false discovery rate (FDR) and was based on the q-value and a decoy database search. The
spectra were searched against UniProt annotated reference proteomes of Homo sapiens and E. coli. The reporter ion quantifier node included a TMT-11-plex quantification method with an
integration window tolerance of 15 ppm and integration method based on the most confident
centroid peak at MS3 level. Quantification was performed using unique peptides only, with
protein groups considered for peptide uniqueness. Proteomics data used for statistical analysis
were scaled and then normalised by log 2 transformation of the fold change to uninfected
control. Statistical analysis All experiments except proteomics were independently repeated at least 3 times as indicated in
the figure legends. Proteomics were independently repeated twice. For qRT-PCR, high-con-
tent imaging analysis and CFU assays, 2 to 3 technical repeats were performed to calculate
means for each biological repeat. For manual cell counting in microscopy images, at least 4
images from randomly selected positions in each coverslip containing a total of 50 to 100 cells
(over 250 cells from 4 biological repeats) were used. For high-content image analysis, 25
images evenly distributed in predefined positions containing a total of over 100 cells in each
well were used. Methods of data transformation are described in their corresponding method
sections. Statistical analysis of all biochemical/biological experimental data was performed using
GraphPad Prism 5.1. Student t test and 1-way or 2-way ANOVA followed by Tukey posttest or
Bonferroni posttest, respectively, were performed on the means as listed in the figure legends. Significant result was defined as having a p-value of <0.05. Statistical analysis, including PCA, ANOVA, t test, and pathway enrichment analysis of the
proteomic data, was performed in Perseus 1.6.2.2 (Max Planck Institute of Biochemistry, Ger-
many) [86]. Pathway enrichment was performed using Uniprot Keywords, Gene Ontology
Biological Process (GOBP) and KEGG pathways [87]. For transcription factor analysis, the
“TF-LOF Expression from GEO” library was downloaded from the Enrichr web tool [88] and
was converted to Perseus-compatible annotation file for 1D annotation enrichment analysis. All
biological terms were filtered for Benjamini–Hochberg FDR < 0.05. Heatmaps were plotted in
Phantasus (https://artyomovlab.wustl.edu/phantasus/), and boxplots were plotted in R-Studio. S1 Data. Excel spreadsheet containing the numerical data used for the main and Support-
ing information figures. S1 Data. Excel spreadsheet containing the numerical data used for the main and Support-
ing information figures. PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986
December 30, 2020 S1 Raw Images. Raw images of the western blots presented in Fig 2E, 2F and 2I–2K and
S2A, S2G and S2I Fig.
(PDF) S1 Fig. Kinetics of Tir-induced cell death throughout 8 h of infection. (A) Gbp2 expression
level was measured by qRT-PCR in IFNγ-primed cells and normalised to the Gbp2 expression
level in the untreated cells. Means ± SEM from n = 3 independent biological repeats. Statistical
significance was determined using 2-tailed t test. p 0.05. (B) PI uptake into unprimed
SNU-C5 cells infected with EPEC WT, EPEC-0 and EPEC-1. Measurements were taken every
10 min. The time-course PI uptake was plotted. PI uptake results were normalised by UI. Means ± SEM from n = 5 independent biological repeats are shown. (C) PI uptake into IFNγ- PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986
December 30, 2020 24 / 31 PLOS BIOLOGY EPEC Tir triggers calcium influx-dependent pyroptosis primed uninfected cells. PI uptake results were normalised to unprimed UI cells. Means ±
SEM from n = 7 independent biological repeats are shown. The underlying data for this figure
can be found in S1 Data. EPEC, enteropathogenic Escherichia coli; IFNγ, interferon gamma;
PI, propidium iodide; qRT-PCR, real-time quantitative PCR; SEM, standard error of the
mean; Tir, translocated intimin receptor; UI, uninfected; WT, wild-type. (TIF) S2 Fig. Apoptosis, NLRP3-dependent pyroptosis and necroptosis do not occur during
EPEC-1 infection. (A) Cell lysates of primed SNU-C5 cells infected with EPEC WT, EPEC-0,
and EPEC-1, or treated by STS for 20 h, were used for PARP1 western blot. Representative
blots were shown. (B) PI uptake into THP1 cells treated by LPS for 3 h followed by nigericin
for 8 h, with or without MCC950 treatment 30 min before nigericin addition. Means ± SEM
from n = 3 independent biological repeats. (C) PI uptake into primed SNU-C5 cells infected
with EPEC-1 treated with MCC950 30 min before infection. Means ± SEM from n = 3 inde-
pendent biological repeats. (D, E) PI uptake into SNU-C5 cells treated by YVAD, Nec1 and
NSA 30 min before LPS transfection (D) or STS and zVAD treatment (E). Means ± SEM from
n = 3 independent biological repeats. (F) PI uptake into SNU-C5 cells infected with EPEC-1
treated with Nec1, NSA, zVAD, and a combination of zVAD, Nec1, and NSA 30 min before
infection. Means ± SEM from n = 3 independent biological repeats. (G) Caspase-8 western
blot of SNU-C5 cells transfected with caspase-8 siRNA. Representative blot from n = 3
independent biological repeats are shown. S1 Raw Images. Raw images of the western blots presented in Fig 2E, 2F and 2I–2K and
S2A, S2G and S2I Fig.
(PDF) (H) PI uptake into SNU-C5 cells transfected by
caspase-4, GSDMD or caspase-8 siRNA or combinations of them and infected with EPEC-1. Means ± SEM from n = 3 independent biological repeats. (I) RIPK3 western blot of SNU-C5
cells transfected with RIPK3 siRNA. Representative blot from n = 3 independent biological
repeats are shown. (J) PI uptake into SNU-C5 cells transfected by RIPK3 siRNA treated by
STS and zVAD. Means ± SEM from n = 3 independent biological repeats. (K) PI uptake into
SNU-C5 cells transfected by caspase-4, GSDMD or RIPK3 siRNA or combinations of them
and infected with EPEC-1. Means ± SEM from n = 3 independent biological repeats. Statistical
significance was determined using 2-tailed t test (B, C, J) and 1-way ANOVA with Tukey post-
test (D, E, F, H, K). p 0.05; p 0.01; p 0.001. The underlying data for this figure
can be found in S1 Data. ANOVA, analysis of variance; EPEC, enteropathogenic Escherichia
coli; GSDMD, gasdermin D; LPS, lipopolysaccharide; NLRP3, NLR family pyrin domain con-
taining 3; NSA, necrosulfonamide; PARP1, poly [ADP-ribose] polymerase 1; PI, propidium
iodide; SEM, standard error of the mean; siRNA, small interfering RNA; STS, staurosporine;
WT, wild-type; YVAD, z-YVAD-fmk; zVAD, z-VAD-fmk. (
) S3 Fig. EPEC adheres to and Tir induces actin polymerisation in SNU-C5, HT-29,
SNU-C2B and COLO-320-HSR. Immunofluorescence staining of SNU-C5, HT-29,
SNU-C2B and COLO-320-HSR cells were infected with EPEC-0 and EPEC-1 for 4 h. DAPI:
blue; EPEC: green; Phalloidin: red. Representative images from n = 3 independent biological
repeats are shown. Error bar: 5 μm. The underlying data for this figure can be found in S1
Data. EPEC, enteropathogenic Escherichia coli; Tir, translocated intimin receptor. (TIF) S3 Fig. EPEC adheres to and Tir induces actin polymerisation in SNU-C5, HT-29,
SNU-C2B and COLO-320-HSR. Immunofluorescence staining of SNU-C5, HT-29,
SNU-C2B and COLO-320-HSR cells were infected with EPEC-0 and EPEC-1 for 4 h. DAPI:
blue; EPEC: green; Phalloidin: red. Representative images from n = 3 independent biological
repeats are shown. Error bar: 5 μm. The underlying data for this figure can be found in S1
Data. EPEC, enteropathogenic Escherichia coli; Tir, translocated intimin receptor. (TIF) S4 Fig. Tir-dependent cell death in unprimed SNU-C5 is inhibited by YVAD and EGTA. (A,
B) PI uptake into unprimed SNU-C5 cells infected with EPEC-1 with 30 min pretreatment with
YVAD (A) or EGTA (B). PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986
December 30, 2020 S1 Raw Images. Raw images of the western blots presented in Fig 2E, 2F and 2I–2K and
S2A, S2G and S2I Fig.
(PDF) Means ± SEM from n = 3 independent biological repeats. (C) PI uptake
into unprimed SNU-C5 cells infected with EPEC-0, EPEC-2, EPEC-1-TirAA-EspZ, EPEC-1 and
EPEC-1-TirAA. Means ± SEM from n = 5 independent biological. Statistical significance was S4 Fig. Tir-dependent cell death in unprimed SNU-C5 is inhibited by YVAD and EGTA. (A,
B) PI uptake into unprimed SNU-C5 cells infected with EPEC-1 with 30 min pretreatment with
YVAD (A) or EGTA (B). Means ± SEM from n = 3 independent biological repeats. (C) PI uptake
into unprimed SNU-C5 cells infected with EPEC-0, EPEC-2, EPEC-1-TirAA-EspZ, EPEC-1 and
EPEC-1-TirAA. Means ± SEM from n = 5 independent biological. Statistical significance was 25 / 31 PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986
December 30, 2020 PLOS BIOLOGY EPEC Tir triggers calcium influx-dependent pyroptosis determined using 2-tailed t test (A, B) and 1-way ANOVA with Tukey posttest (C). p 0.05;
p 0.01; p 0.001. The underlying data for this figure can be found in S1 Data. ANOVA,
analysis of variance; EPEC, enteropathogenic Escherichia coli; PI, propidium iodide; SEM, stan-
dard error of the mean;Tir, translocated intimin receptor; YVAD, z-YVAD-fmk. (TIF) S5 Fig. Illustration of the proteomics workflow used in this study. (A) IFNγ-primed and
unprimed SNU-C5 cells were infected with the indicated EPEC strains for 2 h. Extracted pro-
teins were digested with trypsin and peptides were labelled with the TMT 11plex reagents in 2
separate replicate batches. TMT-labelled peptides were subjected to offline high-pH reversed-
phase fractionation followed by LC–MS analysis. (B) PCA of proteomics data. EPEC, entero-
pathogenic Escherichia coli; IFNγ, interferon gamma; LC–MS, liquid chromatography–mass
spectrometry; PCA, principal component analysis; TMT, tandem mass tag. (TIF) S5 Fig. Illustration of the proteomics workflow used in this study. (A) IFNγ-primed and
unprimed SNU-C5 cells were infected with the indicated EPEC strains for 2 h. Extracted pro-
teins were digested with trypsin and peptides were labelled with the TMT 11plex reagents in 2
separate replicate batches. TMT-labelled peptides were subjected to offline high-pH reversed-
phase fractionation followed by LC–MS analysis. (B) PCA of proteomics data. EPEC, entero-
pathogenic Escherichia coli; IFNγ, interferon gamma; LC–MS, liquid chromatography–mass
spectrometry; PCA, principal component analysis; TMT, tandem mass tag. (TIF) S6 Fig. Proteomic differences of IFNγ-primed and unprimed cells. Heatmap of the differen-
tially regulated proteins between IFNγ-primed and unprimed SNU-C5 cells (t test, FDR <
0.05, absolute log2 ratio versus uninfected > 0.5). S1 Raw Images. Raw images of the western blots presented in Fig 2E, 2F and 2I–2K and
S2A, S2G and S2I Fig.
(PDF) The underlying data for this figure can be
found in S1 Data. FDR, false discovery rate; IFNγ, interferon gamma. (TIF) S7 Fig. Kinetics of Tir-induced Ca2+ influx over 1 h of infection. Fluo-4 assay performed on
primed (A, C) and unprimed (B) SNU-C5 cells infected with EPEC-0 (A, B), EPEC-1 (A-C)
and EPEC-1-TirAA (A, B) with or without 30 min pretreatment with EGTA (A, B) and YVAD
(C). Means ± SEM from n = 3 independent biological repeats. The underlying data for this fig-
ure can be found in S1 Data. EPEC, enteropathogenic Escherichia coli; SEM, standard error of
the mean; Tir, translocated intimin receptor; YVAD, z-YVAD-fmk. (TIF) S8 Fig. EPEC-1 induces cell death independent of ROS- or mitochondrial respiration-
dependent cell death. (A–C) PI uptake into primed SNU-C5 cells infected with EPEC-1, with
30 min pretreatment with DMTU (A), Tiron (B) or oligomycin (C). Means ± SEM from n = 3
independent biological. (D) OCR measurement was performed on SNU-C5 cells before or
after oligomycin treatment. Means ± SEM from n = 3 independent biological repeats are
shown. Statistical significance was determined using 2-tailed t test. ns, nonsignificant;
p 0.05; p 0.01; p 0.001. The underlying data for this figure can be found in S1
Data. DMTU, N,N0-dimethylthiourea; EPEC, enteropathogenic Escherichia coli; OCR, oxygen
consumption rate; PI, propidium iodide; SEM, standard error of the mean. (TIF) S1 Table. Lists of strains, plasmids, oligonucleotide sequences, and antibodies used in this
study. (A) Bacterial strains. (B) Plasmids. (C) Primer sequences. (D) siRNA and miRNA30E
sequences. (E) Immunofluorescence antibodies and reagents. (F) Western blot antibodies. siRNA, small interfering RNA. (DOCX) Author Contributions Author Contributions
Conceptualization: Gad Frankel, Jyoti S. Choudhary. Data curation: Qiyun Zhong, Theodoros I. Roumeliotis, Zuza Kozik. Formal analysis: Qiyun Zhong, Theodoros I. Roumeliotis, Zuza Kozik, Chris Bakal. Funding acquisition: Gad Frankel, Jyoti S. Choudhary. Investigation: Qiyun Zhong, Theodoros I. Roumeliotis, Zuza Kozik. Methodology: Qiyun Zhong, Theodoros I. Roumeliotis, Zuza Kozik, Massiel Cepeda-Molero,
Avinash R. Shenoy, Chris Bakal, Gad Frankel, Jyoti S. Choudhary. Project administration: Gad Frankel, Jyoti S. Choudhary. Supervision: Theodoros I. Roumeliotis, Luis A´ngel Ferna´ndez, Avinash R. Shenoy, Chris
Bakal Gad Frankel Jyoti S Choudhary Conceptualization: Gad Frankel, Jyoti S. Choudhary. Data curation: Qiyun Zhong, Theodoros I. Roumeliotis, Zuza Kozik. Formal analysis: Qiyun Zhong, Theodoros I. Roumeliotis, Zuza Kozik, Chris Bakal. Funding acquisition: Gad Frankel, Jyoti S. Choudhary. Investigation: Qiyun Zhong, Theodoros I. Roumeliotis, Zuza Kozik. Methodology: Qiyun Zhong, Theodoros I. Roumeliotis, Zuza Kozik, Massiel Cepeda-Molero,
Avinash R. Shenoy, Chris Bakal, Gad Frankel, Jyoti S. Choudhary. Project administration: Gad Frankel, Jyoti S. Choudhary. Supervision: Theodoros I. Roumeliotis, Luis A´ngel Ferna´ndez, Avinash R. Shenoy, Chris
Bakal, Gad Frankel, Jyoti S. Choudhary. Validation: Qiyun Zhong. Validation: Qiyun Zhong. Visualization: Qiyun Zhong, Theodoros I. Roumeliotis. Visualization: Qiyun Zhong, Theodoros I. Roumeliotis. Writing – original draft: Qiyun Zhong, Theodoros I. Roumeliotis, Zuza Kozik, Gad Frankel,
Jyoti S. Choudhary. Writing – review & editing: Qiyun Zhong, Theodoros I. Roumeliotis, Zuza Kozik, Avinash R. Shenoy, Chris Bakal, Gad Frankel, Jyoti S. Choudhary. Acknowledgments We thank Enkai Jin (MRC CMBI) for EPEC binding assays, Gerald Larrouy-Maumus for pro-
viding access of the Agilent Seahorse XFp Analyzer, the CMBI High throughput single-cell
analysis facility (HTSCAF), and Pascal Meier for critical reading of the manuscript. PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986
December 30, 2020 26 / 31 PLOS BIOLOGY EPEC Tir triggers calcium influx-dependent pyroptosis Author Contributions
Conceptualization: Gad Frankel, Jyoti S. Choudhary. Data curation: Qiyun Zhong, Theodoros I. Roumeliotis, Zuza Kozik. Formal analysis: Qiyun Zhong, Theodoros I. Roumeliotis, Zuza Kozik, Chris Bakal. Funding acquisition: Gad Frankel, Jyoti S. Choudhary. Investigation: Qiyun Zhong, Theodoros I. Roumeliotis, Zuza Kozik. Methodology: Qiyun Zhong, Theodoros I. Roumeliotis, Zuza Kozik, Massiel Cepeda-Molero,
Avinash R. Shenoy, Chris Bakal, Gad Frankel, Jyoti S. Choudhary. Project administration: Gad Frankel, Jyoti S. Choudhary. Supervision: Theodoros I. Roumeliotis, Luis A´ngel Ferna´ndez, Avinash R. Shenoy, Chris
Bakal, Gad Frankel, Jyoti S. Choudhary. Validation: Qiyun Zhong. Visualization: Qiyun Zhong, Theodoros I. Roumeliotis. Writing – original draft: Qiyun Zhong, Theodoros I. Roumeliotis, Zuza Kozik, Gad Frankel,
Jyoti S. Choudhary. Writing – review & editing: Qiyun Zhong, Theodoros I. Roumeliotis, Zuza Kozik, Avinash R. Shenoy, Chris Bakal, Gad Frankel, Jyoti S. Choudhary. PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986
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Notes ON THE SUPERSTITIONS OF MENSTRUATION.
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public-domain
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ANCIENT VIEWS ON MENSTRUATION. The wise men of old stood baffled before the
mystery of the menses. Pythagoras called menstrual
blood the froth of the blood, and held that it con-
tained the superfluity of the aliment. Aristotle
explained menstruation as a process necessary to
get rid of the excess (7répíTTWJLU) of blood that had
accumulated in the interior of the body of the
woman, and his theory is ardently supported 1500
years later by his great commentator Averrhoes. Can it be honestly said that, apart from some
suggestive evidence as to the interdependence of
ovarian activity and catamenial function, medicine
of to-day has any more satisfying explanation of the
purpose of menstruation than theirs? purpose
Where science has failed to establish a proof
religion has seldom failed to establish a theory. The Levitical ritual was in no doubt that impurities
collected in the woman’s blood were discharged in
the menstrual flux, so that she underwent a recur-
ring series of purifications. To the men of the
monasteries menstruation was a symbol of the
essential
sinfulness and inferiority of woman,
polluted alike and polluting. Men of such widely
different stamp as Paracelsus and de Graaf both
upheld this view of a menstrual purification, and
I seem to hear some echo of it in the modern sug-
gestion that the uterine mucosa, by expulsion of
its surface layer, is preparing a sterilised bed for
the fertilised ovum. Laboriously acquired histo-
logical knowledge competes on unequal terms with
a plausible bacteriological preconception. MAGICAL BELIEFS. Back of all these scientific and religious theories,
and not infrequently casting a shadow upon them,
stands
a congeries of magical beliefs, which
constitute the main subject of our present inquiry. The menstruous woman is possessed by an evil
spirit; the spirit resides in her blood, and by the
medium of her menstrual blood may exert its
infiuence, for good or for harm, on her environ-
ment. The evil spirit may effect its entry into the
woman in the form of a bird, or a, lizard, or a
serpent; hence the folk association
of these
animals with menstruation. It was the serpent
that marred the primordial bliss of Eden. In
Portugal women during menstruation wear drawers
to prevent the access of lizards. On the Pennefather
river the menses are said to be produced by a
curlew operating on woman. ,
PHYSICIAN TO KING’S COLLEGE HOSPITAL AND KING GEORGE’S MILITARY
HOSPITAL; FELLOW OF KING’S COLLEGE, LONDON, ETC. SUPERSTITIONS concerning the menses loom so
large in the customs of primitive peoples and
are so far from extinct among the lower orders
of civilised nations as to be worthy of serious
consideration. Many a farmer’s wife in this country
will still assure you that milk handled by a
menstruous woman cannot be churned to butter
or that hams will not take salt at her hands. That she has demonstrated the contrary in a
series of experiments, month by month, year in
and year out, has left unshaken the pillars of
her faith: the foundations of faith stand firm in
shifting soil. operating
It is of the nature of magic, having postulated an
all-sufficing cause, to take but little account of the
intervening steps in the production of its effect. But in the matter of menstruation it has not been
wholly silent. The natives on the Tully river in
North Queensland attribute it to the breaking and
discharge of the liver; what causes the breakage
they do not know, but believe that menstruation
has nothing to do with pregnancy. They are so
far right in this that it is quite certain repeated
pregnancy may occur in those who have never
menstruated. Segregation of women from the
men during menstruation would lend colour to the
belief that menstruation is not necessarily con-
nected with conception. Among the Sandwich
Islanders menstruation is believed to be the result
of coition. Notes
ON THE
SUPERSTITIONS OF MENSTRUATION. We see how it came about that Aristotle applied
to the menses the self-same explanation of a super-
fluity, that Hippocrates had adopted in the case of
the male semen. DECEMBER 18, 1915. DECEMBER 18, 1915. never takes place. Women of this nature, however, are
incapable of bearing children, because it is of this substance
that the infant is formed. The seed of the male, acting as
a sort of leaven, causes it to unite and assume a form, and
in due time it acquires life and assumes a bodily shape. Notes
ON THE
SUPERSTITIONS OF MENSTRUATION. Read before the Historical Section of the Royal Society of
Medicine, on Dec. 15th, 1915,
BY RAYMOND CRAWFURD, M.A., M.D.OXON.,
F.R.C.P. LOND.,
PHYSICIAN TO KING’S COLLEGE HOSPITAL AND KING GEORGE’S MILITARY
HOSPITAL; FELLOW OF KING’S COLLEGE, LONDON, ETC. SUPERSTITIONS CONNECTED WITH THE MILK-
SUPPLY. Many superstitions centre around the milk-
supply ; much light has been thrown upon them
by the masterly handling of Frazer.3 Among the
kaffir tribes of South Africa the menstruous woman
must not drink milk, for if she do the cows would
die. Even the maidens who attend a girl at her
first menstruation are forbidden to drink milk for
the same reason. Seclusion of the woman from the
cattle and the taboo on milk last a fortnight, double
the period demanded in the case of many other
prohibitions. prohibitions. Among the Bahimas a woman may not drink milk
at her monthly.times, but must eat vegetables and
drink beer; the vegetarian diet implies a taboo on
the flesh of the animals that yield the milk. Exception is made in favour of a girl at her first
menstruation; her father then sets apart for her
use an old cow, which is of small value, as it will
soon pass out of milk. The conception that
underlies these prohibitions is that of a sympathetic
bond between the cow and its milk, which abides
after the milk has left its body. Harm done to the
milk inevitably involves the cow. plausible bacteriological preconception. It must be remembered that till the middle of the
seventeenth century, when the work of Harvey,
de Graaf, and other pioneer embryologists led up to
the discovery of the ovum in the human female, it
was generally believed that the male semen and the
female menses were the constituents out of which
the child was formed, and by most the leading part
was assigned to the menses. Pliny defines the
general belief in quite unequivocal language :- No
4816
g
q
q
g
g
The .discharge, which is productive of such great and
singular effects, occurs in women every thirty days, and in a
greater degree every three months. In some individuals it
occurs oftener than once a month, and in others again it inevitably
Again a menstruous woman is forbidden to cross
those parts of the kraal frequented by the cattle,
for if a drop of blood were to fall on such a path 1332 and in Sumatra they are forbidden the rice fields
for fear of damaging the crop. BELIEFS ABOUT THE CROPS. Superstitions such as we have already cited are
in the nature of things most prevalent among the
unsophisticated children of the soil, and wear a
rural aspect. Others centre about the crops. Thus
Pliny 4 says :- y
y
If a woman strips herself naked while she is menstruating
and walks round a field of wheat, the caterpillars, worms,
beetles, and other vermin will fall off from the ears of corn. Metrodorus, of Scepsos, tells us that the discovery was first
made in Cappadocia, and that, in consequence of such
multitudes of cantharides being found to breed there, it is
the practice for women to walk through the middle of the
fields with their garments tucked up above the thighs. In
other places again it is the usage for women to go barefoot,
with the hair dishevelled and the girdle loose ; due precau-
tion must be taken, however, that this is not done at sunrise,
for, if so, the crop would wither and dry up. y
A mare big with foal, if touched by a woman in this
state, will be sure to miscarry; nay, even more than this,
at the very sight of a woman, though seen even at a
distance, should she happen to be menstruating for the first
time after the loss of her virginity, or for the first time
while in a state of virginity. So pernicious are the properties
of the menstrual discharge that women themselves, the
source from which it is derived, are far from being proof
against its effects : a pregnant woman, for instance, if
touched with it, or if indeed she so much as steps over it,
will be liable to miscarry.15 . Columella and Aelian both testify to the efficacy
of this procedure, which finds a modern echo in
Hiawatha’s injunction to Minnehaha :- Pliny further records its value as an ingredient
of abortifacient remedies and as a means of pre-
venting impregnation. We have the recent testi-
mony of Dr. T. G. Morton, of Philadelphia, that
ignorant female abortionists still vend a nostrum
for defeating pregnancy, one of the component
parts of which is catamenial fluid. SUPERSTITIONS CONNECTED WITH THE MILK-
SUPPLY. The huts of a kafnr kraal are
usually arranged in a circle with the cattle-fold in
the centre, and the circuitous paths taken by the
women, in passing from hut to hut, may be seen
outside the circle of huts, between them and the
palisade which sometimes surrounds the kraal; but
there is no such restriction on the walks of women
who are past child-bearing, as they have ceased to
be a source of danger. Nor is it only menstrual
blood that is feared, for a Zulu custom forbids a
wounded man to drink milk until he has submitted
to a prescribed ritual. p esc
bed
Among many pastoral tribes of South Africa and
India, women are either entirely prohibited from
milking the cows, or are permitted to do so only in
accordance with certain strict rules of ceremonial
purification. Among the Todas of South India the
men alone undertake the operations of milking and
churning. The women may go to the dairy to
fetch butter-milk, but they must approach it by an
appointed path and stand at an appointed place to
receive the milk. Such scrupulous care to shield the
cattle and their milk from harm is commensurate
with their importance in the economy of a pastoral
people. It is interesting to encounter these primi-
tive attempts at the provision of a pure milk-
supply, and to consider the different character of
the impurities to be feared, then and now. SUPERSTITIONS CONNECTED WITH THE MILK-
SUPPLY. At Saigon no woman
is employed in the opium factory lest she should
turn the opium bitter, while Annamese women say
that it is very difficult for them to prepare opium
pipes during the catamenial period."
Even the
lowlier forms of life are activated by this uncanny
influence, so that beer and wine turn sour and
vinegar will not keep. Clearly in this state she
must be secluded from the food-supply. We have
seen that she is forbidden to take milk or meat at
these times for fear of damaging the flocks. Some
Central Australian 10 tribes
will
not allow a
menstruous woman to gather a certain bulb, which
is a staple food, for fear the crop of bulbs should
fail. Again, no woman was formerly admitted to
the great sugar refineries in the North of France
during the boiling or cooling processes on the
ground that the sugar would blacken if a woman
were to enter during her period."
In Syria to this
day women are not allowed to salt or pickle when
their courses are on them, as it is believed that
whatever they salt or pickle will not keep ; and in
Brunswick people think that if a menstruous
woman assists at the killing of a pig the flesh will
putrefy.l any cow passing over it would run great risk of
dying from disease. The huts of a kafnr kraal are
usually arranged in a circle with the cattle-fold in
the centre, and the circuitous paths taken by the
women, in passing from hut to hut, may be seen
outside the circle of huts, between them and the
palisade which sometimes surrounds the kraal; but
there is no such restriction on the walks of women
who are past child-bearing, as they have ceased to
be a source of danger. Nor is it only menstrual
blood that is feared, for a Zulu custom forbids a
wounded man to drink milk until he has submitted
to a prescribed ritual. any cow passing over it would run great risk of
dying from disease. MALIGN INFLUENCE ON ANIMAL LIFE. With an ever-widening circumference the malign
influence affects all living things about the home-
stead. Bees will forsake their hives at her touch,
for they have a special aversion to a thief and a
menstruous woman, and a glance of her eyes
suffices to kill a swarm of bees (Pliny- Among the
Indians an Omaha 14 woman may not approach a
horse during her courses or she will grievously
harm the animal. Pliny says :- BELIEFS ABOUT THE CROPS. That it should
possess this powerful ecbolic faculty is a mere
matter of sympathetic influence, and for the same
reason- j
You shall bless to-night the cornfields,
Draw a magic circle round them
To protect them from destruction,
Blast of mildew, blight of insect. In the night, when all is darkness,
Rise up from your bed in silence,
Lay aside your garments wholly,
Walk around the fields you planted,
Round the borders of the cornfields,
Covered with your tresses only,
Robed with darkness as a garment. co
e ds,
Covered with your tresses only, Reduced to ashes and mixed with oil of roses it is very
useful; applied to the forehead, for allaying headache,
particularly in women (Pliny).17 your
only,
Robed with darkness as a garment. For the most part, however, the influence of
the menstruous woman on vegetation is harmful. According to Pliny- particularly
(Pliny).17
Dogs which may have tasted of the matter so discharged
are seized with madness, and their bite is venomous and
incurable. H Seeds which are touched by her become sterile, grafts
wither away, garden plants are parched up, and the fruit
will fall from the tree beneath which she sits. 6 And again in a later passage Pliny says :- CUSTOMS AMONG PRIMITIVE YEOPLES. (
y
)
The powers of Nature even own the menstrual
sway- CUSTOMS AMONG PRIMITIVE YEOPLES. Among the Australian 26 blacks the boys are
taught from early childhood that if they set eyes
on menstruous blood their hair will turn grey and
their vigour abate prematurely. Consequently, the
women must live apart at these times, and if a boy
3r young man is seen to approach she must call
3ut at once, so that he may avoid her; he must not
3ven cross the tracks made by women at such times. rhe woman is forbidden under pain of death to
jouch anything that the men use or to walk on
ihe paths they frequent. An Australian black
milled his wife when he found that she had lain on
lis blanket during her menstrual period and died
)f terror within a fortnight. Among the Bhinyan
)f South Mirzapur every house has two doors, one
)f which is used exclusively by the women during
nenstruation. These superstitious terrors help
is to appreciate the mentality of those eminent
cientists who find the contiguity of a women an
mpediment to serious work, and who knows but
hat their unconscious influence may explain the
rade unions hostility to the collaboration of
vomen in the workshop. For in the first place hailstorms, they say, whirlwinds and
lightning even will be scared away by a woman uncovering
her body, while her monthly courses are upon her. The
same too with all other kinds of tempestuous weather ; and
out at sea a storm may be lulled by a woman merely
uncovering her body, even though not menstruating at the
time (Pliny 2). Seasonal and other influences are held to heighten
the virulence of the discharge. Aelian lays it down
that during menstruation woman is in communica-
tion with the starry bodies. Aristotle 24 says that
wiseacres assert that the moon is female, because
the menstrual flux and the waning of the moon
both take place towards the end of the month, and
after the wane and the discharge both become
whole again. Yet one more instance may be taken from Pliny
of its influence on animal life :- p
pp
g
The menstruating woman has the divine gift of
kings, for by her touch she can cure not only
scrofulous ulcers, but " imposthumes of the parotid
glands, inflamed tumours, erysipelas, boils, and
defluxions of the eyes." 26 Avicenna and Pliny both
recommend menstrual blood as an application to all
sores. In Bavaria it is held even to have prophy-
lactic power against wounds and to be able to
extinguish
fires. It was given internally for
erysipelas and calculi, and was esteemed a good
cosmetic to drive away pimples. In the Middle
Ages it was a sovereign cure for leprosy.2.> That the ant, an insect so extremely minute, is sensible of
its presence and rejects the grains, which it has been
carrying, and will not return to them again.2" CUSTOMS AMONG PRIMITIVE YEOPLES. According to Pliny- g
g
y
If the menstrual discharge coincides with an eclipse of the
moon or sun, the evils resulting from it are irremediable ;
and no less so, when it happens while the moon is in con-
junction with the sun ; congress with a woman at such a
period being noxious and attended with fatal ejects to the
man. At this period also, the lustre of purple is tarnished
by the touch of a woman, so much more baneful is her
influence at this time than at any other. 23 s
t
1
y
]
a
]
j
l
E
p
Among the people of the Lower Congo 27 a woman,
uring menstruation, may not cook her husband’s
ood nor any other man’s, neither anything belong-
ng to men, and must not return the salutations of
ny man. If a woman in this condition has to pass
ear some men who are likely to give her the
quivalents of "Good morning" or "Good even-
19" " she will deliberately put her pipe in her
iouth as a sign that she cannot answer because
lie is unclean. Virginity too exerts a special power. Pliny say
that ’’ the nature of the discharge is most virulen
in females, whose virginity has been lost solely by
lapse of time."
28
The first menstruation also, as
well as the first after loss of virginity, possess a
special potency. And again in a later passage Pliny says :- g
y
It is universally agreed that, when a person has been
bitten by a dog and manifests a dread of water and of all kinds
of drink, it will be quite sufficient to put under his cup a
strip of cloth that has been dipped in this fluid, the result
heing that hydrophobia will immediately disappear. This
arises, no doubt, from that powerful sympathy which has
been so much spoken of by the Greeks, and the existence of
which is proved by the fact already mentioned, that dogs
become mad on tasting this fluid." Young vines, too, it is said, are injured irremediably by
the touch of a woman in this state ; and both rue and ivy,
plants possessed of highly medicinal virtues, will die instantly
upon being touched by her. "( upon
g
by
All plants will turn of a yellow complexion on the approach
of a woman who has the menstrual discharge upon her. 8 Frazer says that in Galela women at their
monthly periods may not enter a tobacco field, OTHER PHENOMENA ATTRIBUTED TO THE
CATAMENIAL STATE. Household articles enjoy no immunity. Aristotle
,says that the glance of a menstruous woman takes
the polish out of a mirror and that the next person
looking into it will be bewitched. Pliny says
that the tarnished mirror will recover its bright-
ness if the same woman looks steadily upon the
back of it. Her glance also blunts the edge of
steel and takes away the polish from ivory. Lausent 21 enumerates a whole catalogue of un-
canny phenomena attributed to the catamenial
state. Two girls believed that their shoes, stockings,
and clothes stuck to them so closely that they
needed help to remove them. There are many
tales of the snapping of strings of harps and violins
during the menses; also of the readiness with
which women break things at these times, needles
snapping, glasses breaking, and clocks stopping if
touched. Iron wiil become rusty on contact with
her, copper coated with verdigris, razors blunted,
and linen boiling in the cauldron will turn black
(Pliny 22). ges
g
leprosy.2. The wise women of Pliny’s day recommended it
to be placed on a thread or the wool of a black
sheep and worn in a bracelet as an amulet in
tertian or quartan fevers. The midwife Sotira
considered that better results were obtained by
rubbing the soles of the patient’s feet with it, and
if possible this should be done by the woman her-
self without the patient’s knowledge. She had also
proved the efficacy of this method in reviving a
person from an epileptic seizure. For quartan
fever Ictidas, the physician, recommended sexual
intercourse with a woman just beginning to men-
struate. This is but one instance of the profound
influence on males attributed to the menstrual
state. water and laid on the part; the discharge was also
used as a topical application in acute gout. Yet one more instance may be taken from Pliny
of its influence on animal life :- CURATIVE PROPERTIES, when about to compound their medicines, were in
the habit of making a saving clause that the
remedy would be effective, provided no menstruating
woman approached their chamber during the com-
pounding process. Surely nowadays the altogether
inordinate apprehensions of both nurse and surgeon
as to the evils of operation during the menstrual
state are tinctured witli the savour of this same
superstitious fear. superstitious
Among the native Australians 2H the woman may
not eat fish, or cross the water, or even go near it,
or it will spoil the fishermen’s luck; she may not
even fetch water for the camp. Mrs. James Smith,
in her account of the Roandik tribes, says that
men have a special objection to setting eyes on
menstrual blood, because it would enfeeble them
in their fight against enemies and they would be
killed; and the Aruntas 2D of Central Australia
believe that a draught of woman’s blood would kill
the strongest man. In Polynesia the vessels, gar-
ments, and other property of a menstruating woman
may not be used by others on pain of disease and
death. proven
The ordinances of religion, with regard to the
segregation of the menstruous woman, fall into line
with the customs of magic. As Frazer has said,
the divinely inspired law-givers mistook their own
ideas for revelations from heaven. The Hindoo law-giver, Manu, who professed to have
received his institutes from the Creator, Brahman, informs us
that the wisdom, the energy, the strength, the sight, and
the vitality of a man who approaches a woman in her
courses will utterly perish; whereas, if he avoids her, his
wisdom, energy, strength, sight, and vitality, will all
increase. i LOVE CHARMS AND OTHER MAGICAL POWERS. Menstrual fluid has always been reckoned to
possess a powerful influence over the affections of
men, and has held pride of place as an ingredient
of the love-charms administered by girls to would-be
lovers. CURATIVE PROPERTIES, In modern Germany girls have administered
drops of menstrual blood in coffee to their sweet-
hearts to make sure of retaining their affections-
a characteristic display of
" frightfulness." Accord-
ing to Pliny its action is effective on either sex,
for " if a man takes a frog and transfixes it with a
reed entering its body at the sexual parts and coming
out of the mouth, and then dips the reed in the
menstrual discharge of his wife, she will be sure to
conceive an aversion for all paramours." So, too, the Persian law-giver, Zarathustra, who
claims to have derived his code from the mouth
of the Supreme Being, Ahura Mazda, declares
menstruation to be the work of Ahriman, the
Devil, and lays down injunctions similar to those
which the Hebrew law-giver Moses claimed to have
derived from Jehovah. THE PURPOSE OF THE TABOOS. Just as the horror of menstrual blood is merely
a special aspect of the horror of blood in general,
so the taboos by which it is sought to segregate the
menstrual woman are but a special application of
the taboos by which any other supernatural agent
undergoes segregation. The purpose that underlies
them all is the isolation of the tabooed person from
society. The primitive mind discriminates little
between good and evil spirits, between holiness
and pollution, for in each condition there exists a
powerful supernatural force, be it active for good
or for evil. So comes it, as Frazer shows, that the
same taboos as apply to the menstruous woman are
applied to divine kings or priests. Thus in many
primitive races the divine personage may not touch
the ground with his foot, and the sun may not
shine upon him. We find him carried on men’s
shoulders, or stepping only on carpets or mats ; and
in many nations confined in palace, temple, or
underground dwelling: the Mikado of Japan
formerly observed both these taboos. Nowhere is
he so free from danger and so harmless, as
suspended between heaven and earth, in contact
with neither. In electrical language he is insulated. He can neither discharge his supernatural energy
with harmful violence, nor can it be drained from
him to the detriment of that magical power on
which the well-being of his people depends. paramours. Menstrual fluid has other intimate relations to
magic. Thus Pliny says that if it is sprinkled on
the door-posts it will render ineffectual all the
spells of the magicians. Josephus speaks of plants
acquiring magical properties by watering them with
menstruous blood and the urine of a woman-a
radio-active influence, no doubt! and by sym-
pathetic affinity stains made on a garment by
menstrual blood can only be removed by the urine
of the same woman. CURATIVE PROPERTIES, Among the beneficent properties of the menstrua
discharge must be ranked its power to cure various
ailments. The doctrine that the various secretions
and excretions are imbued with the same properties
as the body from which they are liberated led to
their wholesale adoption into the primitive phar-
Diacopoeia. The scatophagy, at which Aristophanes
jibed, was the parent of modern organotherapy. The first napkin used by a healthy virgin was
preserved for use in cases of plague, malignant
carbuncles. and other diseases ; it was damped with ir
J
1
v
f:
c
p
t
k
If in this state she will pollute mere man, still
tore will she pollute a sacred man. A menstruating
swish woman was formerly forbidden to shake
ands with a Rabbi, and women at these times
ere excluded from the Jewish synagogues and
om the communion table of the early Christian
Lurch. The Parsees, who reverence fire, will not
ermit a woman then even to look on a lighted
per ; in Rome, too, it was the vestal virgins who
:pt alive the sacred flame. Medicine men, too, 1334 a fringe of shells or bones hanging from her fore-
head so as to cover her eyes, as she is susceptible
to any malicious sorcerer at such times. Among
the Carrier Indians 132 menstruous women may not
cut up the grebes, which are caught in great
numbers, as by sympathy their blood would cause
an unnaturally prolonged flux in the transgressor. Similarly, Shushap women are forbidden, during
their seclusion at puberty, to eat anything that
bleeds. There are numerous instances of women
refraining, during menstruation, from fish and
flesh, and confining themselves to a vegetable diet
for the same reason. It is probably in obedience
to the dictates of sympathetic magic that the
physician of to-day administers preparations of
blood for the cure of amenorrhcea in place of the old
and proven haematinics. when about to compound their medicines, were in
the habit of making a saving clause that the
remedy would be effective, provided no menstruating
woman approached their chamber during the com-
pounding process. Surely nowadays the altogether
inordinate apprehensions of both nurse and surgeon
as to the evils of operation during the menstrual
state are tinctured witli the savour of this same
superstitious fear. THE UNDERLYING IDEA. Zulu
girls believe that they would shrivel to skeletons if
the sun were to shine on them at puberty, and in
some Brazilian tribes the girls think that a trans-
gression of the rules would entail sores on the neck
and throat. We find the same taboos as apply to
menstruous girls enjoined on warriors who have shed
blood, and on young warriors at the time of their
first campaign. They may not feed themselves;
they may not scratch their heads except with a
stick, for their hands are defiled with blood, and
they need protection from themselves.34
tc intervening stages by which the one has been
transformed into the other. Each condition is due
to ovarian activity, and comes to an end with com-
plete removal of the ovaries and any accessory
ovarian tissue; the medium of stimulation is
almost certainly an internal secretion of the ovary. In each condition the changes that occur in the
external and internal generative organs may be
broadly described as " active congestion," and are
similar in character. The congestive phenomena
in the external genitals are far more marked in
animals than in the human female, though occa-
sionally marked swelling and congestion may also
be seen in the latter. But in this and other points,
when animals have been submitted to ages of
domestic influence, there is a tendency to approxi-
mate to the human type. Domesticity has done for
animals what civilisation has done for man. Speaking generally the human female, in the
absence of pregnancy, menstruates once a month
throughout her sexually effective life, and is
capable of conception at any time. The nearest
approach to this is the case of the monkey, who
has a regular series of monthly periods, as does
the human female, but has a limited breeding
season-i.e., a limited period during which concep-
tion is possible. Both man and monkey have
developed sexual activity to a maximum, but in
the matter of reproductive capacity man seems to
tJ.LLv .LLLQJUUt;JL
V.1. LICJ..I:-’LVUu..B.IU.L Y V
B.IaJ’paJB.I.LIJJ OJ.LL OvvO UV
have outstripped the monkey; thus the latter
occupies a position midway between man and other
animals. Mares, cows, sheep, and pigs exhibit a
series of sexual periods during such limited portion
of the year as constitutes their appropriate sexual
season. The wild cat has one sexual season each
year, but the domestic cat has three or four. THE UNDERLYING IDEA. The idea underlying these customs is that there
resides in the menstrual blood some agent powerful
for good or for harm ; most of all is its power over
the virility of the male, enfeebling him in the
fierce struggle for the survival of the fittest. The spirit of the woman resides in her blood
and is transferable therewith to the male. It
is
a special aspect and
a special
applica-
tion
of the universal primitive blood-horror,
founded on this same belief that the spirit of the
person resides in his blood, and emerges with it
for good or for harm. In addition, there is some-
thing unintelligible about menstruation, something
uncanny, and the primitive mind explains it as due
to some spiritual agency, which may harm the
woman if she does not take certain precautions, and
may pass from her and harm others if they neglect
the prescribed precautions. g
p
p
depe ds. In like manner in some tribes the menstruous
woman is compelled to dwell apart in a dark hut
specially prepared for her use, or even in a hole
in the ground or a dark cage.3s When confined
in holes in the ground they are sometimes covered
with dirt, or sand, or a shelter of branches, or
with mats. Special attendants are told off to wait p
p ecaut o s
As examples of the need of self-protection, among
the Tinneh Indians..31 a menstruating girl may not
touch her own head with her hand, and, if it itches,
she must use a stick to scratch it. In some races
she is not allowed to feed herself. She must wear 1335 upon them and supply their needs, and they may
not eat any food which by sympathy would damage
similar food-supplies. In some races the girls at
these times screen themselves from the sun by
wearing huge hats. Elsewhere they are sewn up in
hammocks, as though dead, leaving only a small
hole for breathing. The rules that the girl may
not touch the ground nor see the sun keep her
suspended between heaven and earth; she can
poison neither of these great sources of life. But
not only the safety of others, but her own safety is
assured, if she be thus perfectly insulated. THE UNDERLYING IDEA. The
wild wolf has but one sexual period a year, but his
kinsman the domestic dog has two or more sexual
seasons each year. In all these animals conception
is strictly limited to the sexual season. It must
be remembered that the monkey, though not
strictly speaking a domestic animal, has developed
a high degree of domesticity of its own, and it is
at least possible that the semi-erect posture may be
an accessory factor in approximating its sexual
features to the human. y
p
We have spoken of the universal horror of blood,
which finds its highest expression in the lowest
orders of civilisation, and which extends also to, at
any rate, the herbivorous group of higher animals. This instinctive horror primitive man explains and
excuses by asserting the presence of a spirit in the
blood; the blood is the life. Blood is therefore
taboo, and taboos all that comes in contact
with it. As one reflects on the horror of menstrual blood,
which in all primitive communities has led to
prohibition, sometimes under penalty of death, of
sexual intercourse during the menstrual period,
the question inevitably suggests itself to one’s
mind whether the horror may not be the parent of
the prohibition. The extreme divergence that sub-
sists between the habit of man and the habit
of animals in the period of sexual intercourse can
hardly have failed to puzzle each of us at one time
or another. Esthetic and hygienic considerations
have come to dominate the mind of highly civilised
man to such an extent that we are apt to look no
further for an explanation. Yet it is just in those
lowly stages of civilisation, in which aesthetic and
hygienic considerations are of least account, that
obedience to the prohibition is most strict; it is,
indeed, a fundamental feature of savage ritual. With the development of civilisation medical
writers, from Hippocrates onward, actually recom-
mended sexual intercourse during menstruation for
sterility and other ailments; and in the Middle
Ages it had become so common that preachers
found it necessary to warn their congregations
against it.3"
. SEXUAL SEASON IN THE HUMAN FEMALE. ]
j
]
{
he
Now there is evidence that in former times the
sexual season of the human female was restricted,
as is the case with most mammals; indeed, it is
not improbable that the original type was universal
in all primitive species and consisted of a single
sexual season each year. Cook noted that not more
than 10 per cent. of Esquimaux women menstruated
during the long winter months; and as though
heightened desire were correlated to less frequent
menstruation, a sexual season of great intensity
set in at the first reappearance of the sun, and little
else was thought of for some time afterwards. Caldwell states that the Queensland natives have a
distinct sexual season in September-i.e., in spring
-and that they cannot be prevailed upon to do any
work for some weeks at that time of the year. Wiltshire iJ6 quotes authorities to show that in a
lowly state of civilisation, such as exists among the
women of Lapland, Greenland, the Faroe Islands,
Tierra del Fuego, and the Guaranis of Paraguay
menstruation takes place only at intervals of several
months. He suggests that the infrequent menstrua-
tion of girls at puberty-at intervals of 3, 4, and 6
months-and the recurrence of a similar periodicity
tt the end of sexual life may be a reversion to a
estricted ancestral type. against
The belief, of very ancient lineage, that coitus
during menstruation led to the birth of monsters
was formerly very widespread. Ambroise Pare
affirmed his belief, and rested it on a dictum of the
non-canonical Book of Esdras, that " menstruous
women shall
bring forth monsters." Ancient
mythology attributed the deformity of Vulcan to
the union of Jupiter with Juno during her men-
strual period. The Talmud went further and
assigned disorders of the mind, such as epilepsy,
cretinism, and insanity to the same cause. The
belief is not wholly dead nowadays. SEXUAL ACTIVITY, HUMAN AND ANIMAL. SEXUAL ACTIVITY, HUMAN AND ANIMAL. The identity of oestrus or " heat " in animals and
of menstruation in the human female admits of no
doubt ; it is, indeed, possible to trace many of the There is evidence also that in former ages the
period during which conception was possible to the
human female in each year was limited. SEXUAL SEASON IN THE HUMAN FEMALE. In
different countries there is a marked excess of
births at some or other period of the year, and
in general terms it may be stated that the
tendency is for women both to commence men-
struation and to bring forth children in the warmer
months. Westermarck considers that the evidence
points to two special seasons of sexual activity-
spring and autumn-and that the activity is greatest
towards the end of spring in the south of Europe
and rather later in the north. for you will find yourself confronted by the
puzzling fact that with some domesticated animals,
notably the bitch, there is a strong tendency, not
more, to reject intercourse during the period of
substantial flux, some preferring the cominence-
ment, but most the actual termination of the period
of heat. - MM!OSMp/;</.—1. Nat. Hist., vii.,
13. 2. Hartland : Primitive
Paternity, ii., 273. 3. Folk-lore in the Old Test,ament ; and Golden
Bough. 4. Nat. Hist., xxviii.,
23. 5. loiigfc,.Ilow:
Hiawatha,
Canto xiii. 6. Nat. Hist., vii., 13. 7. Ibid.. xxviii.. 23. 8. Ibid.,
xix., 57. 9. Havelock Ellis: Psychology of Sex, vol. ii. 10. Frazer:
Gulden Bough. 11. Havelock Ellis. Psvchology ot Sex, vol. ii. 12. Golden Bough, vol. iii., 302. seq. 13. Pliny: Nat. Hist., vii., 13,
and xi., 15. 14. Golden Bough, Ibid. 15. Pliny: Nat. Hist., xxviii., 23. 16. Bourke: Scatologic ]{!tes of All Nations. 17. Nat. Hist., Ibid. 18. Nat. Hist., vii., 13. 19. Ibid., xxviii., 23. 20. Ibid., vii.. 13. 21. Annales des Sciences Psychiques, September-October, 1897. 22. Pliny: Nat. Hist., vii., 13. 23. Ibid., xxviii, 23. 24. Aristotle:
Hist. Anim., vii., 2. 25. H. L. Strack: Der Blutaberglaube in der
Menschheit, eri. 1892, pp. 14-18. 26. Frazer: Golden Bough, iii., 202. 27. Folklore, vol. xix., p. 418; J. H. Weeks: Customs of the Lower
Congo Pfople. 28. Golden Bough, i., 323. 29. Ibid., iii., 331. 30. Nat. Hist., xxiii., 13. 31. Golden Bough, i., ?25. 32. Ibid , iii., 202. 33. Ibid., i., 325. 34. Ibid., iii., 208. 35. Havelock Ellis: Psychology of
Sex, vol. ii. 36. Brit. Med. Jour., 1883, The Comparative Physiology of
Menstruation. 37. Martin : Hirst’s System of Obstetrics. Though closely assodiated, conception and men-
struation are separable functions and not neces-
sarily interdependent, for conception may occur in
the prolonged absence of
menstruation. THE EARLY OPERATIVE TREATMENT OF
GUNSHOT WOUNDS OF THE
ALIMENTARY CANAL.
BY CUTHBERT WALLACE, B.S. LOND.,
F.R.C.S. ENG.,
CONSULTING SURGEON TO THE EXPEDITIONARY FORCE IN FRANCE. The advantage to primitive man and to wild
animals of a limited sexual season is that the
young may be born at the most propitious season of
the year, when food is most abundant and climate
most favourable. Once an artificial supply of food
and of warmth is assured, independent of season,
the necessity for a special season of sexual desire
and a special breeding season disappears, and gives
place to a type of greater frequency, regardless of
season. It is just these things that domestication
and civilisation afford. THE EARLY OPERATIVE TREATMENT OF
GUNSHOT WOUNDS OF THE
ALIMENTARY CANAL. ,
CONSULTING SURGEON TO THE EXPEDITIONARY FORCE IN FR ABDOMINAL wounds have always aroused great,
interest not only in the professional but also in the
lay mind. There has always been a tendency to
demand more care for these cases than for any
other class of injury. It is well, therefore, to inquire
into the frequency of abdominal compared with
other wounds, for in war the medical service must
try to do the greatest good to the greatest number;
q
it must not waste effort on a particular class of
case to the detriment of a larger but possibly less
interesting series of wounded. Under domestication the frequency of cestrus is
increased, and all the evidence goes to show that
food and temperature are the chief efficient agents
in producing the change. As to temperature, it is
a familiar fact that in the bitch winter oestrus does
not last so long as summer oestrus, and in the
human female the menstrual flux is more readily
established in summer than in winter. We may
well look in the same direction for an explanation
of the fact that, other things being equal, town-bred
girls commence menstruation several months in
advance of those reared in the country. Hard fare
and laborious life tend to diminish the menstrual
flow, while luxury and ease tend to increase it. In
some parts of this country the hard-working farm
girls still wear no protection during menstruation. Blood in the discharge is far more abundant in
domesticated animals than in wild. g
Relative Freq2cency of Abdominal Wounds. q
y
The actual number of men wounded in the
abdomen will never be known. The number of
abdominal wounds admitted to field ambulances
and casualty clearing stations are, subject to certain
error, known. The error is caused by the impossi-
bility of accurately diagnosing a perforating wound. The numbers vary with each observer, and the
surgeon who believes in the expectant treatment
will be inclined to reckon as perforating, wounds
which a believer in operative treatment would
class as non-perforating. The following are figures
taken in certain casualty clearing stations by careful
observers :- We see, then, that the circumstances of domesti-
cation and civilisation tend to an increase and a
greater frequency both of oestrus and of menstrua-
tion ; perhaps one other parallel condition may
help us a stage further in the attempt to unravel
the process of transformation. We know that in
most mammals non-satisfaction of sexual desire
by intercourse tends to a quicker return of heat
than normal; and it may be that the greater fre-
quency of menstruation in the human female may
in part be referable to the fact that civilisation
has brought it about that in the human female
sexual desire is often compelled to go unsatisfied.&bgr;7 tage of Abdonzinal Perforatin!1 TJrowuls to Total Wounds. A = 1-88 per cent. C
0 75 per cent. B = 1-5 .,
D = 062
" If we take the statistics from 9 field ambulances
and from 7 casualty clearing stations for a period
of six months we get the following figures :- Percentage of Abdominal Wounds to Total Wounds. Field ambulances
............ 192 per cent. Casualty clearing stations ...... 0-72
" Percentage of Abdominal Wounds to Total Wounds. Field ambulances
............ 192 per cent. Casualty clearing stations ...... 0-72
" SEXUAL SEASON IN THE HUMAN FEMALE. But
statistics show an enormous preponderance of con-
ceptions as occurring immediately after cessation
of the menses, while a minimum is reached just
before their commencement. INFLUENCE OF DOMESTICATION AND CIVILISATION. INFLUENCE OF DOMESTICATION AND CIVILISATION. CONCLUSION. These general considerations, and much more
besides, are a necessary prelude to any reasoned
attempt to answer the question whether the
universal human horror of menstrual blood may
have initiated the practice of abstinence from
intercourse during the menses. Give an affirma. tive reply and you are still little nearer a solution, The difference is probably accounted for in two
ways : 1. The mortality among abdominal cases at
field ambulances is very heavy. 2. There is more
time for accurate diagnosis at the casualty stations. The important fact brought out is the relatively
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https://www.nature.com/articles/s41419-022-04796-8.pdf
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EGFR/MET promotes hepatocellular carcinoma metastasis by stabilizing tumor cells and resisting to RTKs inhibitors in circulating tumor microemboli
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Cell death and disease
| 2,022
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cc-by
| 8,388
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www.nature.com/cddis
ARTICLE
OPEN
EGFR/MET promotes hepatocellular carcinoma metastasis by
stabilizing tumor cells and resisting to RTKs inhibitors in
circulating tumor microemboli
Shouyang Song1, Zhen Yu1, Yajing You1, Chenxi Liu1, Xiaoyu Xie1, Huanran Lv2, Feng Xiao1, Qiang Zhu
✉
Chengyong Qin 1,2,4
1,2,3,4 ✉
and
1234567890();,:
© The Author(s) 2022
The receptor tyrosine kinases (RTKs) family is well-recognized as vital targets for the treatment of hepatocarcinoma cancer (HCC)
clinically, whereas the survival benefit of target therapy sorafenib is not satisfactory for liver cancer patients due to metastasis. EGFR
and MET are two molecules of the RTK family that were related to the survival time of liver cancer patients and resistance to
targeted therapy in clinical reports. However, the mechanism and clinical therapeutic value of EGFR/MET in HCC metastasis are still
not completely clarified. The study confirmed that EGFR/MET was highly expressed in HCC cells and tissues and the
phosphorylation was stable after metastasis. The expression of EGFR/MET was up-regulated in circulating tumor microemboli (CTM)
to accelerate IL-8 production and resistance to the lethal effect of leukocytes. Meanwhile, highly expressed EGFR/MET effectively
regulated the Ras/MAPK pathway and stabilized suspended HCC cells by facilitating proliferation and inhibiting apoptosis.
Moreover, EGFR/MET promoted phosphorylation of hetero-RTKs, which was dependent on high-energy phosphoric acid
compounds rather than their direct interactions. In conclusion, highly expressed EGFR/MET could be used in CTM identification and
suitable for preventing metastasis of HCC in clinical practice.
Cell Death and Disease (2022)13:351 ; https://doi.org/10.1038/s41419-022-04796-8
INTRODUCTION
Liver cancer is one of the most lethal cancers, causing the 6th
carcinoma incidence and 4th cancer mortality in the world [1].
Hepatocellular carcinoma (HCC) is the most common type of liver
cancer in clinical with high relapse and mortality rates [2].
Respectively, as the first-line drug for the treatment of liver cancer
targeted to PDGFR/VEGFR family, sorafenib extends the survival
time of patients by 2.8 or 2.3 months in two clinical trials among
Caucasians and Asians, providing modest survival benefit [3, 4].
Recurrence and metastasis are responsible for the death of HCC
patients [5]. Research has confirmed that the lung was the most
common site of extrahepatic metastasis and significantly reduces
the survival time of patients [6]. However, the mechanism in
extrahepatic metastases of HCC remains unclear. According to
several studies, tumor cells in the circulating system are precursors
of metastasis and may contribute to tumor progression or
metastasis [7, 8]. Also, anoikis resistance is essential for tumor
metastasis [9, 10]. Multiply molecules and pathways including the
Ras/MAPK pathway, contribute to stabilizing tumor cells in
suspension [11]. However, mechanisms of survival and hematogenous metastasis of suspended HCC cells need to be completely
explored.
Receptor tyrosine kinases (RTKs) are currently considered as
critical roles in malignant transformation and cancer metastasis
[12]. The human genome encodes for 58 RTKs, which can be
divided into 20 subfamilies, and certain RTKs like VEGFR/PDGFR
families, are used for clinical target therapy of primary HCC [13].
They belong to the same membrane protein family which can
catalyze transferring of the γ phosphate of ATP to hydroxyl groups
of tyrosinases on target proteins and play important roles in the
control of most fundamental cellular processes including cell
cycle, migration, metabolism, proliferation, differentiation and
survival [14]. We have confirmed activation of VEGFR-1, one of the
RTKs which leads to tumor angiogenesis and induces MMP-9dependent invasion in HCC [15]. Some RTKs namely EGFR/MET can
participate in metastasis of tumors by forming a molecular
complex with integrin, mutation, amplification, and drug resistance [16–18]. RTKs may be vital for tumor cell survival in the
circulating system and distal metastasis. For example, overphosphorylation of EGFR in tumor cells cultured in suspension
was observed [7, 10]. EGFR and Her2 are also identified as a
biomarker on the surface of circulating tumor cells [19].
Additionally, MET-amplification has been discovered in the CTC
of NSCLC patients [20]. However, the function of RTKs expressed in
CTM is not completely clear. Expression and phosphorylation of
1
Shandong Provincial Hospital, Cheeloo College of Medicine, Shandong University, Jinan 250021 Shandong, P.R. China. 2Shandong Provincial Hospital Affiliated to Shandong First
Medical University, Jinan 250021 Shandong, P.R. China. 3The First Affiliated Hospital of Xinjiang Medical University, Urumqi 830054 Xinjiang, P.R. China. 4Shandong Provincial
Engineering and Technological Research Center for Liver Diseases Prevention and Control, Jinan 250021 Shandong, P.R. China. ✉email: zhuqiang@sdu.edu.cn;
chengyong_qin@126.com
Received: 2 November 2021 Revised: 25 March 2022 Accepted: 29 March 2022
Official journal of CDDpress
S. Song et al.
2
Fig. 1 EGFR/MET were highly expressed in HCC tissue and HCC cell lines compared to other RTKs. A, B Luminex and ELISA assay were used
for detection of expression of RTKs in HCC tissues and HCC cell lines. Data of tissues are shown as the mean ± SD. C Expression and
phosphorylation of EGFR/MET in several HCC cell lines were determined by Western blot. D Expression of EGF and HGF in carcinoma tissue
and HCC cells. Tissues and cells were lysis and Luminex was used for detection of concentration. Data of tissues are shown as the mean ± SD.
Patient information for detecting concentration of RTKs and EGF/HGF in tissues is suppled in Table 4.
RTKs are quite different in various tumors [21]. It is therefore that
targeting the most expressional and functional RTKs is the key to
the treatment of HCC.
In the present study, we determined the expression of RTKs
proteins in CTM and cells and verified the important roles of EGFR/
MET and the possibility of being used as a clinical treatment
target. Furthermore, we identified the mechanism that EGFR/MET
promotes metastasis and stabilizes HCC cell lines in hematogenous metastasis. These findings provide mechanistic evidence for
us to design HCC medications targeting EGFR/MET, thereby
benefiting patients with HCC in reducing the incidence of
metastasis and prolonging survival time.
RESULTS
EGFR/MET is highly expressed in human HCC tissues and cell
lines compared to other RTKs families
We firstly explored the expressions of RTKs in HCC tissues and
HCC cell lines. Interestingly, the expressions of RTKs in HCC
tissues and HCC cell lines varied greatly. Certain RTKs namely
M-CSFR were highly expressed in HCC tissues whereas poorly
expressed in cell lines. However, other RTKs namely the HER
family were highly expressed in both HCC tissues and cell lines.
Compared to the other RTKs we detected, the concentration of
EGFR/MET was high expressional RTKs both in HCC tissues and
HCC cell lines. Especially in HCC cells, expressions of EGFR and
MET in MHCC97H were 54,548.83 and 607,470.8 pg/mg, respectively, and those of SK-HEP1 were 103,099.9 and 38,652.84 pg/mg,
respectively, higher in expression than other RTKs (Fig. 1A, B).
Additionally, both EGFR/MET in MHCC97H/LM3 cell lines with MET
amplification were highly phosphorylated. In contrast, low
phosphorylation of EGFR/MET was detected in PLC/PRF/5 and
SK-HEP1 (Fig. 1C). Interestingly, the high expression level of HGF
and low expression of EGF were observed in HCC tissue, though
low concentration of EGF and HGF were expressed in HCC cells of
tissues (Fig. 1D, Supplementary Fig. 1A). Low concentrations of
EGF and HGF were observed in HCC cell lines, while HGF
expression in MHCC97H was higher than SK-Hep 1 (Fig. 1D,
Supplementary Fig. 1B). In brief, the EGFR/MET of the RTKs family
was significantly expressed in HCC tissues and cells, and
phosphorylation of EGFR/MET was related to the expression of
MET.
Expression of EGFR/MET is up-regulated in CTM but downregulated in lung metastasis
Next, we verified whether the expressions of EGFR/MET using a
xenograft model for lung metastasis of mice were stable during
metastasis. In this model, Hepa1-6 cells were successfully
metastasized from the left lobe of the liver to the lungs (Fig.
2A). Compared with the primary tumor, there was no significant
difference in expression and phosphorylation levels of EGFR/MET
in metastatic tumors (Fig. 2B). The expression of EGFR in HCC was
markedly lower than that in peritumor tissues, while the
expression of MET was substantially higher. However, the
expression in lung metastases was just reversed compared with
normal lung tissues (Fig. 2C). Interestingly, we found the presence
of CTM in mice with widespread lung metastasis, and the
expression level of EGFR/MET was higher than that in primary
tumor and lung metastasis (Fig. 2D). In scRNA-seq, cells were
divided into clusters for analysis (Fig. 2E). We found EGFR/MET
were mainly expressed in HCC cells of carcinoma tissue, which
differed from some RTKs for clinical targets expressed mainly in
peritumor tissue such as the PDGFR family (Fig. 2F). However,
expression of EGFR/MET in lung metastasis was lower than that in
primary tumor, which was in agreement with most of the highly
expressed RTKs (Fig. 2G). The ratios of RTKs expression in
carcinoma tissue compared to whole tissues in primary tumor
and lung metastasis were provided in Table 1. Collectively,
expression of EGFR/MET increased during metastasis and
decreased after metastasis, but phosphorylation of EGFR/MET
kept stable after metastasis. It suggested that EGFR/MET was an
important marker for the detection of CTM. As stable phosphorylation of EGFR/MET is suitable for the treatment of lung
metastasis, targeting EGFR/MET in CTM may be more effective
in the treatment of HCC.
Anoikis resistance is related to up-regulation in expression
and phosphorylation of EGFR/MET
Since metastasis of CTM was closely related to anoikis resistance,
we next observed the role of EGFR/MET in HCC cell lines of
Cell Death and Disease (2022)13:351
S. Song et al.
3
Fig. 2 Expression of EGFR/MET is higher in CTM but lower in lung metastasis. A Gross image of HCC and lung metastasis in mice 60 days
after injection of Hepa1-6 cell line. B Mice Phosphorylation Array was used for detection of RTK phosphorylation of HCC tissue separated from
liver and lung. C Immunofluorescence analyzed the differences of EGFR/MET expression in cancer tissues and peritumor tissues in liver and
lung. D Immunohistochemistry analyzed the differences of EGFR/MET expression in cancer tissues and CTM. E UMAP showed different clusters
in carcinoma tissues of liver and lung. F Distribution of RTKs were showed in each cell of primary tumor and lung metastasis with Single cell
RNA sequence by heatmap. G Difference of RTKs with high expression in carcinoma tissue between primary tumor and lung metastasis were
showed. Data are shown as the mean ± SD, *P < 0.05.
suspension. Phosphorylation RTKs array reported that phosphorylation of EGFR/MET in suspended HCC cell lines was enhanced
and Western blot assays indicated that peak point appeared at
24–48 h (Fig. 3A, B). IL-8 synthesis/secretion could be interrupted
by EGFR/MET inhibitor, which was observed in MHCC97H cell lines
with high phosphorylation of EGFR/MET. However, in PLC/PRF/5
HCC cell lines with lower phosphorylation of EGFR/MET, the
Cell Death and Disease (2022)13:351
inhibitory effect of EGFR/MET inhibitors was greatly reduced, and
secretion of IL-8 could interrupt by EGFR inhibitor was relatively
obvious (Fig. 3C). Also, inhibition of EGFR/MET made HCCLM3 cell
lines more easily killed by leukocytes and more leukocytes could
survive (Fig. 3D), which were not beneficial to hematogenous
metastasis of HCC cell lines. The findings suggested that EGFR/
MET played an important role in the metastasis of HCC cells.
S. Song et al.
4
Table 1.
Expression of RTKs in carcinoma tissue.
Liver
Lung
GENE
Expressed in tumor
Expressed totally
Ratio
Expressed in tumor
Expressed totally
Ratio
P Value
****EGFR
4536
4722
0.96061
1780
2703
0.658528
<0.0001
****MET
5143
5396
0.953113
1766
2174
0.812328
<0.0001
AXL
3536
5604
0.630978
1991
8497
0.234318
*DDR1
2276
2641
0.861795
1259
1997
0.630446
0.107143
EPHA1
16
29
0.551724
3
28
****EPHA2
9029
9507
0.949721
3394
4141
0.819609
EPHA3
0
14
0
3
114
0.026316
EPHA4
10
41
0.243902
30
285
0.105263
EPHA5
2
6
0.333333
5
8
0.625
EPHB1
0
6
0
0
37
0
EPHB2
148
159
0.930818
53
318
0.166667
EPHB3
66
75
0.88
113
262
0.431298
****EPHB4
5616
6007
0.934909
2236
2613
0.855721
EPHB6
847
914
0.926696
378
485
0.779381
**ERBB2
1031
1085
0.95023
489
632
0.773734
ERBB3
174
271
0.642066
192
306
0.627451
ERBB4
0
6
0
0
3
0
****FGFR1
3603
4064
0.886565
1512
2961
0.510638
FGFR2
165
199
0.829146
79
265
0.298113
FGFR3
135
230
0.586957
96
280
0.342857
****FGFR4
1257
1326
0.947964
555
606
0.915842
FLT1
119
1597
0.074515
108
2701
0.039985
FLT3
31
882
0.035147
26
890
0.029213
FLT4
1415
2320
0.609914
916
1255
0.72988
IGF1R
20
761
0.026281
45
2199
0.020464
IGF2R
660
1477
0.446852
464
2272
0.204225
INSR
2362
3133
0.75391
1022
2073
0.493005
KDR
32
2086
0.01534
17
707
0.024045
MCSFR
186
3752
0.049574
228
7354
0.031004
MST1R
22
23
0.956522
65
161
0.403727
MUSK
11
12
0.916667
28
39
0.717949
NGFR
45
77
0.584416
431
499
0.863727
PDGFRA
191
235
0.812766
119
2844
0.041842
PDGFRB
5
200
0.025
23
1477
0.015572
RET
0
28
0
2
73
0.027397
ROR1
463
496
0.933468
360
755
0.476821
ROR2
41
47
0.87234
110
263
0.418251
***RYK
3315
3880
0.854381
1766
2935
0.601704
TIE1
11
345
0.031884
15
435
0.034483
0.0317
<0.0001
<0.0001
0.0011
<0.0001
<0.0001
0.0007
RTKs Ratio > 85% expression in carcinoma cells of liver and copy > 1000 was used for comparing difference in carcinoma tissue before and after metastasis.
Mann-Whitney test was used for analysis. * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001.
EGFR/MET stabilizes suspended HCC cells and avoids the
killing of leukocytes by regulating the Ras/MAPK pathway
As the Ras/MAPK pathway regulated by RTKs played an important
role in tumor metastasis, the function of EGFR/MET in regulating
the Ras/MAPK pathway will be further investigated in our
subsequent research. Firstly, like phosphorylation of EGFR/MET,
we found Ras-GTP was up-regulated in HCC cell lines in
suspension (Fig. 4A). Both EGF and HGF could activate the
corresponded receptor and lead to the activation of the Ras/MAPK
pathway in HCC cell lines with low phosphorylation of EGFR/MET
and activation of EGFR were was substantially promoted in the
regulation of the Ras/MAPK pathway. However, HCC cell lines with
high phosphorylation of EGFR/MET were not sensitive to EGF and
HGF. In contrast, the inhibitor of EGFR/MET could down-regulate
the Ras/MAPK pathway in HCC cell lines with high phosphorylation of EGFR/MET instead of a condition of low phosphorylation of
EGFR/MET. In brief, the Ras/MAPK pathway could be regulated by
the basal phosphorylation levels of EGFR/MET (Fig. 4B, C). While
fully activating the corresponding RTKs by a sufficient amount of
ligand, we found that MET worked more effectively inactivating
Cell Death and Disease (2022)13:351
S. Song et al.
5
Fig. 3 Expression and phosphorylation of EGFR/MET was up-regulated in suspended HCC cells. A RTK array was used to measure
phosphorylation of RTKs in suspended HCC cell lines. B Western blot was used to analyze phosphorylation of EGFR/MET in HCC cell lines with
0, 24, 48, 72 h of suspension. Low concentration of suspended HCC cells was used in prevention of cluster formation. C ELISA was used to
detect IL-8 secretion of MHCC97H and PLC/PRF/5 cell lines with EGFR/MET inhibitor. D Immunofluorescence analyzed the interactions
between MHCC97H cells and leukocyte with or without EGFR/MET inhibitor. Data are shown as the mean ± SD, n = 3. *P < 0.05.
the Ras/MAPK pathway than other RTKs in treating clinical HCC
(Fig. 4D). However, simple knockdown of EGFR/MET, EGFR in
particular, could directly inhibit the Ras/MAPK pathway, which
could barely be restored by activating other RTKs (FGFR, VEGFR,
and PDGFR) based on their low expression. Moreover, the Ras/
MAPK pathway could not respond to activation of RTKs (PDGFR
and VEGFR) poorly expressed (Fig. 4E–G). However, based on high
expression and activation of EGFR or MET could offset the effect of
knockdown in each other on the Ras/MAPK pathway to some
degree (Fig. 4H, I). EGFR and MET synergistically regulated the Ras/
MAPK pathway. EGFR inhibitors could decrease the phosphorylation level of MET that was activated endogenously or exogenously
in HCC cell lines which were sensitive to EGFR inhibitors, but it did
not work effectively in HCC cell lines with low phosphorylation of
EGFR (Fig. 4J, K). Moreover, MET knockdown could weaken the
effect of EGFR inhibitors on the interruption of the Ras/MAPK
pathway (Fig. 4L). Different expressions of EGF/HGF indicated that
activation of EGFR/MET relied on different ways in vivo and higher
levels of HGF in HCC tissue allowed MET to play an important role
in the regulation of the Ras/MAPK pathway.
Next, we analyzed the activation of ERK in suspended HCC cell
lines. The findings were identical to Ras-GTP indicating that the
phosphorylation level of ERK was up-regulated in suspended
HCC cell lines (Fig. 5A). Following the inhibition of the Ras/MAPK
pathway, DNA synthesis and G2/S-phase of the cell cycle were
Cell Death and Disease (2022)13:351
decreased and the G1-phase of the cell cycle increased in HCC
with or without suspension. Moreover, S-phase increased in
suspended HCC cells, indicating that the Ras/MAPK pathway
maintained the growth of HCC cell lines (Fig. 5B, C). Flow
cytometry confirmed that inhibition of the Ras/MAPK pathway in
HCC cells with or without suspension could promote the
increase of apoptosis in HCC cell lines, but the effect in HCC
cells with suspension was significant (Fig. 5D). We also found all
cyclin proteins were down-regulated followed by Ras/MAPK
inhibition. In detail, Cyclin A/B were less interfered with by Ras/
MAPK inhibitors and poorly expressed in suspended HCC cell
lines. Conversely, Cyclin E was highly expressed in suspended
HCC cell lines and could be easily down-regulated by Ras/MAPK
inhibitors. Both cultured in suspension and Ras/MAPK inhibitors
could activate PARP, a protein major in DNA repairing in HCC
cell lines [22]. Moreover, a combination of Ras/MAPK inhibitor
and suspension caused higher activation of PARP in HCC cell
lines, which was related to higher apoptosis of HCC cell lines
(Fig. 5E). Also, inhibition of the Ras/MAPK pathway made
suspended HCC cells more easily to be killed by leukocytes
(Fig. 5F). Collectively, the data indicated that the Ras/MAPK
pathway induced by EGFR/MET stabilized suspended HCC cell
lines and prevented the lethal effect of leukocytes. Moreover, it
was more difficult for suspended HCC cells to survive under a
condition of poorly phosphorylated ERK.
S. Song et al.
6
Fig. 4 EGFR/MET were powerful in regulation of Ras/MAPK pathway based on high expression. A Ras Activation Assay Kit was used for
detection of Ras-GTP in suspended HCC cell lines. Low concentration of suspended HCC cells was prevented from cluster formation and used
for detection of Ras activation in 0, 24, 48 and 72 h. B Western Blot was used to confirm the regulation of EGFR/MET in Ras/MAPK pathway,
including sensitivity of Ras/MAPK pathway to EGFR/MET inhibitors and C EGF/HGF in activation of Ras/MAPK pathway in different HCC cell
lines. D 50 ng/ml of recombined ligands was used for RTKs phosphorylation and cells were harvested after 1 h for comparing activation of Ras/
MAPK pathways. (E) VEGF, (F) PDGF-BB and (G) FGF2 were verified in restoration of Ras/MAPK pathway activation in PLC/PRF/5 cell line with
EGFR/MET knock-down. H EGF/HGF was detected in activation of Ras/MAPK pathway of PLC/PRF/5 with MET knock-down and I EGFR knockdown. J Western Blot showed the effect of EGFR on MET down-regulation in EGFR inhibitor in PLC/PRF/5 cell line with exogenous/
endogenous HGF stimulation. K EGFR inhibitor was used in detection of MET inhibition in SK-Hep1 cell line, which is not sensitive to EGFR
inhibitor. L Effect of EGFR inhibitor was verified on PLC/PRF/5 cell line with MET knock-down.
EGFR/MET participates in the phosphorylation of multiple
RTKs family members
Based on the high expression of EGFR/MET in HCC tissue/cell lines
and stable expression in metastasis, the regulatory effects of
EGFR/MET on the phosphorylation level of other RTKs were
investigated. In this study, Lenvatinib, as an inhibitor of PDGFRβ/
VEGFR/FGFR/RET, was effective in suppressing EGFR/MET and
targeting RTKs in MHCC97H and HCCLM3, but less effective on
PLC/PRF/5. In contrast, Sorafenib, as an inhibitor of PDGFRβ/
VEGFR/Raf, had less effect on EGFR/MET, and barely suppressed
the function of PDGFRβ in MHCC97H and LM3 (Fig. 6A). Also, we
found that high phosphorylation of EGFR/MET could maintain the
hyperphosphorylation status of some RTKs which were clinical
targets for HCC treatment, such as PDGFRβ and FGFR1, and it was
obvious in HCC cell lines with MET amplification. Briefly, both
EGFR/MET inhibitors could reduce phosphorylation of ErbB family,
MET family, and RET, and MET inhibitors could additionally
interfere with the InsR family (Fig. 6B). MET knockdown exhibited
the same effect as MET inhibitors in the suppression of
phosphorylation of multiple RTKs (Fig. 6C). Additionally, EGFR/
MET inhibitors could effectively interrupt phosphorylation of FGFR,
VEGFR, and PDGFRβ which were for clinical therapy, even
stimulated by RTK ligand (Fig. 6D). Similarly, MET knock-down
also inhibited phosphorylation of those RTKs (Fig. 6E). EGFR/MET
had an impaired ability to influence the phosphorylation of
hetero-RTKs in HCC cell lines with low phosphorylation (Fig. 6F). In
brief, EGFR/MET also affected the inhibitory effect of clinical antiHCC drugs on RTKs, which might lead to treatment failure and
extrahepatic metastasis.
Phosphorylation of EGFR/MET is related to high-energy
phosphate compounds, not EGFR/MET hetero-dimer
Next, we focused on possible mechanisms related to the
phosphorylation of EGFR/MET. Interestingly, we found that phosphorylation of MET was affected by MET amplification (Fig. 7A).
Phosphorylated EGFR/MET, either endogenous or induced by
ligands, tended to form homo-dimers and it was almost impossible
for EGFR/MET to form hetero-dimer, whether EGFR/MET was
phosphorylated or not (Fig. 7B, C). It suggested that phosphorylation
of EGFR/MET was not affected by hetero-dimer. In further research,
we found sufficient ATP in the solution could effectively promote
phosphorylation of most RTKs, even in HCC cell lines with METamplification, but MET was not affected (Fig. 7D). MET phosphorylation was affected by GTP rather than ATP (Fig. 7E). Phosphorylation
of EGFR induced by ATP was not affected by MET, but the factor
creatine kinase that degraded ATP could reduce the level of EGFR
phosphorylation (Fig. 7F). It suggested that EGFR could be directly
phosphorylated by high-energy phosphate compounds in the
absence of ligands, but MET relied on GTP, MET amplification, or
ligand induction (Fig. 8).
Cell Death and Disease (2022)13:351
S. Song et al.
7
Fig. 5 Ras/MAPK pathway stabilized suspended HCC cell lines and avoid killing of leukocytes. Cells were divided in 4 groups: (a) cultured
without suspension (b) cultured with suspension (c) cultured without suspension, 10 µM of AZ628 were used for Ras/MAPK pathway inhibition
(d) cultured with suspension, 10 µM of AZ628 were used for Ras/MAPK pathway inhibition. Cells in different group were detected in (A)
phosphorylation of ERK by Western blot, (B) cell cycle by flow cytometry, (C) DNA synthesis by EDU, (D) apoptosis by flow cytometry and (E)
Cyclin, CDK4 and PARP by Western blot. F Immunofluorescence was used in determining interactions between MHCC97H and leukocyte with
or without Ras/MAPK inhibitor. Data of flow cytometry are shown as the mean ± SD, n = 3. *P < 0.05.
DISCUSSION
RTKs nowadays are considered to be critical in tumorigenesis,
intrahepatic metastasis, and distal metastasis of liver cancer [23].
The correlation exists between multiply RTKs and recurrence times
of liver cancer [13]. Currently, the clinical targeted drugs for liver
cancer are mainly aimed at RTKs. For example, sorafenib, as a
multi-RTKs inhibitor that exerts an antiangiogenic effect by VEGFR
2/3 and PDGFR-β, is typically used as the first-line therapy in
patients with primary advanced HCC [24, 25]. However, sorafenib
resistance can cause therapeutic failure and metastasis of HCC
[26]. Research has shown that RTKs EGFR/MET are responsible for
sorafenib resistance [27, 28] and they were highly phosphorylated
in multiply carcinoma cells including the lungs, breasts, and
pancreas [21]. Interestingly, highly expressed RTKs VEGFR1 and
PDGFRβ in HCC tissues were barely expressed in HCC cell lines in
the present study. Instead, RTKs EGFR/MET, which was targeted for
HCC treatment, were highly expressed in both HCC tissues and
HCC cell lines. Especially in HCC cell lines, the EGFR/MET levels
were higher than other common RTKs. It suggested that some
RTKs inhibitors sorafenib, tended to be effective to primary HCC
mass by interruption of neoplastic angiogenesis, but low
Cell Death and Disease (2022)13:351
expression of VEGFR/PDGFRβ or compensation effect from
EGFR/MET in HCC cell lines were invalid in metastasis. Thus,
discovering the mechanism of EGFR/MET promoting distal
metastasis may be helpful for the treatment and recurrence
of HCC.
Extrahepatic metastasis is easily observed in patients with HCC
[29]. Because of the high level of expression, EGFR/MET has been
reported as a hopeful target for HCC and combination therapy
may benefit patients with prolonged survival time by interaction
with multiply molecules like PARP [22]. Lung metastasis is most
frequently observed in extrahepatic metastasis [6]. In the C57
xenograft model, CTM with high EGFR/MET expression was
detected in the blood vessels of lung metastasis mice, which
was higher than that in tumor tissues, indicating EGFR/MET as a
suitable marker for CTC/CTM detection. Meanwhile, stable
phosphorylation of EGFR/MET makes targeted therapy possible
for lung metastases. However, expression of EGFR/MET in CTM is
higher than that in lung metastasis, suggesting that it is necessary
to target CTM. EGFR/MET may participate in the metastasis of
carcinoma through circulating tumor cells. For example, EGFR has
been identified as a biomarker on the surface of circulating tumor
S. Song et al.
8
Fig. 6 EGFR/MET participated in regulation of phosphorylation of hetero-RTKs. A Western Blot was used to observe phosphorylation of
RTKs in HCCLM3, MHCC97H and PLC/PRF/5 cell lines with Lenvatinib or Sorafenib added. B Western blot was used to determine the effect of
down-regulation of EGFR/MET on hetero-RTKs, including: Phosphorylation of EGFR, MET, Her3, RON, IR/IGF-1R and RET in HCC cell lines with
EGFR/MET inhibitor added. C Phosphorylation of EGFR, Her3, RON, IR/IGF-1R and RET in HCC cell lines with MET knock-down.
D Phosphorylation of FGFR1, VEGFR2 and PDGFRβ with ligand stimulated in HCC cell lines with EGFR/MET inhibitor added. E Phosphorylation
of FGFR1, VEGFR2 and PDGFRβ with ligand stimulated in HCC cell lines with MET knock-down. F Phosphorylation of RTKs in PLC/PRF/5 cell line
with EGFR or MET knock-down.
cells [19]. Over phosphorylation of EGFR has been observed in
suspended carcinoma cells such as lung and gastric cancer cells
[7, 10]. Also, a clinical trial has shown that MET inhibition is related
to reducing CTC [30]. Cancer cells with anoikis resistance are
suitable for entering the circulating or lymphatic system, which is
essential for successful cancer metastasis [31], and this may be
one mechanism of tumor metastasis promotion. Interference
function of EGFR/MET attenuated anoikis resistance of tumor cells
[32, 33]. Similarly, our data showed the phosphorylation of EGFR/
MET phosphorylation was keeping stable or up-regulated in HCC
cell lines cultured at beginning of suspension in this study. As the
suspension time passed by, phosphorylated EGFR/MET decreased.
Most tumor cells in the circulating system survive for only a few
minutes and living cells are prone to combine with other tumor
cells and non-tumor cell types in peripheral blood, which are more
aggressive than single cells in cancer metastasis [34, 35]. EGFR/
MET was also related to cytokines synthesis/secretion and
resistance to leukocytes of HCC cell lines and inhibition of EGFR/
MET could interrupt metastasis of HCC cell lines, especially in CTM
with higher expression of EGFR/MET.
The Ras/MAPK pathway is considered a survival factor for
carcinoma cells, and RTKs produce a similar mechanism
in regulating the Ras/MAPK pathway [14, 36]. Increased expression
and activity of the Ras/MAPK pathway have been observed
in mouse HCC models and human HCC tissues and they have
been reported as important factors for anoikis resistance and
metastasis [36, 37]. Over-activated Ras/MAPK pathway is considered to be associated with tumorigenesis and metastasis [38].
We found that the ability of RTKs to regulate the Ras/MAPK
pathway was correlated with phosphorylation and expression
levels in cells. Moreover, the RTKs family has cross-regulation on
the Ras/MAPK pathway and RTKs inhibitors can sometimes cause
Ras/MAPK re-activation, namely IR/IGF-1R knockdown leading to
up-regulation of EGFR [39]. On the other hand, for RTKs with
extremely low expression levels, even sufficient ligands cannot
trigger the regulation of the Ras/MAPK pathway. Thus, EGFR and
MET play important roles in the regulation of the Ras/MAPK
pathway based on their high expression in the RTKs family. Same
as the result of phosphorylation of EGFR/MET, we found that ERK
was highly phosphorylated in suspended HCC cell lines. Inhibition
of Ras/MAPK pathway promoted apoptosis, declination of DNA
synthesis, and interfered cell cycle, which was observed in HCC
cell lines both cultured without and with suspension. Also, overactivation of ERKs in suspended HCC cell lines could offset but not
completely reverse the damage from the suspension environment, thereby maintaining the stability of suspended cells and
preventing lethal effect of leukocytes in degree with mild
activation of PARP. Based on the previously described results,
EGFR/MET stabilized HCC cell lines and promoted distal metastasis
by activating the Ras/MAPK pathway.
RTKs have a similar effect on the activation of downstream
signaling pathway Ras/MAPK pathway [14]. Our previous experiments have confirmed the advantage in the concentration of
EGFR/MET in highly aggressive HCC cell lines and MET amplification is one of the important reasons for the hyper-phosphorylation
of EGFR. It has been reported that EGFR/MET may be involved in
resistance of RTKs target therapy [40, 41], and MET amplification is
important in tumor metastasis [42]. In our result, EGFR/MET could
indirectly regulate the phosphorylation of multiple RTKs and the
effect was superior to their ligands in cells with MET amplification.
Thus, highly phosphorylated EGFR or MET could easily induce
resistance to RTKs inhibitors. Interestingly, EGFR/MET didn’t
directly induce phosphorylation of hetero-RTKs via the action of
kinases. High-energy phosphate compounds represented by ATP
played important roles in RTKs phosphorylation [13]. Also, EGFR/
MET over-phosphorylation might cause regulation disorder of
high-energy phosphate compounds, thereby promoting heteroRTKs phosphorylation. EGFR could be easily phosphorylated by
ATP/GTP in the absence of EGF, that is, EGFR might be more
dependent on high-energy phosphate compounds in the cell than
Cell Death and Disease (2022)13:351
S. Song et al.
9
Fig. 7 Phosphorylation of EGFR/MET was related to high-energy phosphate compounds but not EGFR/MET hetero-dimer. A MET
phosphorylation ratio was compared in different HCC cell. Co-immunoprecipitation was used for detection of crosstalk in (B) EGFR/p-EGFR or
MET/p-MET after stimulation of ligand and (C) EGFR-EGFR, MET-MET and EGFR-MET in cells with MET amplification. D RTKs Phosphorylation
Array was used for detection of phosphorylation of RTKs after incubation with 0.2 mM ATP in PLC/PRF/5 and MHCC97H cell lines. E Western
Blot was used for detection of phosphorylation of EGFR/MET stimulated by ATP/GTP. 0.2 mM of ATP/GTP was incubated with cell lysate of PLC/
PRF/5 with kinase buffer for 1 h at 37 °C. F Phosphorylation of EGFR in PLC/PRF/5 cell lysis with 0.2 mM of ATP with creatine kinase or MET
knock-down was detected by Western Blot.
Table 2.
Sequences of EGFR/MET SiRNAs.
Gene
Target sequence
EGFR
GCAGUCUUAUCUAACUAUGAUGCAA
MET SiRNA1
CUGGUUUUGUCGACGUAAA
MET SiRNA2
CGAGGGAAUCAUCAUGAAA
MATERIALS AND METHODS
Cell preparation
Human HCC cell lines MHCC-97H (Zhong Qiao Xin Zhou Biotechnology Co.,
Ltd, China), PLC/PRF/5 (Hongbo Biotechnology, China), HCCLM3, SK-Hep1,
and mouse HCC cell line (Procell, China) were cultured in DMEM containing
high glucose (Biological Industries, Israel) with 10% fetal bovine serum
(Biological Industries, Israel) at 37 °C in a humidified atmosphere containing 5% CO2. 100 mg/mL penicillin G and 50 μg/mL streptomycin (Biological
Industries, Israel) were added. All cell lines were identified by STR and
mycoplasma tests and all were used within 3 months after thawing early
passage cells.
Fig. 8 A proposed model of EGFR/MET-mediated in HCC and
metastasis. After metastasis to blood, both expression and
phosphorylation of EGFR/MET was up-regulated in CTM. Based on
high expression, EGFR/MET could induce phosphorylation of other
RTKs via ATP production, and over-phosphorylation of RTKs were
independent to RTKs ligand and resistant to RTKs inhibitors.
Compared to other RTKs, EGFR/MET could more effectively activate
Ras/MAPK pathway. Activation of Ras/MAPK pathway participate in
cell proliferation, resistance to apoptosis and stabilization of
suspended HCC cells. Also, EGFR/MET mediated in helping CTM to
avoid killing effect of leukocytes.
ligands in phosphorylation. Moreover, MET was not sensitive to
ATP, and HGF not EGF was over-expressed in cell lines with MET
amplification and HCC tissues, suggesting that MET was easier to
perform biological functions than EGFR in terms of liganddependent phosphorylation. Thus, dysfunctional RTKs could
definitely affect the functions of the Ras/MAPK pathway.
In conclusion, we have demonstrated that EGFR/MET are as the
two of the most expressed in the RTKs family in both HCC tissue
and cells. The study also clarifies the relationship between EGFR/
MET and CTM. Activation of EGFR/MET caused by complicated
factors helps suspended HCC cell lines to maintain cell stability
and distal metastasis. Targeting EGFR/MET may provide opportunities for the prevention of metastasis of HCC.
Cell Death and Disease (2022)13:351
Suspension culture
Of 3% Poly-HEMA (Sigma, MO, USA) was dissolved in ethanol for
preventing HCC cell lines adhesion. Tissue culture plates were coated
with 1 ml of Poly-HEMA solution and dried overnight at room temperature
in a laminar flow hood. All cells were sub-cultured, re-plated in suspension
condition, and harvested at indicated time points for detection.
EGFR/MET knockdown cell construction
For EGFR/MET knockdown, siRNAs were transfected into MHCC97H/
HCCLM3/PLC-PRF-5 cells using Lipofectamine 3000 (Thermo Fisher, MD,
USA). Cells were cultured for further experiments after 48 h. The sequences
of siRNAs were listed in Table 2.
RTKs and Ras/MAPK inhibitors and RTKs ligands
Sorafenib (VEGFR and PDGFRβ), Lenvatinib (RET, VEGFR, PDGFRβ, and
FGFR), Erlotinib (EGFR), Afatinib (EGFR), Crizotinib (MET), and AZ628 (Ras/
MAPK) were purchased from Selleck (TX, USA) and dissolved in DMSO.
Recombined EGF, HGF, FGF2, PDGF-BB, and VEGF were purchased from
PeproTech (NJ, USA), and recombined insulin was bought from Selleck
(TX, USA).
Western blot and co-immunoprecipitation (Co-IP)
The composition of Co-IP buffer was 50 mM Tris, 150 mM NaCl, 1% NP40,
0.5% sodium deoxycholate, 1 mM EDTA, 1 mM PMSF and 1× protease
inhibitor cocktail (CWBIO, China). Co-IP and Western blot were performed
S. Song et al.
10
Table 3.
Purchase source and catalog ID of antibodies used.
Antibody for Western blot
Assay
Table 4. Clinicopathologic characteristics of HCC patients in cohorts
for IHC and RTKs concentrations.
Cohort 1 (n = 23,
for IHC) No. of
patients (%)
Cohort 2 (n = 16, for
Luminex detecting
RTKs and EGF/HGF)
No. of patients (%)
22 (95.7%)
14 (87.5%)
1 (4.3%)
2 (12.5%)
<60
13 (56.5%)
10 (62.5%)
≥60
10 (43.5%)
6 (37.5%)
Characteristics
Enterprise
Detail
EGFR
Abcam
ab52894
pEGFR
CST
3777T
MET
CST/Abcam
8198T/ab51067
Gender
pMET
CST
3077T
Male
pHer3
Abcam
ab255607
Female
pMST1R
Abcam
Ab124671
Age
pIGF1R/pInsR
CST
3021T
pRET
Abcam
ab51103
pFGFR
Abcam
ab173305
HBV infection
pVEGFR2
CST
2478T
Yes
12 (52.2%)
12 (75%)
pPDGFRβ
Abcam
ab218534
No
11 (47.8%)
4 (25%)
MEK
Abcam
ab178876
Tumor size
p-MEK
CST
9154T
<5 cm
5 (21.7%)
10 (62.5%)
ERK
Abcam
ab184699
≥5 cm
18 (78.3%)
6 (37.5%)
p-ERK
CST
4370T
Receiving anti-HBV treatment before resection
Ras
CST
3339T
Entecavir
0 (0%)
2 (12.5%)
CDK4
CST
12790T
Adefovir Dipivoxil
0 (0%)
1 (6.3%)
CyclinA
CST
4656T
Lamivudine
1 (4.3%)
0 (0%)
CyclinB
CST
12231T
Recall lacking
2 (8.6%)
0 (0%)
CyclinD
CST
2978T
No
CyclinE
CST
4129T
Receiving non-resection treatment before resection
C-PARP
Abcam
ab32064
TACE
1 (4.3%)
2 (12.5%)
B-Actin
ProteinTech
Cat No. 66009-1-Ig
0 (0%)
ProteinTech
Cat No. 60004-1-Ig
Microwave
Ablation
1 (4.3%)
GAPDH
20 (87%)
21 (91.4%)
13 (81.3%)
Antibody For Co-immunoprecipitation
No
Assay
Enterprise
Detail
Receiving any RTKs inhibitors treatment before resection
14 (87.5%)
EGFR
santa
sc-373746
Yes
0 (0%)
0 (0%)
23 (100%)
16 (100%)
MET
CST
sc-8057
No
IgG
santa(M)/CST(R)
sc-2025/3900S
HCC metastasis existence before resection
p-EGFR
CST
3777T
Intrahepatic
thrombus
0 (0%)
1 (6.3%)
pMET
CST
3077T
Retroperitoneal
0 (0%)
1 (6.3%)
1 (4.3%)
0 (0%)
Antibody For Immunofluorescence
CD45
Abcam
52894
Common
bile duct
EpCAM
Abcam
187372
No
22 (95.7%)
14 (87.5%)
Antibody For Immunohistochemistry
EGFR
santa
sc-373746
MET
santa
sc-514148
Flow cytometry
as described [15] and all antibodies used were listed in Table 3. Markers
were used for indicating protein lower than 180 KD (PageRuler 26616,
Thermo Scientific, MA, USA) and more than 180 KD (PL00003, ProteinTech,
China).
All HCC cell lines were harvested and resuspended at a concentration of
105/ml. Trypsin digestion was performed in cells with or without
suspension at the same time to dissociate clusters into single cells and
reduce interference from trypsin. PI/RNAse was used for detecting cell
cycle and Annexin V/PI was used for detecting apoptosis of HCC cell lines.
Flow cytometry was performed according to the manufacturer’s
instructions.
Luminex, ELISA, Array kit, and Ras Activation Assay Kit
Luminex Kit (R&D system, CA, USA) was coated with RTKs (VEGFR1-3,
ErbB2-3, Tie, EphA2, and MCSFR) and RTKs ligands (EGF and HGF). ELISAs
(EGFR, MET, PDGFRβ, IGF-1R, and InsR) were used to detect the
concentration of RTKs which Luminex Kit didn’t contain. Phosphorylation
of RTKs was performed by Human/Mice Phospho-RTKs Array Kit (R&D
system, CA, USA) according to the manufacturer’s instructions. Image pro
plus 6.0 was used for intensity quantification. Ras Activation Assay Kit
(Sigma, MO, USA) was used for the detection of Ras activation.
Immunohistochemistry/Immunofluorescence
Paraffin specimens from patients with HCC and carcinoma tissues of
mice were performed according to the manufacturer’s instructions.
Immunofluorescence assays were conducted in a manner blinded to the
sample identity. Cells cultured with or without suspension were
harvested and dried on coverslips with Poly-L-lysine (Beyotime, China)
coated. EDU assay (KeyGEN, China) was used for detecting DNA
synthesis of HCC cell lines.
Cell Death and Disease (2022)13:351
S. Song et al.
11
Co-culture of HCC and leukocytes
Leukocytes were collected from healthy donor blood by gradient
centrifugation with leukocytes separation (Solarbio Technologies, China).
After being cultured with DMSO or inhibitors for 24 h, HCC cells lines were
re-suspended and the supernatant was discarded. Then, equal leukocytes
were added to the culture medium with MHCC97H cell line and were cocultured for 24 h in suspension. Cell mixtures were collected, dried on
coverslips with Poly-L-lysine coated, and observed by immunofluorescence
with EpCAM + EGFR and CD45 staining.
Animal xenograft model
5-week-old C57 male mice (Charles River Ltd, Beijing, China) were
employed to construct xenograft tumor models. The mice were
anesthetized with 50 mg/kg Zoletil®50 (VIRBAC, France) combined with
5 mg/kg Xylazine hydrochloride (Selleck, TX, USA) by intraperitoneal
injection, and 105 Hepa1-6 cells were injected into the left lobe of the
liver in each mouse. The animals were sacrificed after 60 days and the
presence of any extrahepatic metastasis except lungs was considered to
be a failure, even lung metastasis model was successfully established.
Tumor tissues from the liver and lung in the same mouse were collected
for phosphorylation detection, immunohistochemistry, immunofluorescence, and scRNA-seq.
Single-cell RNA sequence (scRNA-seq) and analysis
ScRNA-seq was performed by Annoroad Gene Tech (Beijing, China).
Carcinoma tissues were separated from the xenograft model in 48 h
and a library was constructed as manufacturer’s instructions. CellRanger was applied to identify cell barcodes in reads1 and UMI marks in
different transcripts, and the corresponding reads2 were compared
referring to genome through STAR to determine source gene of the
reads and complete quantification of gene expression. RBC in
cells containing <200 expressed genes or mitochondria UMI rate
>80% were excluded. Then, mitochondrial genes were removed in the
expression table. After normalizing UMI data of gene expression in
each cell by Seurat analysis and UMI data dimension reduction by PCA,
the top 20 principal components were selected for cell classification
and UMAP construction based on graph-based clustering method.
FindAllMarkers function by the Wilcox rank-sum test algorithm (lnFC >
0.25; p-value < 0.05; min. pct > 0.1) was used for marker gene
identification.
Clinical HCC specimens
Paraffin specimens and HCC resections of HCC tissues were collected
from the Department of Hepatobiliary Surgery, Shandong Provincial
Hospital, Cheeloo College of Medicine, Shandong University. Paraffin
specimens of HCC resection for IHC were obtained from patients with
further HCC metastasis within ten years. RTKs concentration of HCC
resections was determined by Luminex and Elisa. Details of the
clinicopathologic characteristics of the recruited HCC patients were
shown in Table 4.
Statistical analysis
GraphPad Prism 8 software (GraphPad, CA, USA) was used for data analysis.
The statistical significance of differences between different experimental
cell groups was determined by t-tests. Except results of scRNA-seq failed in
conforming to Gaussian distribution, all data were estimated of variation
within each group of data, and P-value used in all analyses was two-tailed,
and the value <0.05 was considered statistically significant. As for the
results of scRNA-seq, a Mann-Whitney test was used for analyzing gene
expression differences among samples.
DATA AVAILABILITY
The datasets used and/or analyzed during the current study are available from the
corresponding author on reasonable request.
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AUTHOR CONTRIBUTIONS
CQ performed study concept and design; QZ performed methodology and guided
writing, review of the paper; SS performed the major experiment and statistical
analysis; ZY established mice xenograft model; YY, CL, XX, HL and FX provided
technical and material support. All authors read and approved the final paper.
FUNDING
This work was supported in part by grants from the National Natural Science
Foundation of China (81772626, 82160124 and 81770607), the Major Special Plan of
Science and Technology of Shandong Province (2015ZDXX0802A01), the Clinical
Medical Science and Technology Innovation Program (202019094), and WBE Liver
Fibrosis Foundation (CFHPC2021011).
COMPETING INTERESTS
The authors declare no competing interests.
CONSENT TO PARTICIPATE
Protocols dealing were in accordance with guidelines of the Institutional Animal Care
and Use Committee and were approved by the Medical Ethics Committee of
Shandong Provincial Hospital, Cheeloo College of Medicine, Shandong University.
ETHICS APPROVAL
Written informed consents were obtained from all patients before the research was
initiated. Protocols were in accordance with the Helsinki Declaration and approved
by the Medical Ethics Committee of Shandong Provincial Hospital, Cheeloo College of
Medicine, Shandong University.
ADDITIONAL INFORMATION
Supplementary information The online version contains supplementary material
available at https://doi.org/10.1038/s41419-022-04796-8.
Correspondence and requests for materials should be addressed to Qiang Zhu or
Chengyong Qin.
Reprints and permission information is available at http://www.nature.com/
reprints
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© The Author(s) 2022
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